Mechanism Studies of Antitubulin Agents-Mediated MMP Down-Regulation and Nitroxoline Repurposing in Human Prostate Cancer Cells

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Mechanism Studies of Antitubulin Agents-mediated MMP Down-regulation and Nitroxoline Repurposing in Human Prostate Cancer Cells

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Wei-Ling Chang, M.S.

Graduate Program in Pharmacy

The Ohio State University

2015

Dissertation Committee:

Ching-Shih Chen, Ph.D., Advisor

Kari R. Hoyt, Ph.D.

Karl Werbovetz, Ph.D.

Jack C. Yalowich, Ph.D

Wei-Ling Chang

2015

Abstract

Prostate cancer is a common male tumor worldwide. Tubulin-binding drugs demonstrated palliative responses of decreasing prostate-specific antigen (PSA), improvement of life quality, and extension of survival in advanced hormone-refractory prostate cancer patients. Because metastasis accounts for the majority of prostate cancer-related deaths, targeting the critical step of tumor metastasis is a potential therapeutic approach. Several tubulin-binding agents show the ability to inhibit matrix metalloproteinases (MMPs), which are the zinc-dependent endopeptidases and involved in angiogenesis and cancer invasion. However, their mechanisms remain to be further elucidated. In the first study, three tubulin-binding agents, paclitaxel, vincristine, and evodiamine, were used to explore the mechanism of the down-regulation of MMP-2 and MMP-9 protein expression.

Tubulin-binding agents decreased the protein levels of MMP-2 and -9 but not their mRNA levels. Treatment with tubulin-binding agents caused an increase of mitotic proteins, including cyclin B1, MPM2 (mitosis-specific phosphoprotein), and phosphorylation of Plk1. The inversed correlations were shown between the reduced

MMP-2 and -9 expression and the elevated expression of mitotic proteins. Blocking the mitotic entry via treatment with MG132 prevented the down-regulation of MMP-2 and -9 expression. Further, synchronization of cell cycle by thymidine block or nocodazole treatment demonstrated that the exit from mitotic phase rescued the down-regulation of

MMP-2 and -9 expression. Moreover, inhibition or knockdown of cyclin-dependent

ii kinase1 (Cdk1), a key regulator in mitotic phase, significantly inhibited the reduction of

MMP-2 and -9 expression induced by tubulin-binding agents. Taken together, the data suggest that the down-regulation of MMP-2 and -9 expression induced by tubulin- binding agents is associated with the mitotic entry of the cell cycle and, furthermore,

Cdk1 may play a central role in this down-regulatory mechanism.

Despite taxanes-based chemotherapies showing benefits in patients with advanced prostate cancer, the disease are progresses in the majority of the patients with metastatic cancers. Currently, drug development faces the challenge of high failure rates and huge expenditures. Drug repurposing is an alternative approach for drug development.

Nitroxoline is an old antibiotic that is used in treatment of urinary tract infection. In the second study, nitroxoline induced cell cycle arrest in G1 phase and resulted in apoptosis in both hormone-sensitive and hormone-refractory prostate cancers. Nitroxaline induced the inhibition of cyclin D1-Rb-Cdc25A axis. The decrease of cyclin D1 protein expression and induction of apoptosis were related to nitroxoline-mediated AMPK activation since knockdown of AMPK prevented the down-regulatory effect of cyclin D1 as well as the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Besides, nitroxoline induced DNA damage and Chk2 activation, but did not result in the formation of -H2AX. The activation of Chk2 was associated with AMPK activation, and it also participated in nitroxoline-induced apoptosis. In summary, the data suggest that nitroxoline is a potential candidate for treatment of prostate cancer. The anticancer activity of nitroxoline is mediated by AMPK-dependent inhibition of cyclin D1-Rb-

Cdc25A axis, leading to cell cycle arrest as well as apoptosis. AMPK-dependent activation of Chk2, which acted as a pro-apoptosis inducer, partially contributes to apoptosis in prostate cancer cells.

iii

Dedication

This document is dedicated to my family.

Acknowledgments

I would like to thank to Dr. Ching-Shih Chen, Dr. Jih-Hwa Guh, and the members of both laboratories for the advice, support and help.

Vita

2006...... B.S. Pharmacy, National Taiwan University

2008...... M.S. Pharmacy, National Taiwan University

2008 to 2009 ...... Research Associate, Institute of Biomedical

Science, Academia Sinica, Taiwan

2009 to present ...... Graduate Research Associate, Department

of Pharmacy, The Ohio State University

Publications

Chang WL, Yu CC, Chen CS, Guh JH, Tubulin-binding agents down-regulate matrix metalloproteinase-2 and-9 in human hormone-refractory prostate cancer cells-a critical role of Cdk1 in mitotic entry. Biochemical Pharmacology. 2015; 94, 1, 12-21.

Guh JH, Chang WL, Yang J, Lee SL, Wei S, Wang D, Kulp SK, Chen CS, Development of novel adenosine monophosphate-activated protein kinase activators, J. Med. Chem.

2010; 53, 2552-61.

Chang WL, Chang CS, Chiang PC, Ho YF, Liu JF, Chang KW, Guh JH.. 2-Phenyl- 5-

(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl) -1H-benzimidazole, a benzimidazole derivative, inhibits growth of human prostate cancer cells by affecting tubulin and c-Jun

N-terminal kinase. Br J Pharmacol. 2010; 160:1677-89

Hsiao CJ, Ho YF, Hsu JT, Chang WL, Chen YC, Shen YC, Lyu PC, Guh JH. Mana-Hox displays anticancer activity against prostate cancer cells through tubulin depolymerization and DNA damage stress. Naunyn Schmiedebergs Arch Pharmacol 2008;

378:599-608.

Fields of Study

Major Field: Pharmacy

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vi

Table of Contents ...... viii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1 introduction ...... 1

1.1 Prostate cancer ...... 1

1.2 Molecular pathogenesis in prostate cancer cells ...... 2

1.3 Cytotoxic chemotherapy for prostate cancer ...... 3

1.4 Novel identified therapies for prostate cancer ...... 5

Chapter 2 Anti-tubulin agents down-regulate protein levels of matrix metalloproteinase-2 and 9 in hormone refractory prostate cancer cells ...... 7

2.1 Matrix metalloproteinases (MMPs) ...... 7

2.2 MMP-2 and MMP-9 (Gelatinase A and Gelatinase B) ...... 9

viii

2.3 Tubulin-binding agents ...... 11

2.4 Hypothesis and specific aims ...... 15

2.5 Materials and methods ...... 16

2.6 Experimental results...... 20

2.6.1 Tubulin-binding agents down-regulate the protein levels of MMP-2 and -9 .20

2.6.2 Inhibition of several kinases does not contribute to the down-regulation of

MMP-2 and -9 protein expression ...... 21

2.6.3 Mitotic protein levels inversely correlate to protein expression of MMP-2 and

MMP-9 ...... 22

2.6.4 MG-132 prevents mitotic entry and inhibits down-regulation of MMP-2 and -9

expression ...... 23

2.6.5 Recovery of protein expression of MMP-2 and -9 after mitotic exit ...... 24

2.6.6 Cdk1 phosphorylation at Tyr15 shows a high correlation to MMP-2 and -9

protein levels ...... 25

2.7 Discussion ...... 26

Chapter 3 : Repurposing of nitroxoline as a potential anticancer agent against human prostate cancer cells ...... 33

3.1 Drug reposition ...... 33

3.2 5' AMP-activated protein kinase (AMPK), mTOR, and autophagy ...... 37

3.3 DNA check point and Checkpoint kinase 2 (Chk2)...... 40

3.4 Hypothesis and specific aims ...... 41

3.5 Materials and Methods ...... 42

3.6 Experimental results...... 49

3.6.1 Nitroxoline shows the most effective anti-proliferative and cytotoxic effects

compared to other antibiotics...... 49

3.6.2 Nitroxoline induces cell cycle arrest and apoptosis in prostate cancer cells. ...49

3.6.3 Inhibition of cyclin D1-Rb-Cdc25A axis contributes to nitroxoline-induced G1

arrest...... 50

3.6.4 Nitroxoline induces AMPK activation and cyto-protective autophagy...... 51

3.6.5 Nitroxoline increases the phosphorylation of Chk2 and induces DNA damage.

...... 53

3.6.6 ZnCl2 supplements do not affect nitroxoline-mediated anti-proliferative effect

and cellular signaling...... 55

3.7 Discussion ...... 56

Chapter 4: Conclusion...... 63

References ...... 105

List of Tables

Table 1. The correlation coefficient (r) between two variables in DU-145 cells...... 65

Table 2. Effect of selected antibiotics on cell proliferation in human prostate cancer cells.

...... 66

Table 3. Effect of nitroxoline on cell proliferation in human prostate cancer cells...... 67

List of Figures

Figure 1. Tubulin-binding agents cause down-regulation of MMP-2 and MMP-9 protein expression...... 68

Figure 2. Tubulin-binding agents do not change the mRNA levels of MMP-2 and -9. ... 69

Figure 3. Tubulin-binding agents do not alter the extracellular levels of MMP-2 and -9. 70

Figure 4. The effects of tubulin-binding agents on cell apoptosis ...... 71

Figure 5. The reduction of MMP-2 and -9 levels are not related to phosphorylation...... 72

Figure 6. Effects of tubulin-binding agents on the phosphorylations of NF-kB and c-Jun.

...... 73

Figure 7. Tubulin-binding agents decrease the phosphorylations of mTOR, Akt and p38.

...... 74

Figure 8. Effects of several inhibitors on the phosphorylations of mTOR, Akt and p38 and the expression of MMP-2 and -9...... 75

Figure 9. The expression or phosphorylation of mitotic proteins induced by tubulin- binding agents correlate to MMP-2 and -9 down-regulations...... 76

Figure 10. Co-treatment with MG-132 blocks the mitotic entry and prevents the down- regulations of MMP-2 and MMP-9...... 78

Figure 11. Mitotic exit rescues the reduction of the MMP-2 and -9 protein expression. . 80

xii

Figure 12. The expression of MMP-2 and -9 are decreased when cell cycle progress into mitotic phase...... 81

Figure 13. The increase of phosphorylation at Thr161 and decrease of phosphorylation at

Y15 of Cdk1induced by tubulin-binding agents correlate to MMP-2 and -9 down regulations...... 82

Figure 14. Treatment with roscovitine or transfection with Cdk1 siRNA prevent the down-regulation of MMP-2 and MMP-9 ...... 83

Figure 15. Effects of selected antibiotics on cell viability in PC-3 cells...... 84

Figure 16. Effects of selected antibiotics on cell cycle distribution in PC-3 cells...... 85

Figure 17. Effects of nitroxoline on anti-proliferation in prostate cancer cells...... 86

Figure 18. Effect of nitroxoline on anti-proliferation...... 89

Figure 19. The long-term exposure effect of nitroxoline on cell proliferation...... 88

Figure 20. Effects of nitroxoline on cell cycle profile in prostate cancer cells...... 89

Figure 21. Effect of nitroxoline on the protein expression of cell cycle progression regulators in PC-3 cells...... 90

Figure 22. Effect of nitroxoline on the protein expression of cell cycle progression regulators in LNCaP cells...... 91

Figure 23. Effects of nitroxoline on the protein expression of mTOR, AMPK, Chk2 and autophagy pathway in PC-3 cells...... 92

Figure 24. Effects of nitroxoline on the protein expression of mTOR, AMPK, Chk2, and autophagy pathways in LNCaP cells...... 93

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Figure 25. Examination the effects on protein expression of nitroxoline-mediated AMPK activation...... 94

Figure 26. Effects of nitroxoline on autophagy-mediated cyto-protection...... 95

Figure 27. Effect of nitroxoline on DNA damage response...... 96

Figure 28. Effect of nitroxoline on ROS production...... 97

Figure 29. Effects of nitroxoline on the formation of TOPO-DNA cleavable complexes.

...... 98

Figure 30. Examination of the effects on protein expression of nitroxoline-mediated Chk2 activation...... 100

Figure 31. Effects of nitroxoline on protein expression of p-Chk2 and -H2AX...... 102

Figure 32. Effect of ZnCl2 supplement on nitroxoline-related anti-cancer effects...... 103

Figure 33. Summary of the anti-cancer effects of nitroxoline...... 104

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Chapter 1

Introduction

1.1 Prostate cancer

Prostate cancer is a common cancer among men. In 2014, there were 233,000 estimated new prostate cancer cases, accounting for 27% of all newly diagnosed cancers [1].

Prostate cancer was the second leading cause of cancer-related death in the United States in 2014. The majority of prostate cancer patients are elderly men (>60 years old) and the disease is also related to ethnicity and heredity [1]. African Americans and the people with family history of either prostate or breast cancers have an increased risk of prostate cancer [1,2]. The early stage of prostate cancer is asymptomatic. Prostate specific antigen

(PSA), which is a glycoprotein secreted by benign or malignant prostate gland tissue, is widely used in screening the incidence of prostate cancer. It is also useful for categorizing the stage of prostate cancer, monitoring the response of treatment, and evaluating the prognosis [3,4]. Because PSA is not only specific to enlarged or malignant prostate tissue but also elevated by other factors, other histological tests are applied together to confirm the diagnosis of prostate cancer [2]. The major treatments of primary prostate cancer are radiation or surgery to remove the tumor. However, about 10-20% of prostate cancer patients develop metastasis [4]. Usually, androgen ablation is able to

1 improve the symptoms and to decrease PSA. However, the disease usually progresses and becomes resistant to hormone treatment, referred as hormone-refractory prostate cancer

(HRPC). Once tumor progresses, the estimated median survival time for HRPC patients is approximately 16 months [3]. The majority goals of HRPC treatment are symptom palliation and life quality improvement. The treatment options of HRPC include supportive care, radiation, second line hormonal therapy, cytotoxic chemotherapy, and novel discovered target therapy [3].

1.2 Molecular pathogenesis in prostate cancer cells

Several chromosomal abnormalities have been suggested to associate with development of prostate tumors. Mutation or over-expression of oncogenes contributes to the initial transformation of preneoplastic lesions [5]. Amplification of anti-apoptotic Bcl-2 is shown in many HRPC cells and is associated with lower survival rate in prostate cancer patients [6]. Approximately 30-70% of prostate cancer has Her2/neu oncogene over- expression, which is associated with the growth of HRPC and the recurrence of the disease [5]. Tumor suppressor genes, including p53, Rb, p16 and PTEN, are more frequently detected in prostate cancer cells and are highly related to advanced tumors. It has been reported that p53 abnormality is found in 70% of metastatic tumors, which occurs approximately twice of the frequency than in primary prostate tumors [7]. Further, p16 reduction and PTEN mutation are two of the most frequently altered genes in prostate cancer [5]. Evidence indicates that 43% of untreated prostate cancer exhibit a decrease of p16 mRNA expression [8], and 60% PTEN mutation has been demonstrated

2 in prostate cancer xenografts or cell lines [9]. For example, three established prostate cancer lines, LNCaP, DU145, and PC-3, display different expression profiles of these oncogenes or tumor suppressor genes. PC-3, a hormone-refractory prostate cancer cell line derived from bone metastasis, has been widely used in many laboratories. PC-3 shows p53 and PTEN null, mutation of p16, and over-expression of Bcl-2. Another frequently used HRPC, DU145, demonstrates mutation of p53 and Rb. A hormone- sensitive prostate cancer cell line, LNCaP, shows normal expression of p53, low expression of p16 and over-expression of Bcl-2 [3,5]. Moreover, alterations of adhesion molecules and proteases, including integrins, cadherin and matrix metalloproteinase

(MMPs), have been reported in a variety of prostate cancer cells such as reduced expression of E-cadherin and over-expression of MMPs [3].

1.3 Cytotoxic chemotherapy for prostate cancer

Metastatic prostate cancer has been regarded as chemoresistant malignancy. Many early chemotherapy trials, including 5-fluorouracil, gemcitabine, irinotecan, and oral etoposide, have demonstrated low response rates in HRPC patients [3]. Clinical trials of mitoxantrone have revealed the palliative benefit, in which mitoxantrone improves the quality of life and relieves the symptoms although it does not show significant prolongation of survival [3]. Estramustine, which targets microtubule-associated proteins and causes microtubule disassembly, shows palliative benefit in a clinical trial with increased response rates [3,10]. Combination of estramustine with other agents shows inconsistent results in clinical trials. Some of them have shown clinical benefits on decreasing PSA or improving survival in estramustine combined with etoposide or anti- 3 tubulin agents (i.e. vinca alkaloids) [3,10,11]. Taxanes have been widely studied in clinical trials either used as a single agent or combination with estramustine in HRPC patients. Several clinical trials of paclitaxel with estramustine have demonstrated PSA response rates of 37%-56% and 42%-49% response rates for measureable diseases [3]. In addition to the response rates, two clinical studies (TAX327 and SWOG9916) have demonstrated that docetaxel is able to significantly prolong the survival in patients with

HRPC [11,12]. SWOG9916 is a study in comparison of two combined treatments, docetaxel/estramustine versus mitoxantrone/prednisolone. It shows that docetaxel/estramustine not only exhibits a higher PSA response rate but also improves both overall survival and disease-free survival. TAX327 is a randomized phase III trial, comparing the survival rates of mitoxantrone and two different schedules of docetaxel administration with prednisolone in HRPC patients. For the regimens of the docetaxel administration, one is weekly docetaxel (30 mg/m2) for five out of six weeks and the other is docetaxel (75 mg/m2) for every 3 weeks. Response rates of both PSA and quality of life are higher in the docetaxel arms than those in the mitoxantrone arm. A significant increase of survival has been shown in three weekly docetaxel group but not in weekly docetaxel group compared to mitoxantrone [3,4]. Although the first-line docetaxel-based chemotherapy shows therapeutic benefit in HRPC patients, the cancer progresses eventually. Docetaxel is also an option for HRPC patients, who received mitoxantrone or other agents as first-line therapy. In addition to docetaxel, mitoxantrone, satraplatin, and ixabepilone are also used as second-line chemotherapy agents [4].

1.4 Novel identified therapies for prostate cancer

So far, there is no curative treatment for HRPC. Although docetaxel-based therapies demonstrate benefits for HRPC patients, the improvement of survival remains unsatisfied.

The development of new agents, new combination regimens or new approaches such as vaccines and immunotherapies are still under study and several clinical trials are ongoing.

Many small molecule inhibitors for novel cellular targets have been identified. The proteasome inhibitor, bortezomib, induces growth arrest and apoptosis in prostate cancer cells. It inhibits IκBα degradation and results in suppression of cytoplasmic NF-κB [13].

Phase I clinical trial in combination of bortezomib and other cytotoxic agent is ongoing.

Flavopiridol, which targets cyclin-dependent kinase, shows growth inhibition effect against prostate cancer cells [14]. Phase I study of the flavopiridol in combination with paclitaxel has shown responses in patients with advanced prostate tumors. [15]

Atrasentan, a antagonist of endothelin A receptor, enhances the anti-cancer effects of docetaxel in vitro and in vivo [16]. It has shown that PSA progression was delayed in a clinical trial of atrasentan in prostate cancer patients [3,16]. Clinical trial of combination with atrasentan and docetaxel is ongoing. BMS-247550, an epothilone B analogue and causing microtubule stabilization, has demonstrated the anti-cancer activity in taxane- resistant cancer cells. Phase II trial of BMS-247550 revealed an acceptable toxicity profile, and the activity of single-agent was observed in patients with metastatic prostate cancer [17]. Inhibiting metastasis through targeting MMPs is under investigation.

Although phase III clinical trial of the MMP inhibitor, prinomastat, combined with mitoxantrone/prednisolone does not show a significant response on survival [18],

5 preclinical results of more specific MMP inhibitors show clinical potential [3,19].

In recent decades, the response rates of HRPC patients have been improved because of the development of PSA screening and more effective chemotherapy regimen. However, the advanced prostate cancer is still the leading cause of cancer-associated death showing that discoveries of new cellular targets, identification of novel anticancer mechanisms, and development of new drugs are in urgent need.

Chapter 2

Anti-tubulin agents down-regulate protein levels of matrix metalloproteinase-2 and -9 in hormone refractory prostate cancer cells

2.1 Matrix metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases, which have been identified by the function of inducing breakdown of extracellular matrix

(ECM). ECM degradation is an important process for tumor cells to invade neighboring blood vessels and to migrate to other locations. Therefore, excessive activation of MMPs is frequently associated with tumor metastasis [20–22]. Currently, 23 human MMPs have been discovered [20,21]. According to the structure and substrate specificity, human

MMPs have been categorized into several groups: collagenases, gelatinases, membrane- type (MT) metalloproteinases, stromelysins, and others [23,24]. The structure of MMPs contains three conserved domains: propeptide domain, catalytic domain and hemopexin- like C-terminal domain (PEX) [20]. The catalytic domain contains a conserved zinc binding site and is responsible for the hydrolysis of substrates. The propeptide domain, or

N-terminal domain, has a cysteine residue that regulates the latency of MMPs. The C- terminal domain, responsible for substrate specificity, is able to bind MMP substrates, to interact with tissue inhibitor of metalloproteinases (TIMPs), and to involve in activation

7 of MMPs [25–28].

MMPs are secreted as inactivate forms, in which the activities are prohibited by an interaction of the catalytic zinc and the cysteine in propeptide domain. The zinc and cysteine residue form a covalent bond, which blocks the entrance of water molecule in the catalytic site. Disruption of the zinc-cysteine interaction, called cysteine switch, is essential for the activation of MMPs, which can be achieved by either cleaving the propeptide domain via tissue- or plasma-serine proteases or destroying the cysteine-zinc interaction using non-physiological reagents [22,23,29].

Active MMPs selectively degrade the structural component of ECM and enable cell migration or invasion. Recent studies demonstrate that the functions of MMPs not only cleave ECM components but also involve in many signaling pathways. MMPs cleave and release many bioactive molecules such as cytokines and chemokines that are embedded within ECM [20,21,23,30]. Furthermore, MMPs induces the proteolytic activation of several latent growth factors, pro-inflammatory cytokines, chemokines, and cell surface proteins such as pro-TNFα, pro-TGF-β, pro-IL-1β, EGFR ligand, and E-cadherin [21].

MMP-2 and -9 activate TGF-β1 by proteolysis and promote cancer invasion as well as angiogenesis [31]. Therefore, MMPs participate in regulation of many important cellular physiological events, including cell growth, survival, metastasis, invasion, angiogenesis and inflammation. Because of the multiple functions of MMPs, aberrant regulations of

MMPs are associated with many diseases such as arthritis, coronary artery and heart disease, lung disease, cancer, and brain disorders [32].

2.2 MMP-2 and MMP-9 (Gelatinase A and Gelatinase B)

Increasing evidence shows that MMPs, especially MMP-2 and -9, are potential cancer biomarkers which are closely involved in cancer progression [20,21,33]. The structures of

MMP-2 and -9 contain an additional collagen binging domain (CBD) that is bound by collagenous substrates, elastin, fatty acids and thrombospondins [20]. The expression, post-modification, and substrate specificity between MMP-2 and -9 are not identical.

MMP-2 is ubiquitously and constitutively expressed and is modestly increased or decreased in various conditions [34,35]. MMP-9 expression is induced by growth factors, chemokines, pro-inflammatory cytokines and other signals [35,36]. MMP-9 contains two

N-glycosylation and several O-glycosylation sites, which are important for protein interaction and contribute to substrate specificity [36]. In contrast, MMP-2 is non- glycosylated. Recent studies have shown that MMP-2 activity is regulated by S- glutathiolation with exposure to peroxynitrite (ONOO2), or by phosphorylation which is catalyzed by protein kinase C (PKC) [37,38].

MMP-2 and -9 are involved in many physiological and pathological processes and participate in reproduction, development, inflammation, leukocyte mobilization, and wound healing [35]. Both MMP-9 and -2 knockout mice are viable but show some abnormalities. Mice with MMP-9 deficiency exhibit bone-development defects and slower healing of bone fracture [28]. MMP-2 knockout mice show relatively minor defects, including an osteolytic syndrome, as well as reduced body size and lung saccular development [28].

Up-regulation of MMP-2 and -9 are reported in many types of cancer cells, including cancers of prostate, breast, ovary, pancreas, colon, and brain. The activities and expression of MMP-2 and -9 are related to poor prognosis and tumor aggressiveness

[20,33]. Both MMPs play multiple roles during different stages of tumor progression, including initiation of primary tumorigenesis, angiogenesis, metastasis as well as establishment of supportive microenvironments for metastasis [35]. Evidence shows that

MMP-2 is an important regulator for early step of metastasis in ovarian cancer cells, in which MMP-2 enhances peritoneal adhesion via cleavage of ECM proteins. Inhibition of

MMP-2 through siRNA transfection or treatment with antibody against MMP-2 reduces the attachment of cancer cells [39]. In addition, inhibition of MMP-2 also suppresses tumor growth and metastasis. The survival in the in vitro mouse model is prolonged as well [39]. Itoh and colleagues found that the angiogenesis and tumor progression were decreased in mice with MMP-2 deficiency [40]. MMP-9 deficient mouse model also shows an ability to reduce the formation of adenoma in heterozygous APC (APC-min) knock out mice [41]. Knockdown of MMP-9 by siRNA transfection induces proteolytic activation of caspase-8 and caspase-3, and cleavage of poly (ADP-ribose) polymerase, leading to apoptosis in human glioblastoma xenograft cell lines [42].

Because MMPs are involved in many cellular functions in cancer biology and increasing evidence supports that inhibition of MMPs induces inhibitory activity in cancers, several

MMP inhibitors (MMPIs) have been designed and developed. However, a lot of clinical trials based on MMPIs have ended up with unsatisfying results. MMPI-mediated musculoskeletal toxicity in the phase I clinical trial limits the dosages administrated in

10 subsequent trials, and it may account for the failure of MMPI [20,23,43]. Furthermore,

MMPs are involved in complicated regulation during tumor progression, showing pro- or anti-tumor activities in different conditions. The lack of comprehensive understanding about MMPs and the use of broad-spectrum MMPI are also associated with the failure of the trials [35]. Notably, the patients enrolled in some MMPI clinical trials were in terminal cancer stage, which involves more complicated interaction in related signaling pathways [20,35]. It has been demonstrated that batimastat, an MMPI, is less effective at the later stage of tumor in mouse model [44]. Therefore, the anti-tumor potential of

MMPIs may be underestimated because of the inappropriate design of clinical trials. In addition to discovering more selective and potent MMPIs, it is important to further elucidate the role of MMPs in specific tumor stage in order to develop optimal trials for application of MMPIs in cancer therapy [23,43].

2.3 Tubulin-binding agents

Tubulin-binding agents as single or combined treatment are used for cancer chemotherapy. This group of agents have been categorized into two groups, microtubule- stabilizing agents and microtubule-destabilizing agents, according to the effects on microtubule dynamics [45]. Taxaens, such as paclitaxel and docetaxel, are well-known microtubule-stabilizing agents for the treatment of cancers of prostate, breast, liver and lung. Vinca alkaloids, including vinblastine and vincristine, are microtubule-destabilizing agents for haematological malignancies [45,46]. Microtubule is a component of the cytoskeletons, which is critical in maintaining cell shapes and in participating in many

11 cellular processes. The regulation of dynamic assembly or disassembly of microtubules is crucial for mitosis, intracellular trafficking, exocytosis, and cell mobility. The anticancer effect of tubulin-binding agents is predominantly through interference with the function of microtubules. After binding to tubulin, the agents induce the disruption of microtubule dynamics, leading to mitotic arrest associated with abnormal mitotic spindle formation and a subsequent apoptosis [45]. In addition to inducing cancer cell death, the agents also suppress tumor progression by anti-vascular effects. They induce antiangiogenic activities not only through the inhibition of cellular functions in endothelial cells, including proliferation, migration and tube formation, but also through the suppression of HIF-1 expression and the secretion of pro-angiogenesis factors [45,46].

Paclitaxel

Paclitaxel was isolated from the bark of Taxus brevifolia (Pacific Yew Tree) and identified in 1967 to show a broad anticancer spectrum against various cancers. Paclitaxel stabilizes microtubule by binding β-tubulin within the lumen of microtubule and prevents microtubule disassembly. Besides anti-tubulin properties, paclitaxel can also decreases the viability of cancer cells through inhibition of glycolysis by decreasing the level of important glycolysis regulators, leading to the decrease of cellular ATP [47,48].

Furthermore, paclitaxel inhibits androgen receptor (AR) activity via inducing the accumulation of FOXO1, a transcriptional repressor of AR, in nucleus in PTEN-positive castration-resistant prostate cancer cells [49].

The ability of paclitaxel to inhibit tumor metastasis has been widely studied. The antiangiogenic effect of paclitaxel has been demonstrated in both in vivo mouse models [50]

12 and in chick embryo chorioallantoic membrane (CAM) models [51]. Paclitaxel inhibits proliferation, invasion, and cord formation on matrix gel as well as reduces the secretion of MMP-2, MMP-9, and u-PA in endothelial cells [50,51]. Paclitaxel also suppresses corneal neovascularization induced by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in mouse models [52]. Furthermore, paclitaxel suppresses the attachment and migratory activity of the cancer cells and alters the location of MMP-2 [53].

Vinca alkaloids

Vinca alkaloids are a group of molecules, isolated from Catharanthus roseus G. Don

(Madagascar periwinkle plant). They are used in several diseases such as diabetes, high blood pressure, and cancer. Currently, vincristine, vinblastine, vinorelbine, and vindesine are used in cancer treatment. Only vincristine, vinblastine, and vinorelbine have been approved in the United States [54]. Binding to tubulin by vinca alkaloids results in disruption of microtubule network. Different from paclitaxel binding site, vinca alkaloids bind to central portion of β-tubulin subunit of α/β-tubulin heterodimer and cause microtubule disassembly [55]. Vinca alkaloids also display anticancer activities, which are unrelated to the anti-tubulin property. It has been reported that vinca alkaloids inhibit calmodulin-dependent ATPase by binding to calmodulin and Ca2+-dependent ATPase

[56]. Vincristine treatment induces ROS production and AMPK activation, leading to cell apoptosis [57]. Furthermore, it has been suggested that NF-B/IB signaling pathway also participates in vinca alkaloid-mediated apoptosis in the cancer cells [58].

The potentials of inhibiting metastasis of vinca alkaloids have been extensively studied.

Exposure of cancer cells to vinblastine, vincristine or vindesine reduces the invasiveness in three-dimensional culture in mouse fibrosarcoma cells [59]. The anti-angiogenic effect of vinca alkaloids also has been reported in CAM model [60]. Low dose of vinblastine treatment in the mouse model with neuroblastoma cell xenografts has demonstrated a significant delay of tumor growth, decrease of tumor vascularity, and inhibition of angiogenesis [61].

Evodiamine

Evodiamine is a major active component of the traditional Chinese herb, Evodia rutaecarpa (Wu-Chu-Yu), which has been used in treatment of headache, amenorrhea, postpartum hemorrhage, gastrointestinal disorders and dysentery [62]. The anticancer effect of evodiamine has been demonstrated in many studies. It inhibits tumor development by suppressing cell proliferation, metastasis and invasion, and inducing apoptosis in various cancer cells [63–73]. Several studies reveal that evodiamine treatment induces cell cycle arrest in G2/M phase in cancer cells of colon, prostate, thyroid, gastric, and lung [63,64,66,67,73]. Evodiamine interrupts microtubule function, results in mitotic arrest and ultimately induces cell apoptosis [72,73]. In addition to the anti-tubulin function, evodiamine displays various activities such as inhibition of NF-B pathway and topoisomerase I, induction of endoplasmic reticulum (ER) stress, ROS production and iNOS expression. [67–69,71] The ability of evodiamine to inhibit angiogenesis has been demonstrated by reducing tube formation in HUVEC cells and in

CAM model [74]. Evodiamine therefore has been considered as a potential chemotherapeutic agent for the treatment of cancer cells.

2.4 Hypothesis and specific aims

MMP-2 and -9 are promising anticancer targets based on a variety of in vitro and in vivo studies. Because of their complicated mechanism, it is important to further understand the regulation of MMP-2 and MMP-9. Many studies have suggested that tubulin-binding agents, including taxanes and vinca alkaloids induce the down-regulation of MMP-2 and

-9 protein levels that contributes to the anti-metastatic effects by these agents. However, the mechanism has not yet been fully explored. In this study, we attempt to test the hypothesis that the down-regulation of MMP-2 and -9 protein levels is a common effect induced by tubulin-binding agents. We aim to identify the mechanism and to discover the key regulators in human hormone-refractory prostate cancer (HRPC) cells with the compound treatment.

Specific aim 1: To use three anti-tubulin agents, paclitaxel, vincristine and evodiamine, to

test their effects on MMP-2 and -9 expression in HRPC cell lines, PC-3

and DU145.

Specific aim 2: To identify the mechanism of MMP down-regulation and to examine the

role of mitotic arrest on the signaling pathways. Approaches for cell

synchronization have been performed to understand how cell cycle

progression regulates MMP expression.

Specific aim 3: To perform siRNA transfection to determine the functional role of target

protein (or kinase).

2.5 Materials and methods

Materials

RPMI 1640 medium and fetal bovine serum (FBS) were purchased from GIBCO/BRL

Life Technologies (Grand Island, NY). Chemical compounds were from Sigma-Aldrich

(St. Louis, MO). Primary antibody against cyclin B1 as well as horseradish peroxidase conjugated secondary antibodies including anti-mouse and anti-rabbit IgGs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to MMP-2 and MMP-9 were purchased from Millipore (Bedford, MA.). Antibody against mitotic protein monoclonal 2 (MPM2) was obtained from BD Biosciences PharMingen (San

Diego, CA). Antibodies against p-Ser473-Akt, p-Ser2448-mTOR (mammalian target of rapamycin), pThr180/Tyr182-p38, p-Thr210-Plk1 (polo like kinase), Cdk-1, p-Tyr15-

Cdk1, p-Thr161-Cdk1 and GAPDH were obtained from Cell Signaling Technologies

(Boston, MA).

Cell lines and cell culture

PC-3 and DU145 cells were obtained from American Type Culture Collection (ATCC,

Rockville, MD). Both cells were cultured in RPMI 1640 medium containing 10% FBS

(v/v), penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells were cultured at 37oC in an incubator with 5% CO2/95% air.

Cell cycle synchronization

Thymidine block and nocodazole were used to synchronize the cell cycle at late G1 or the mitotic phase. Cells were treated with 2 mM thymidine or 0.25 μM nocodazole for 24 hour and then washed by PBS once followed by incubation of fresh RPMI medium with

10% FBS. After release from synchronization, cells were harvested at the indicated time and fixed for propidium iodide (PI) staining to analyze the cell cycle progression or processed for Western blotting to detect the protein expression.

Flow cytometric assay of DNA content

Cells treated with the indicated agents were collected with 0.05% trypsin at the indicated time point and fixed with 70 % (v/v) alcohol at -20 oC for 30 min. Fixed cells were washed by PBS and resuspended by propidium iodide solution (80 μg/ml propidium iodide, 0.1 % v/v Triton X-100, and 100 μg/ml RNase). DNA content of cells were detected by flow cytometry and analyzed by CellQuest software (Becton Dickinson,

Mountain View, CA).

Western blotting and quantifications of protein expression

Cells were collected with trypsinization after treatment with indicated agents, and then washed with PBS. The collected cell pellets were lysed on the ice for 30 minutes by lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EGTA, 1 % Triton X-100) containing protease inhibitors and phosphatase inhibitors (1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 100 μM sodium orthovanadate and 50 mM NaF). The

17 lysed samples were centrifuged at 13,000 rpm for 30 minutes and the protein concentrations were determined using Bradford assay (Bio-Rad Laboratories, Inc.,

Hercules, CA). Protein samples were mixed with 5X sample buffer and boiled at 90 oC for 5 minutes. Samples with equal amount of proteins were separated by electrophoresis using 10 % SDS-polyacrylamide gels, transferred to PVDF membranes, and incubated with 5% non-fat milk. Membranes were probed with specific primary antibodies and then incubated with appropriate horseradish peroxidase conjugated-secondary antibodies, followed by visualization with an enhanced chemiluminescence detection kit (Amersham,

Buckinghamshire, UK). Bands were quantified using ImageQuant software (GE

Healthcare). The intensities of detected bands were corrected by average background and normalized to loading control (GAPDH or actin). The percentage of protein expression was calculated relative to the control group.

Small interfering RNA (siRNA) transfection

Cdk1 siRNA was purchased from Dharmacon Inc. (Chicago, IL, USA). The transfection was performed in 60-mm tissue culture dishes. PC-3 cells were plated in dishes with 30% confluence for 24 hours and transfected with siRNA according to manufacturer’s instructions. Mixture of siRNA and Dharmacon reagent in serum-free Opti-MEM (Life

Technologies, Ground Island, NY) were added into each dish containing 4 ml antibiotic- free RPMI/10%FBS medium. After 24 hours incubation, medium was replaced with fresh

RPMI-1640/10% FBS medium with presence or absence of indicated agents.

Reverse transcription polymerase chain reaction (RT-PCR)

The treated cells were collected from 60-mm tissue culture dishes and the RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA) based on manufacturer’s instructions. 1 μg of RNA from each sample was reverse-transcribed into cDNA using

M-MLV reverse transcriptase (Promega Corporation, Madison, WI). PCR amplification was performed with 5X Taq master mix (Protech, Taiwan) containing 1 μl of cDNA in a final volume of 25 μl. The sequences of primers are: MMP-2: forward, 5’-

GTATTTGATGGCATCGCTCA-3’, reverse, 5’-CATTCCCTGCAAAGAACACA-3’;

MMP-9: forward, 5’-CGCTACCACCTCGAACTTTG-3’, reverse 5’-

GCCATTCACGTCGTCCTTAT. Reaction mixtures were pre-denaturized at 95°C for 5 minutes and then amplified with optimized cycles (denatured at 95°C for 30 seconds, renatured at 55~60°C for 20 seconds and extended at 72°C for 30 seconds), followed by

72°C for 10 minutes. The PCR products were loaded on a 2% agarose gel, electrophoresis with 100V for 40 minutes and visualized with SYBR Green staining.

Detection of the releases of MMP-2 and -9

Cells were seeded in 96-well plates and incubated with tubulin-binding agents for 24 hours. The medium of each well was collected and centrifuged to remove cell debris.

Amount of MMP-2 and -9 in conditioned medium of each treatment was determined in triplicate by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc.,

Minneapolis, MN) according to manufacturer’s instruction and detected by spectrometer at 450 nm.

Data analysis

All compounds in this study were dissolved in DMSO with the final concentration of

0.1% in incubation medium. One-way analysis of variance (ANOVA) analysis was used for statistical analysis. The comparison of two groups was performed by Student’s t-test.

P-values < 0.05 were considered statistically significant.

2.6 Experimental results

2.6.1 Tubulin-binding agents down-regulate protein levels of MMP-2 and -9.

We examined the effects of three tubulin-binding agents, paclitaxel, vincristine and evodiamine, on the expression of MMP-2 and MMP-9 in PC-3 cells by Western blotting.

The data showed that the protein levels of MMP-2 and MMP-9 were decreased concentration-dependently after compound treatment for 24 hours (Figure 1). Similar results were shown in DU145 cells (data not shown). Further, mRNA levels of MMP-2 and MMP-9 were determined by RT-PCR, showing that there was no significant alteration in the mRNA expression in compound-treated cells (Figure 2). Latent MMP-2 and MMP-9 are secreted, activated and functioned as peptideases to cleave ECM component. We tested the extracellular levels of MMP-2 and MMP-9 using colorimetric

ELISA assays. The data showed no increase of released MMP-2 and -9 after compound treatment (Figure 3).

The apoptosis induced by tubulin-binding agents was examined using PI staining and

FACS analysis. Paclitaxel, vincristine and evodiamine induced significant cell apoptosis at high concentrations (Figure 4). However, the down-regulation of MMP-2 and -9

20 protein expression occurred in relatively lower concentrations. In this regard, paclitaxel and vincristine, which decreased MMP-2 and -9 protein expression starting from 10 nM, did not cause an excess increase of apoptosis (Figure 4). The data indicate that the MMP down-regulation does not resulted from severe cytotoxic effect. In addition to down- regulation of MMP-2 and -9 protein expression, we also observed band shift of MMP-2 and MMP-9 in Western blotting, suggesting the regulation of MMP-2 and -9 protein levels through post-transcriptional modifications after exposure to tubulin-binding agents

(Figure 5). Phosphorylation is a well-known protein modification, and one of its effects is recruited and recognized by ubiquitin E3 ligases in protein degradation. In order to identify if the band shift of MMP-2 and -9 are due to phosphorylation, phosphatase inhibitors were applied to prevent dephosphorylation. As shown in Figure 5, absence of phosphatase inhibitors significantly reduced the phosphorylation level of c-Jun, which was used as a positive control. However, both absence and presence of the phosphatase inhibitors neither altered the band shift nor prevented the down-regulation of MMP-2 and

-9 protein expression. These results suggest that the reduction of MMP-2 and -9 protein levels is not associated with protein phosphorylation.

2.6.2 Inhibition of several kinases does not contribute to the down-regulation of

MMP-2 and -9 protein expression.

The protein expression of MMP-2 and -9 is regulated by many signaling pathways such as MAPK pathway (p38 and JNK), NF-κB, and phosphatidylinositide 3-kinase

(PI3K)/Akt/mTOR pathway. The activation of these pathways induce the expression of

MMP-2 and -9 [35,75,76]. We evaluated the effects of these pathways on PC-3 cells treated with tubulin-binding agents. The data showed that paclitaxel, vincristine, and evodiamine did not significantly modify the phosphorylation and activation of NF-κB but dramatically increased the phosphorylation of c-Jun, a downstream substrate of JNK

(Figure 6). In contrast, tubulin-binding agents significantly decreased the phosphorylations of mTOR at Ser2448, Akt at Ser473, and p38 MAPK at Thr180/Tyr-

182, indicating the inhibition of these kinases (Figure 7). We further applied selective kinase inhibitors, SB203580 (a p38 MAPK inhibitor), rapamycin (an mTOR inhibitor) and MK2206 (an Akt inhibitor), to block the activities of these kinases in order to determine whether these kinases play important roles in the down-regulation of MMP-2 and -9 protein expression. The data in Figure 8 showed that the phosphorylations of these kinases were inhibited by the respective inhibitors; however, the protein levels of MMP-2 and -9 were not affected (Figure 8). The data in the parallel experiments indicated that the down-regulation of MMP-2 and -9 protein expression by tubulin-binding agents did not result from the inhibition of the indicated kinases.

2.6.3 Mitotic protein levels inversely correlate to protein expression of MMP-2 and

MMP-9.

The well-known cellular event caused by tubulin-binding agents is mitotic arrest, which results in the increase of expression or phosphorylation of mitotic proteins and leads to subsequent cell apoptosis [45,46]. As expected, tubulin-binding agents caused a concentration-dependent increase of protein expression of cyclin B1 and MPM2 and

22 phosphorylation of PLK1 in PC-3 cells (Figure 9). Cyclin B1 is essential for cell mitosis.

It binds to Cdk1 and coordinates the transition of G2-M phase. PLK1 participates in regulation of multiple processes during mitosis including centrosome maturation, mitotic spindle formation, and mitotic exit [77]. The correlation coefficients (r) demonstrated the inverse relationships between mitotic-related proteins and MMP-2 and -9 expression in

PC-3 cells (Figure 9). Similar results were also found in DU-145 cells (Table 1). The data suggest that mitotic arrest of cell cycle might associate with the reduction of MMP-2 and

-9 protein expression.

2.6.4 MG-132 prevents mitotic entry and inhibits down-regulation of MMP-2 and -9 expression

In order to further identify if the down-regulation of MMP-2 and -9 expression was related to mitotic arrest, MG132, a proteasome inhibitor and causing blockade of mitotic entry [78], was used to prevent cell cycle progression into mitotic phase. Flow cytometric analysis showed that the cell cycle was accumulated at 4N stage in the presence of

MG132 (Figure 10A). Furthermore, Western blotting showed that the expression of

MPM2 (Mitotic phase marker) was almost completely abolished by MG132. These results indicated that the cells were in the G2 phase instead of mitotic phase and demonstrated the inhibition of mitotic entry in the presence of MG132 (Figure 10B). It is noteworthy that the decrease of MMP-2 and -9 expression induced by tubulin-binding agents was significantly inhibited by MG132 (Figure 10B). It suggests that the prevention

23 of mitotic entry rescues the down-regulation of MMP-2 and -9 expression induced by tubulin-binding agents.

2.6.5 Recovery of protein expression of MMP-2 and -9 after mitotic exit

In order to further confirm the decrease of MMP-2 and -9 only occurred when cells were arrested in mitotic phase, we used nocodazole to synchronize cell cycle in mitotic phase and then washed out the compound to release the cells into the sequential progression of the cell cycle. As shown in Figure 11A and 11B, PC-3 cells exposed to nocodazole resulted in cell cycle arrest in the mitotic phase (0 hour), indicated by cell accumulation in 4N stage and the high expression levels of cyclin B, MPM2 as well as the phosphorylation of PLK1. Furthermore, the low phosphorylation at Tyr15 of Cdk1 indicated the activation of Cdk1 during the mitotic phase (Figure 11C). Consistent to the effects of the tubulin-binding agents treatment, MMP-2 and -9 protein expression were down-regulated during mitotic arrest (Figure 11C). After the release from nocodazole synchronization, cells were time-dependently progressing into G1 phase, evidenced by the increase of cell population in G1 phase and reduced expression of mitosis-related proteins (Figure 11C). Morphology of the cells showed rounded shapes in the mitotic phase at the beginning of release and then re-attached as well as extended after mitotic exit (Figure 11B). Notably, MMP-2 and -9 protein levels were recovered after mitotic exit (Figure 11C). In addition, we used high concentration of thymidine, which interfered

DNA synthesis and arrested cell cycle at late G1 to S phases, to synchronize the cell

24 cycle and found similar results of inverse relationships between MMP-2 and -9 expression and mitosis events (Figure 12).

2.6.6 Cdk1 phosphorylation at Tyr15 shows a high correlation to MMP-2 and -9 protein levels

Progression of cell cycle is sophisticatedly regulated by Cdks, including Cdk1, Cdk2,

Cdk4 and Cdk6. Cdks are phosphorylated by upstream kinases and bound with their partner cyclins for fully activation. Different Cdk/cyclin complexes coordinate the progression of cell cycle. The transition from G2 to M phase is precisely regulated by

Cdk1/cyclin B complex, including nuclear membrane breakdown, chromatin condensation, and mitosis-specific microtubule reorganization. The activation of Cdk1 is controlled by multiple processes, including the binding of cyclin B, phosphorylation of

Cdk1 on Thr161 (a activative residue), and removal of phosphorylation on Thr14 and

Tyr15 (inhibitory residues). The phosphorylations of Cdk1 at Thr14 and Tyr15 are mediated by Wee1 kinase, resulting in inhibition of Cdk1 activity during G2 phase of the cell cycle [79]. Tubulin-binding agents induced the phosphorylation of Cdk1 on Thr161 residue and caused de-phosphorylation at Tyr15 residue (Figure 13A). The correlation coefficients between the phosphorylation at Tyr15 of Cdk1 and protein expression of

MMP-2 and -9 were determined. High correlations were shown in both PC-3 (Figure 13B) and DU145 cells (Table 1). The good correlations between the expression of Cdk1Tyr15 and MMP-2 and -9 were also demonstrated in the result of MG132-mediated inhibition of mitotic entry (Figure 10B) and cell cycle synchronization by nocodazole or high

25 thymidine treatment (Figure 11C and 12). It suggests that Cdk1 activity inversely correlated with the protein expression of MMP-2 and -9. Furthermore, inhibition of Cdk1 activity by using roscovitine (a Cdk1 inhibitor) significantly prevented the decrease of

MMP-2 and -9 protein expression induced by tubulin-binding agents (Figure 14A). This result was further confirmed by using Cdk1 siRNA to block the expression of Cdk1

(Figure 14B). The data suggest the important role of Cdk1 in the down-regulation of

MMP-2 and -9 expression induced by tubulin-binding agents.

2.6 Discussion

Several mechanisms of suppressing tumor metastasis by tubulin-binding agents have been reported, including the inhibition of angiogenesis, decrease of related cytokine production and increase of expression levels of angiogenesis inhibitors [45,46]. It has been reported that metastatic tumors show higher MMP-2 levels compared to non- metastatic cells and paclitaxel treatment can reduce MMP-2 production in the advanced tumors [80]. Other studies also show that anti-tubulin agents cause down-regulation of

MMP-2 and -9 expression as well as inhibit tumor invasion. For example, CHM-1, which is a quinolone derivative and triggers microtubule disassembly, causes the decrease of

MMP-2 and -9 protein expression, induction of cell apoptosis, and inhibition of metastasis in osterogenic sarcoma cells [81,82]. MJ-29, a tubulin de-polymerization agent, inhibits invasion of cancer cells and suppresses MMP-2 and -9 protein expression in oral cancer cells [83,84]. Although many tubulin-binding agents display activities on decreasing MMP-2 and -9 expression, the mechanism has not been fully understood. In

26 the present study, we have used paclitaxel, vincristine, and evodiamine, three tubulin- binding agents with different effects on microtubule dynamic, to study the mechanism of the down-regulation of MMP-2 and -9 expression induced by this class of agents.

Paclitaxel, vincristine, and evodiamine treatment resulted in significant reduction of

MMP-2 and -9 protein expression in both PC-3 and DU-145 cells in this study. Although these agents induced cell apoptosis, the down-regulation of MMP-2 and -9 expression occurred at lower concentrations without showing dramatic cytotoxicities. Yu and the colleagues examined the effect of MMP-2 and -9 expression on the treatment of KUD773

(a tubulin de-polymerization agent), camptothecin (a topoisomerase I poison), and etoposide (a topoisomerase II poison) in PC-3 cells. The data show that the decrease of

MMP-2 and -9 protein levels is observed in KUD773-treated cells but not in those treated with camptothecin and etoposide [85]. The study suggests that the decrease of MMP-2 and -9 expression induced by tubulin-binding agents is mediated by particular mechanism but not by cytotoxic effects.

MMP-9 expression can be induced by cytokines and growth factors as well as regulated by many signaling pathways, including the activation of PI3K/Akt, mTOR and MAPK

(e.g., ERK, JNK and p-38) pathways [35,75]. NF-B, activated by various cytokines, also plays an important role in regulation of MMP-9 expression [36,75]. MMP-2 is less inducible compared to MMP-9. It is usually regulated by post-transcriptional regulation

[23,35,75]. In spite of lack of well-defined transcriptional factor binding sites on its promoter, recent studies show that JNK pathway can induce protein and mRNA expressions of MMP-2 [76,86]. Paclitaxel, vincristine, and evodiamine did not inhibit

JNK activity (data not shown) but increased the phosphorylation of c-jun (a substrate of

JNK). Furthermore, these tubulin-binding agents did not modify the phosphorylation and activity of NK-κB. Altogether, the results suggest that the down-regulation of MMP-2 and -9 expression is not caused by the inhibition of these transcription factors.

PI3K/AKT/mTOR signaling pathway regulates a lot of important cellular functions. They are frequently activated or over-expressed in many types of cancers and involve in cancer progression as well as resistance to anticancer drugs [87]. PI3K activation triggers the phosphorylation and activation of Akt, leading to mTOR activation [87]. There is evidence showing that PI3K/AKT/mTOR pathway is activated and involved in cancer invasion and metastasis, which are associated with up-regulation of MMP-9 expression

[88]. Using kinase inhibitors, including LY294002 (a PI3K inhibitor), SH-5 (an Akt inhibitor) or rapamycin (an mTOR inhibitor), can reduce the expression levels or secretion of MMP-2 and -9 in various cancer cell lines [76,88,89]. Besides, p38 MAPK signaling pathway is activated in response to cellular stresses (e.g., oxidative stress, UV radiation, and heat shock) or by proinflammatory cytokines (e.g., TNFα , IL-1, and IL-6).

This pathway is involved in cell growth, proliferation, survival, apoptosis and differentiation [90]. Biosynthesis of MMPs is also regulated by p38 MAPK. Inhibition of p38 MAPK using selective inhibitor (e.g., SB203580) blocks the secretion of MMP-9 and inhibits cell invasion in squamous carcinoma cells [91]. In the present study, tubulin- binding agents reduced the phosphorylation of Akt, mTOR and p38. In order to know whether inhibition of mTOR, Akt or p38 MAPK is crucial for the effect of MMP-2 and

MMP-9 down-regulation, several selective inhibitors were used in this study. The data

28 showed that these inhibitors, including rapamycin, MK2206 and SB203580, displayed inhibitory activities against the respective kinases, but did not alter the protein expression of MMP-2 and -9. It suggests that although paclitaxel, vincristine and evodiamine inhibit the PI3K/Akt/mTOR and p38 MAPK signaling pathways, the decrease of MMP-2 and -9 expression were not associated with the inhibition of these kinases.

There are various post-transcriptional modifications of MMP proteins such as phosphorylation, glycosylation, oxidation and nitrosylation [13, 14, 65, 82]. Post- modification may participate in regulating the activity or stability of modified proteins.

For example, phosphorylation is ubiquitously involved in ubiquitin-mediated degradation pathways. The target protein is phosphorylated and then recognized by E3-ligase, leading to ubiquitination and degradation. The present study showed that the tubulin-binding agents induced band shift of MMP-2 and -9. It suggests that there might be an increase of molecular weight in these MMPs due to un-identified modifications. MMP-9 contains O- and N-glycosylated modification. The O-glycosylation is related to substrate specificity of MMP-9 and is important for molecular interactions with exogenous proteins [36]. The function of N-glycosylation is not fully elucidated but may relate to maturation of MMP-

9 [92]. MMP-2 is a non-glycosylated protein but contains several phosphorylation sites

[35,75]. We used inhibitors to test if MMP-2 and MMP-9 were modified by phosphorylation or glycosylation under the treatment with tubulin-binding agents.

However, incubation with or without phosphatase inhibitors or tunicamycin (a glycosylation inhibitor, data not shown) neither prevented the band shift nor rescued the decrease of MMP-2 and -9 expression. It suggests that neither phosphorylation nor

29 glycosylation contributes to these regulatory events. Other types of modifications need to be further tested.

The association between MMP-2 and -9 expression and mitotic regulation has not been clearly identified. Schnaeker and the colleagues have demonstrated that MMP-2 and -9 distribute along with the microtubule in melanoma cells by using immunofluorescent labeling [93]. It suggests that microtubule-dependent trafficking of MMP-2 and -9 is involved in cancer invasion. Furthermore, MMP-9 has been reported to localize around mitotic spindle at different stages of cell division in human neuroblastoma cells and bone marrow macrophages[94]. Inhibition of MMP-9 by selective inhibitor suppresses cell growth and S phase cell cycle arrest, leading to block mitotic entry [94]. It suggests that

MMP-9 may participate in cell division process. In the present study, the increases of cyclin B1, MPM2 and p-PLK1 expression induced by tubulin-binding agents was inversely correlated to the protein expression of MMP-2 and -9. The data suggests cell mitosis is associated with the down-regulation of MMP-2 and -9 expression. MG132 was used to prevent mitotic entry for further study. Wee1 kinase phosphorylates the inhibitory residues at Tyr-15 of Cdk1, resulting in inhibition of Cdk1/cyclin B complex activity and blocking the mitotic entry [95]. MG132, a proteasome inhibitor, is able to prevent the degradation of Cdc6 and Wee1. Accumulation of Wee1 cause the delay of mitotic entry

[78,96]. Our data showed that cell cycle was arrested in G2 phase in the presence of

MG132. Accordingly, the effect on the down-regulation of MMP-2 and -9 expression induced by tubulin-binding agents was almost completely abolished. The regulation of protein levels of MMP-2 and -9 during cell cycle progression was further examined by

30 using cell cycle synchronization approaches. Nocodazole treatment and thymidine block synchronized the cells in mitotic phase and S phase. Both methods confirmed that the levels of MMP-2 and -9 protein expression were decreased in the mitotic phase, which is similar to the mitotic arrest induced by tubulin-binding agents, and the levels of MMPs were recovered after mitotic exit.

Cdk1 and the partner cyclin B1 form an active complex, playing a key role in mitotic phase of the cell cycle. Doxorubucin, a DNA damage-agent, induces cell cycle arrest predominantly in G2 phase that is associated with a decreased expression of Cdk1 and

Cdk2 [97]. Fitzner and the colleagues have reported that doxorubicin inhibits Cdk1 expression but increases protein expression of MMP-9 in pancreatic cancer cells [98].

However, the relationship between Cdk1 activity and MMP expression has not yet been identified. The Cdk1 activity is regulated by phosphorylation at several residues. The phosphorylation at Thr161 contributes to Cdk1 activation, and the phosphorylation at

Tyr15 is critical to the inhibition of this kinase [79]. In the current study, tubulin-binding agents increased the phosphorylation at Thr161 and decreased the phosphorylation at

Tyr15 of Cdk1. It suggests that the activation of Cdk1 was also correlated with mitotic arrest of the cell cycle. The high correlations between the phosphorylation of Cdk1 at

Tyr15 and the expression of MMP-2 and -9 suggest the inhibition of Cdk1 activity is closely associated with the prevention of the down-regulation of MMP-2 and -9 protein expression. Treatment with roscovitine, a Cdk1 inhibitor, blocked the Cdk1 activity and inhibited the down-regulation of MMP-2 and -9 expression. Since roscovitine not only inhibits Cdk1 activity but also impedes the activities of other Cdks [99], siRNA was

31 applied to knockdown Cdk1 for further validation. The similar data were shown, and it confirmed the key role of Cdk1. However, there may be other proteins and/or kinases that participate in the regulation of MMP-2 and -9 expression during mitosis and need to further elucidation.

In conclusion, the data suggest that tubulin-binding agents, such as paclitaxel, vincristine, and evodiamine, cause the down-regulation of MMP-2 and -9 protein expression. The mitotic entry is responsible for determining the down-regulatory effect. Furthermore,

Cdk1 plays a central role in mitotic phase and inversely regulates the MMP-2 and -9 protein levels during the cellular stresses caused by tubulin-binding agents.

Chapter 3

Repurposing of nitroxoline as a potential anticancer agent against human prostate cancer cells

3.1 Drug repurposing

Drug development is a high risk and both time- and cost-consuming process. It takes an average of 13 years to research and expends about US$1.8 billion to develop a new drug.

However, only few of developed compounds get approved by FDA. Therefore, establishment of an alternative way for drug discovery is important [100]. Drug repurposing is a strategy for development of drugs that applies existing drugs to new indications. On one hand, the existing drugs may have off-target effects, which are distinct from the claimed actions and can be developed for new treatment. On the other hand, the mechanism of different diseases may share common molecular pathways.

Therefore, the drug may have the potential to treat secondary disease based on the similar pathological mechanism. Because the pharmacodynamics, pharmacokinetics, and toxic profiles of currently existing drugs are well-established in preclinical and clinical studies, they can be moved to advanced clinical studies more rapidly with reduced time and cost

[100].

Two approaches are used in repurposing the existing drugs for cancers. One is screening the drug library for specific targets. The other is based on biological mechanisms of these 33 drugs, in which their cellular targets have been identified with the potential for anticancer activities. There are many successful cases of drug repurposing. For example, aspirin, which has been approved for relief of pain, reduction of fever, and prevention of stroke, has demonstrated the growth inhibitory effects in several cancer cells [100].

Recently, some widely-used antibiotics have been repurposed to treat cancers [100].

Evidence has shown that several nitrogen-containing heterocyclic antibiotics exhibit anti- proliferative or cytotoxic effects in numerous cancer cells. Anticancer mechanisms of these agents are diverse, such as alkylation of biomolecules, production of ROS, DNA intercalation and inhibition of topoisomerases [101]. These nitrogen-containing heterocycles may work as prodrugs, in which the generation of electrophilic species is essential for their anticancer effects. Different expression levels of the activating enzymes between normal and tumor tissue contribute to selectivity [101].

Metronidazole (MTZ), 1-[2-hydroxyethyl]-2-methyl-5-nitroimidazole, is used as an antiparasitic and antibacterial agent against protozoa and anaerobic bacteria. It has been applied in treatment of anaerobic bacterial infections, amebiasis, trichomoniasis, and giardiasis. MTZ has been considered as a carcinogenic because it has been reported that

Swiss mice exposed to MTZ developed lung tumors and malignant lymphomas [102,103].

However, whether MTZ is carcinogenic to humans has not been clearly identified [103].

Although MTZ shows mutagenic potential, recent studies have demonstrated the anticancer activities of MTZ and its derivatives.[104,105] It has been reported that MTZ decreases the viability and induces apoptosis and necrosis in colorectal cancer cells [102].

Nifurtimox, a nitrofuran compound, is widely used for the treatment of trypanosomal

34 diseases. Recent studies demonstrate the anticancer effect of nifurtimox in neuroblastoma cells. It shows that nifurtimox inhibits cell viability, reduces Akt phosphorylation and activates caspase-3. Moreover, nifurtimox also suppresses tumor growth and induces apoptosis in mouse model [106]. Therefore, nifurtimox has been regarded as a promising therapeutic candidate for neuroblastoma. Phase I clinical trials of nifurtimox has been completed, and it shows nifurtimox is well-tolerated in multiple-relapsed or refractory neuroblastoma pediatric patients [107].

Nitrofurantoin is used to treat urinary tract infections (UTIs), which targets to bacterial ribosomal proteins and results in inhibition of protein synthesis [108,109]. Nitrofurans and its derivatives show both in vitro and in vivo anticancer activities. Nitrofurantoin inhibits cancer growth in FANFT-induced murine bladder tumor and a human transitional cell carcinoma cell line. It also demonstrates growth inhibition of tumors in xenograft mouse models [110].

Ciprofloxacin, a fluoroquinolone (FQ) antibiotic, shows broad-spectrum anti-bacterial activity against gram-positive and gram-negative bacteria. It has been used to treat various infections, including urinary tract, pulmonary tract, and prostate gland infections

[111]. Evidence of anticancer activity of ciprofloxacin has been demonstrated in several cancer cell lines, including prostrate, colon, lung, and ovarian cancer cells [111–115].

Ciprofloxacin induces cell cycle arrest in G2/M phase, causes membrane potential loss of mitochondria, and triggers apoptosis [113,115]. Furthermore, ciprofloxacin inhibits DNA synthesis, which may associate with inhibition of DNA topoisomerase I and II activities[112–115] .

Ofloxacin, similar to ciprofloxacin, is a 4-quinolones derivative used in UTI treatment.

Ofloxacin shows anticancer activity in high concentrations [116,117]. Similar to ciprofloxacin, ofloxacin also inhibits DNA topoisomerase I and II, and results in inhibition of DNA synthesis [112].

Nitroxoline (5-nitro-8-hydroxyquinoline) is an oral antibiotic that has been used in

Europe since 1962 for treatment of acute or recurrent UTIs [118–120]. The drug is metabolized in liver and excreted into urine rapidly [89]. Nitroxoline shows a bacteriostatic activity against E. coli as well as other urophathogens by inhibiting RNA synthesis [121]. At sub-inhibitory concentrations, nitroxoline inhibits bacterial adherence of E. coli to epithelial cells [122]. In addition to the antibacterial activity, nitroxoline shows the activity against several microorganisms such as fungi, mycoplasma, and trichomonas [89].

Recent studies have shown that nitroxoline displays an anticancer activity against a variety of cancer cells in vitro and in vivo [27-30]. Compared to structure-related analogues, clioquinol and other 8-hydroxyquinoline derivatives, nitroxoline shows the most potent cytotoxic effect in pancreatic cancer, ovarian cancer, leukemia, and lymphoma cells. Nitroxoline induces the activation of caspase-3 and produces more reactive oxygen species (ROS) compared to other 8-hydroxyquinoline derivatives [123].

In addition, nitroxoline induces cell cycle arrest in G0/G1 phase and PARP-1 cleavage

[124]. The in vivo anticancer efficacy of nitroxoline has been demonstrated in several mouse models, including bladder cancer, breast cancer as well as glioma [124–126].

There are evidence indicate that nitroxoline is able to suppress cancer cell invasion

[124,126]. It shows that nitroxoline reduced invasiveness of U87 cells in matrigel invasion assay [124]. Furthermore, nitroxoline has been identified as a potent and reversible inhibitor of cathepsin B that participates in degrading of extracellular matrix and enabling migration, invasion, and metastasis of cancer cells. The effect of nitroxoline on tumor invasion also has been evaluated in transformed human breast epithelial cell line. The results show that nitroxoline reduces the invasive activity in two- and three- dimensional in vitro invasion assay [126]. In addition, nitroxoline displays an antiangiogenic effect. It inhibits methionine aminopeptidase 2 activity, and suppressing the proliferation by inducing senescence in human umbilical vein endothelial cells (HUVEC)

[125].

3.2 5' AMP-activated protein kinase (AMPK), mTOR, and autophagy

AMPK is a serine/threonine kinase that maintains intracellular energy status by sensing the ratio of AMP/ATP and it also regulates cellular catabolic as well as anabolic pathways. Many cellular stresses, including oxidative stress, nutrient deficiency, heat shock, ischaemia and hypoxia, are associated with ATP depletion and cause AMPK activation [127]. AMPK is composed of three subunits: a catalytic subunit α, containing a phosphorylation residue of Thr172 and two regulatory subunits, β and γ. Binding of AMP to γ subunit induces allosteric activation of AMPK, which promotes upstream kinases such as LKB1 to phosphorylate the Thr172 residue. The phosphorylation of Thr172 results in 50 to 100-fold increase of AMPK activity [128]. AMPK participates in management of many cellular functions by phosphorylation of downstream substrates and

37 regulating gene expression as well as mRNA stability [128]. Activation of AMPK turns off anabolic pathways, including the synthesis of protein, fatty acid, and cholesterol, and it also activates catabolic pathways such as increases of lipid oxidation, glycolysis, and glucose uptake. Aberrant regulation of AMPK is associated with many diseases such as type II diabetes, atherosclerosis, and obesity [127,128]. Recent studies demonstrate that

AMPK acts as a tumor suppressor by coordinating several cellular events such as cell growth, cell cycle progression, and production of protein and fatty acid [127]. mTOR pathway, which regulates mRNA and protein synthesis, plays a critical role in cell growth and cell division. mTOR pathway is stimulated by growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin.

Activated mTOR sequentially phosphorylates the important downstream effectors, 70 kDa ribosomal protein S6 kinase 1 (p70S6K1 or S6K1) and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Both of them participate in the initiation step of translation [127,128]. AMPK suppresses mTOR pathway resulting in a decrease of protein synthesis with direct and indirect mechanisms Evidence shows that

AMPK inhibits phosphorylation at Thr2446 of mTOR and, therefore, reduces p70S6K1 phosphorylation [127]. In addition, the activation of mTOR is regulated by TSC1/TSC2 complex, which suppresses mTOR activity in the absence of growth factors stimulation.

AMPK phosphorylates TSC2 at Thr1227 and Ser1345, increasing the activity of

TSC1/TSC2 complex, and results in enhancing the suppression of mTOR [127,128].

Autophagy is a self-digesting process, which is characterized by formation of vesicles to engulf cellular components for degradation or recycle. Autophagy is a response to

38 nutrient deficiency, and it leads to breakdown of cellular components to generate energy.

However, excessive degradation also results in cell death. Therefore, autophagy has been classified as type II programmed cell death [129] and it is different from classic caspase- dependent apoptosis pathway. The process of autophagy is initiated by forming a phagophore, an isolated membrane derived from endoplasmic reticulum (ER). After phagophore biogenesis, also called nucleation, multiple steps are involved in sequential processes. The phagophore expands to form a double-membrane vacuoles

(autophagosomes), which engulf cellular components, including protein aggregates, ribosomes, and organelles. Then, autophagosome fuses with lysosome into autophagolysosomes, in which the content of vacuoles is degraded by lysosomal hydrolases [129]. Autophagy is regulated by autophagy-related (Atg) proteins. The formation of phagophore is triggered by composition of Ulk1-Atg13-Atg17 complex

[130]. mTOR, an important negative regulator of autophagy, induces the phosphorylation of Atg13, preventing the binding of Atg13 to Ulk1, and results in inhibition of autophagy

[130]. Lack of energy suppresses PI3K/Akt pathway and activates AMPK pathway. Both effects lead to inhibition of mTOR signaling pathway, improving the binding of Atg13 to

Ulk1, and sequentially inducing the initiation of autophagy [130]. In addition, evidence suggests that AMPK also directly activates Ulk1-Atg13-Atg17 complex by phosphorylation of Ser317 and Ser777 on Ulk1 [131]. The crosstalk between autophagy and apoptosis is complicated. In some cases, autophagy functions as a protector, which generates energy to prevent cell from apoptosis [129]. However, in other cases, autophagy leads to cell apoptosis. For example, inhibition of autophagy by using

39 autophagy inhibitor, 3-MA, or knocking down the expression of essential Atg7 or Beclin-

1 reduces caspases activation and cell death [129,132]. The autophagy and apoptosis are not mutually exclusive. They may happen at the same time to cause cell death. Therefore, the eventual consequence of the cells with autophagy induction is determined by the cellular condition and signaling interactions [129].

3.3 DNA check point and Checkpoint kinase 2 (Chk2)

DNA damage happens frequently, which can be caused by diverse endogenous or exogenous sources such as ROS, ultraviolet (UV) light, ionizing radiation, and several

DNA damage agents. In order to maintain genome integrity and to prevent the permanent or lethal genetic damages, cells have developed a complex mechanism in response to

DNA lesion. In whole cell cycle, there are several checkpoints and repair mechanisms to fix damaged DNA. Impairment of DNA activates checkpoint signaling and results in either cell cycle arrest to enable DNA repair or senescence or death if the damage is irreparable [133]. When sensor proteins detect damaged DNA, ATM/ATR-Chk1/Chk2 pathway is activated to coordinate sequential activation of checkpoints and DNA repair processes [134]. Chk1 and Chk2 are structurally similar and functionally redundant.

However, there are several differences between Chk1 and Chk2. Chk2 expression is all throughout the cell cycle and is activated mostly in response to double-strand DNA breaks. Instead, Chk1 expresses mainly through S to G2 phase and responds not only to

DNA lesion but also to other impaired DNA replication [135]. In addition to DNA damage, Chk2 is also involved in telomere shortening-mediated senescence, in which

40 telomere erosion results in Chk2 activation, an increase of p21 expression, and promotion of cells entering into G0 phase [136]. Chk2 plays an important role in DNA damage pathway, in which Chk2 accepts the signals from ATM and delivers to downstream regulators [133,134]. Activation of Chk2 by DNA damage results in different cellular events such as DNA repair, cell cycle arrest, senescence, or apoptosis depending on the severity of DNA damage [133].

3.4 Hypothesis and specific aims

Drug repurposing is a promising strategy for drug development and many successful cases have been reported. Since many antibiotics have demonstrated potential anticancer effects, we have hypothesized that some nitroheterocyclic antibiotics or gyrase inhibitors are able to display anti-proliferative and apoptotic activities in prostate cancer cells. One of the most effective analogues will be processed for mechanism study.

Specific aim 1: To find the potential analogue from six antibiotics by using anti-

proliferation assay and flow cytometric analysis in prostate cancer cells.

Specific aim 2: To identify the anticancer mechanism of the potential antibiotic using

biochemical and cellular biological approaches.

3.5 Materials and Methods

Materials

RPMI 1640 medium, fetal bovine serum (FBS), Lipofetamine 2000, and OPTI-MEM were from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies against Cdk1,

Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D1, cyclin E, Cdc25A, p-Ser807/811

Retinoblastoma (Rb), PARP-1, GAPDH and AMPK siRNA and secondary antibodies against mouse and rabbit IgGs were purchased from Santa Cruz Biotechnology, Inc.

(Santa Cruz, CA). Antibodies to p62, p-Ser345-Chk1, p-Thr68-Chk2, p-Thr172-AMPKα, p-Ser2448-mTOR, pThr389-p70S6K (p70 ribosomal S6 kinase), p-Ser139-H2AX were purchased from Cell Signaling Technologies (Boston, MA). Antibody against LC3 II was from Novus Biologicals (Littleton, CO). The Chk2 siRNA was purchased from

Dharmacon (Lafayette, CO). Nitroxoline, metronidazole, nifurtimox, nitrofurantoin, ciprofloxacin, ofloxacin, Chk2 inhibitor, etoposide, sulforhodamine B (SRB), propidium iodide (PI), carboxyfluorescein succinimidyl ester (CFSE), 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and all other chemical compounds were purchased from Sigma-

Aldrich (St. Louis, MO).

Cell lines and cell culture

The prostate cancer cell lines, PC-3 and DU145 (hormone-refractory cancer cells) and

LNCaP cells (hormone-sensitive cancer cells), were from American Type Culture

Collection (ATCC, Rockville, MD). Cells were maintained in RPMI 1640 medium

42 containing 10% FBS (v/v), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cell cultures were incubated in a 37 °C incubator with 5% CO2/95% air supply.

SRB assay

The SRB assay is used for determination of cell viability by staining and detecting cellular protein. Cells were plated with optimal density in 96-well plates in RPMI/10%

FBS medium. After 24 hours attachment, cells were fixed by trichloroacetic acid (TCA) with final concentration 10 % (v/v), which represented as cell population at the time of adding agents (T0). After incubation of indicated agents or DMSO for 24, 48 or 72 hours, cells were fixed with 10 % TCA for 10 minutes and washed by tap water. SRB solution

(0.4 % (w/v) in 1 % acetic acid) was added to each well to stain cells for 30 minutes.

Unbound SRB was removed by 1 % acetic acid. Stained cells were dissolved by 10 mM

Trizma base and the absorbance was determined by spectrometer at 515 nm wavelength.

Percentage of growth inhibition determined by using the following absorbance values, such as time zero (T0), growth of control group (C), and cell growth of incubation with the indicated agent (Tx), the percentage of growth was calculated at each of the agent concentrations levels. Percentage of growth inhibition was calculated according to following equation: [1- (Tx-T0)/(C-T0)] x 100%. 50% growth inhibition (IC50) is determined at the concentration of the indicated agent that causes 50% reduction of total protein increase compared to control cells during the agent incubation.

Cell proliferation assay with CFSE labeling

Cell suspensions were labeled with CFSE dye for 10 minutes at 37°C in PBS and plated into 6-cm dishes. After incubation for 48 hours, the medium were replaced with fresh

RPMI/ 10% FBS medium with 10 μM nitroxoline or DMSO. The fluorescent intensity of treated cells was detected by flow cytometry at 0, 24, 48 and 72 hours.

Colony formation assay

PC-3 cells were plated in each well with density 100 cells/well in 6-well plates and incubated with indicated concentration of nitroxoline for 10 days. After treatment, cells were washed by PBS and fixed by 100% methanol. The fixed cells were stained with crystal violet (0.25% crystal violet/20% methanol) and photographed by a scanner. For measurement of colony content, the colonies were dissolved by 50mM sodium citrate/

50% ethanol and read at the wavelength of 595 nm.

Flow cytometric analysis of PI staining

Cells treated with the indicated agents were collected with 0.05% trypsin at the indicated time point and fixed with 70 % (v/v) alcohol at -20oC for 30 min. Fixed cells were washed by PBS and resuspended by PI solution (80 μg/ml PI, 0.1 % v/v Triton X-100, and 100 μg/ml RNase). DNA content of cells were detected by flow cytometry and analyzed by CellQuest software (Becton Dickinson, Mountain View, CA).

Western blotting and quantifications of protein expression

After treatment, the protein expression were examined by Western blot analysis. The cells were lysed by ice-cold lysis buffer and determined the protein concentrations. For electrophoresis, equal amount of proteins were separated by optimal percentage of polyacrylamide gels, transferred to PVDF membranes, and incubated with 5% non-fat milk. Membranes were probed with specific primary antibodies and then incubated with appropriate horseradish peroxidase conjugated-secondary antibodies, followed by visualization with an enhanced chemiluminescence detection kit (Amersham,

Buckinghamshire, UK). Bands were quantified using ImageQuant software (GE

Healthcare). The intensities of detected bands were corrected for average background and normalized to loading control (GAPDH or actin). The percentage of protein expression was calculated relative to the indicated control group.

Small interfering RNA (siRNA) transfection

The transfection was performed in 60-cm tissue culture dishes. PC-3 cells were seeded in dishes with 30% confluence and attached for 24 hours. For transfection, the siRNA were mixed with Lipofectamine (AMPK siRNA) or Dharmacon reagent (Chk2 siRNA) with recommended ratio based on manufacturer’s instructions. Si-RNA mixture was added into each dishes containing antibiotic-free RPMI/10%FBS medium. After 24 or 48 hours incubation, medium was replaced by fresh RPMI-1640/10% FBS medium with presence or absence of indicated agents and performed sequential experiments.

Comet assay

After treatment, the cells were collected and resuspended in ice-cold PBS. The resuspended cells were mixed with two-fold volume of 1.5% low melting point agarose at

37°C. The mixture was loaded onto a fully frosted slide, which was pre-covered by 0.7% normal melting agarose. The slides were immersed in pre-chilled lysis solution (1%

Triton X-100, 2.5 M NaCl, and 10 mM EDTA in PBS solution, pH 10.5) at 4 °C for 1 hour and than soaked with pre-chilled unwinding and electrophoresis buffer (0.3 N

NaOH and 1 mM EDTA) at 4 °C for 30 minutes. The slides were applied to electrophoresis with 10 mV for 15 minutes. After electrophoresis, 1X SYBR Green was added to slides and the fluorescent images of stained nuclei were visualized at 400X magnifications with a fluorescence microscope (Axioplan 2, Zeiss, Germany) and captured with a CCD camera (Optronics, Goleta, CA). Over one-hundred of cells were scored and percentage of cellular DNA contents in tails cells was determined by

OPENComet software.

Detection of ROS

PC-3 cells were plated in 6-well dishes and treated with or without nitroxoline for 1 or 3 hours. The fluorescent dye, CM-H2DCFDA, was added into medium 15 minutes before the harvesting, and the cells were kept in the dark. After this incubation, the cells were collected by trypsinization, washed by PBS and analyzed by flow cytometry FL1 channel.

ICE assay

TOPO-DNA cleavage complexes were measured using ImmunoComplex of Enzyme

(ICE) assay in PC-3 cells. Cells were seeded in 6-cm dished, incubated with indicated agents, and then lysed by pre-warmed 37 °C 1% sarkosyl buffer (1g/100ml sodium lauroyl sarcosinate in 1mM Tris-HCl, pH 8.0). Lysed cells were carefully added onto

CsCl2 gradient solution (density ranged from 1.37 to 1.82 g/ml) and centrifuged in 31,000 r.p.m at room temperature for 24 hours. After CsCl2 gradient partition, gradient fractions were taken out carefully from the top of tube. TOPO-free form was partitioned into fractions 1 to 4 and TOPO-DNA complex form was partitioned into fractions 5 to 8. Each fraction was mixed with equal volume of SP buffer (25mM Na3PO4) and blotted on the

PVDF membrane. TOP-DNA complexes were visualized by immunoblotting using the primary antibodies against topoisomerases and incubated with secondary antibodies. The membranes signal was developed with a chemiluminescence detection kit (Amersham).

Immunofluorescence microscopic examination

Cell were plated in a chamber slide and treated with indicated agents. After treatment, the cells were fixed with 100 % methanol for 20 minutes at -20°C. Fixed cells were blocked and permeated with 1% bovine serum albumin (BSA) containing 0.1% Triton X-100 for

30 minutes at 37°C. For double staining of p-Chk2 and γ-H2AX, the fixed cells were first performed the single staining procedure. The cells were washed by PBS and incubated with the p-Chk2 antibody for 1 hour at room temperature. After PBS wash, cells were stained with Cyt3-conjugated secondary antibody for an hour at room temperature and

47 protected from light. After washed with PBS, cells were blocked with 1% BSA and then repeated the staining steps for γ-H2AX with FITC-conjugated secondary antibody. After staining, 1 μg/ml DAPI was added for nuclear counterstaining. The cells were visualized and captured by an Axio Imager A1 microscope (Carl Zeiss).

Flow cytometric assay of γ -H2AX

The cells were collected and fixed with 70% (v/v) ethanol at -20°C for 30 minutes and washed once by PBS. Cells were blocked and permeated with 1% BSA/0.1% Triton X-

100/PBS for 30 minutes and then probed with primary antibodies against γ-H2AX for 1 hour at room temperature. The cells were washed by PBS and incubated with FITC- conjugated anti-rabbit secondary antibody for 1 hour at room temperature. Stained cells were washed, re-suspended in PBS for FACScan analysis. The fluorescent intensity was determined by FL1 channel of flow cytometry and analyzed by CellQuest software

(Becton Dickinson, Mountain View, CA).

Data analysis

All compounds in this study were dissolved in DMSO (final concentration of 0.1% in media). Data are expressed as mean±SEM for the indicated number of individual experiments. One-way analysis of variance (ANOVA) analysis was used for statistical analysis. The comparison of two groups was performed by Student’s t-test. P-values <

0.05 were regarded as statistical significant.

3.6 Experimental results

3.6.1 Nitroxoline shows the most effective anti-proliferative and cytotoxic effects compared to other antibiotics.

The anti-proliferative and cytotoxic effects of six nitrogen-containing heterocyclic antibiotics (metronidazole, nifurtimox, nitrofurantoin, nitroxoline, ciprofloxacin and ofloxacin) were examined using SRB assay and FACS flow cytometric analysis in PC-3 cells. As shown in Figure 15, nitroxoline demonstrated the most effective anti- proliferative effect with an IC50 of 5.6±0.1 μM in a 48-hour treatment. Other compounds showed the growth inhibition effects at high concentrations (50 to100 μM) after treatment for 48 and 72 hours. The IC50 of each compound in 48 and 72-hour treatment in PC-3 cells were calculated (Table 2). Apoptotic populations of antibiotic-treated PC-3 cells were examined by PI staining and analyzed by FACS flow cytometry (Figure 16).

Nitroxoline, but not the other compounds, caused a significant increase of sub-G1 peak, which is indicative of apoptotic cells.

3.6.2 Nitroxoline induces cell cycle arrest and apoptosis in prostate cancer cells.

In addition to PC-3 prostate cancer cells, the anti-proliferative effects were examined in both hormone-sensitive (LNCaP) and hormone-refractory prostate cancer cells (DU145)

(Figure 17). The results of SRB assay showed that LNCaP cells were more susceptible to nitroxoline (IC50 = 6 μM) than that in DU145 cells (IC50 = 16 μM) after 24 hours of exposure. The anti-proliferative effect of nitroxoline was further evaluated by CFSE staining. CFSE dye is able to stably label the cellular proteins by covalently conjugation,

49 and it distributes evenly when the parent cell divides into two daughter cells with the reduction of fluorescent intensity. The data showed that nitroxoline treatment delayed the decrease of fluorescent intensity in PC-3 cells (Figure 18). Similar results were shown in both DU145 and LNCaP cells. The averages of CFSE fluorescent intensity at 72 hours of control vs. nitroxoline were 66±1 vs. 267±33 (P < 0.001) in PC-3 cells, 144±4 vs. 326±9

(P < 0.001) in DU145 cells, and 258±24 vs. 498±15 (P < 0.001) in LNCaP cells.

Furthermore, clonogenic assay was used to examine the long-term effect of nitroxoline for a 10-day exposure in PC-3 cells. It showed that nitroxoline treatment inhibited colony formation with an IC50 of 3.2±0.6 μM (Figure 19). Flow cytometric analysis was used to detect the effect of nitroxoline on cell cycle profiles in three prostate cancer cell lines. It demonstrated that nitroxoline induced cell cycle arrest in G1 phase in all cell lines

(Figure 20).

3.6.3 Inhibition of Cyclin D1-Rb-Cdc25A axis contributes to nitroxoline-induced G1 arrest.

Since nitroxoline induced G1 arrest of the cell cycle, several cell cycle regulators were examined for further study. Cdk4/Cyclin D complex and Cdk2/Cyclin E complex coordinate G1-S transition of cell cycle by regulating the transcription of essential regulators for DNA syntheses and the degradation of S phase inhibitors. The expression of cell cycle regulatory proteins was examined in nitroxoline-treated PC-3 and LNCaP cells. Cyclin D1, but not other Cyclins, was significantly down-regulated in time- and concentration-dependent manner (Figure 21 and 22). While in the late G1 phase, cells

50 receive mitogenic stimulation to activate Cdk/Cyclin complexes and promote cells to pass restriction point (R) to enter S phase [137]. Retinoblastoma gene (Rb), an important regulator for controlling R point progression, is hypophosphorylated when cells are in G1 phase. It is gradually phosphorylated during cells pass through the R point.

Hypophosphorylated Rb functions as a repressor that binds and sequesters E2F, a transcriptional activator essential for DNA replication. Cdk4/Cyclin D complex initiates the phosphorylation of Rb and partially suppresses Rb functions. The following phosphorylation on multiple sites of Rb results in dissociation of Rb/E2F complex [138].

In the present study, as the down-regulation of Cyclin D1, the phosphorylation of Rb was also decreased after nitroxoline treatment. Furthermore, nitroxoline caused the decrease of Cdc25A expression (Figure 21 and 22). Cdc25 is a family of serine and threonine phosphatases and it regulates the cell cycle by removing inhibitory phosphorylations of cyclin-dependent kinases. Cdc25A controls the entry into S phase by activating

Cdk2/Cyclin E complex [139]. Overall, it suggests that nitroxoline inhibits Cyclin D1-

Rb-Cdc25A axis and results in G1 arrest.

3.6.4 Nitroxoline induces AMPK activation and cyto-protective autophagy.

AMPK activation-mediated anticancer effects have been reported in many studies. It causes cell cycle arrest, autophagy, and apoptosis. Exposure to nitroxoline induced concentration- and time-dependent AMPK activation by increasing the phosphorylation at Thr172 of AMPK and further reducing the phosphorylation at Ser2448 of mTOR. It also inhibited phosphorylation of p70S6K at Thr389, which is the downstream target of

AMPK-mTOR pathway, in both PC-3 and LNCaP cells (Figure 23 and 24). To identify the functional effects of nitroxoline-induced AMPK activation, AMPK siRNA was used to block the expression of AMPK (Figure 25). It showed that AMPK knockdown significantly prevented nitroxoline-mediated effects, including the down-regulation of

Cyclin D1, the reduction of p70S6K phosphorylation and the cleavage of PARP.

Autophagy, which is triggered by nutrient stress, results in a self-digestion of cellular organelles either to protect cells from energy deficiency or to cause cell death [27]. LC-3 is required for autophagosome formation. It is cleaved into LC-3 I and further converted into LC-3 II with phosphatidylethanolamine (PE) conjugation. LC-3 II associates tightly in the membrane of autophagosome and has been regarded as a marker of autophagy

[130]. Sequestosome 1 (SQSTM1), or p62, is an ubiquitin-binding scaffold protein. It co- localizes with ubiquitinated protein aggregates and connects these protein aggregates with autophagic process to enable their degradation in the lysosome [130]. p62 is also degraded by autophagolysosome. Inhibition of autophagy results in accumulation of p62.

Since induction of autophagy results in the decrease of p62, it can be used as a marker of autophagy. Nitroxoline caused the increase of LC-3 II and decrease of p62 expression in

PC-3 cells (Figure 23), which indicated nitroxoline treatment induced autophagy.

However, these effects were not shown in LNCaP cells (Figure 24). To identify the consequence of nitroxoline-triggered autophagy, we co-treated PC-3 cells with nitroxoline and autophagy inhibitor, chloroquine, which inhibits the fusion of autophagosome and lysosome, resulting in blockade of the process of autophagy [141].

The co-treatment caused the increases of apoptosis, as demonstrated by the enhancement

52 of PARP cleavage and a significant increase of apoptosis using flow cytometric analysis

(Figure 26). These results suggest that nitroxoline-induced autophagy is cyto-protective rather than pro-apoptotic. The induction of autophagy frequently follows AMPK activation. As expected, transfection with AMPK siRNA reduced the conversion of LC3

II in nitroxoline-treated PC-3 cells (Figure 25). Taken together, these data indicate that

AMPK plays a critical role in nitroxoline-mediated cell cycle arrest, autophagic activity and apoptosis.

3.6.5 Nitroxoline increases the phosphorylation of Chk2 and induces DNA damage.

Nitroxoline, a nitroheterocyclic agent, which may be bio-activated by cellular enzymes and act as an electrophile to attack biomolecules such as DNA [101]. Comet assay, also called single cell gel electrophoresis assay, was used to detect the DNA damage in nitroxoline-treated PC-3 cells. It showed that nitroxoline caused DNA damage with an increase of fluorescence in comet tail, representing the fraction of DNA in cells (Figure

27). ROS is a common source to induce DNA damage. Evidence shows that potent cytotoxic effect of nitroxoline is related to an increase of ROS production in Raji cells

[123]. However, in the present study, exposure to nitroxoline showed little increase of

ROS level in PC-3 cells (Figure 28). Furthermore, the structure of nitroxoline is related to quinolone antibiotics, which may act as an enzyme poison of bacterial gyrase and topoisomerase IV to inhibit bacterial DNA synthesis [142,143]. ICE assay was performed to determine if nitroxoline could inhibit DNA synthesis by causing the formation of topoisomerase covalent complexes, in which TOPO-free form was presence in low-

53 density of fractions (fraction 1 to 4) and TOPO-DNA complex form was presence in high-density fractions (fraction 5 to 8). As shown in Figure 29, both camptothecin and etoposide (as positive controls) induced an increase of TOPO-DNA complex formation.

However, there was no increase of TOPO-DNA complex in nitroxoline-treated cells.

Chk2 and Chk1 are important mediators of DNA damage signaling pathways triggered by

DNA double-strand breaks and single-stranded DNA. Both of them coordinate downstream effects in response to DNA damage [134]. Nitroxoline induced Chk2 activation with the increase of Chk2 phosphorylation at Thr68. Notably, nitroxoline- mediated Chk2 activation was AMPK-dependent. Knockdown of AMPK by siRNA significantly reduced the nitroxoline-induced Chk2 phosphorylation although AMPK siRNA transfection, by itself, caused a moderate increase of Chk2 phosphorylation

(Figure 25). Chk2 is an important signal distributor in DNA damage cascade. It transduces DNA damage signal to downstream substrates such as Cdc25A, Cdc25C and

E2F1, and induces DNA repair pathways, cell cycle arrest, or apoptosis based on the severity of DNA damage and cellular circumstances [133]. Because Chk2 may display either pro-apoptotic or anti-apoptotic activity, the functional role of Chk2 activation was determined by using Chk2 inhibitors or siRNA transfection. Treatment with Chk2 inhibitor or transfection with Chk2 siRNA did not prevent nitroxoline-induced down- regulation of cell cycle regulatory proteins, including Cyclin D1, phospho-p70S6K and

Cdc25A in PC-3 cells (Figure 30). It indicates that Chk2 activation has no contribution to nitroxoline-induced G1 cell cycle arrest. However, both Chk2 inhibitor and siRNA significantly prevented nitroxoline-induced PARP cleavage (Figure 30B and 30C). Flow

54 cytometric analysis also demonstrated that Chk2 inhibitor significantly reduced nitroxoline-mediated increase of apoptosis (sub-G1 population, Figure 30A).

Histone H2AX plays a crucial role in double strand break of DNA damage. It is phosphorylated by ATM and ATR in response to double strand break formation. The phosphorylated H2AX, also called γH2AX, is required for recruitment of DNA repair proteins at the damaged site and for activation of checkpoint proteins. Western blotting demonstrated that nitroxoline exposure caused Chk2 activation without increase of γ-

H2AX expression (data not shown). Fluorescent microscope and flow cytometric analysis revealed that DNA damage inducers, camptothecin, and etoposide, increased the protein levels of both γ-H2AX and Chk2 phosphorylation. In contrast, nitroxoline only triggered an increase of Chk2 phosphorylation but not γ-H2AX formation. (Figure 31A and 31B).

It suggests that -H2AX-related DNA repair mechanism was absent in nitroxoline- induced effects.

3.6.6 ZnCl2 supplements do not affect nitroxoline-mediated anti-proliferative effect and cellular signaling.

Evidence shows that the antibiotic function of nitroxoline involves chelation of Fe2+ and

Zn2+, two important ions that regulate the formation of bacterial biofilms [144]. Zinc is an essential element for more than 300 proteins, such as superoxide dismutase, DNA- binding proteins with zinc fingers and other proteins involved in DNA repair. Hence, zinc participates in various cellular events, including cell proliferation, immune function, antioxidant mechanism and DNA repair [145]. In order to determine if nitroxoline-

55 mediated anticancer effects are associated with the alteration of intracellular zinc level, the protein expression were examined with presence or absence of ZnCl2. The results demonstrated that the addition of ZnCl2 did not significantly prevent the reductions of

Cyclin D1 and p70S6K phosphorylation, the activations of Chk2 and AMPK, and the cleavage of PARP (Figure 32). SRB assay also showed that ZnCl2 supplements did not significantly affect nitroxoline-induced anti-proliferative effect, in which the IC50 of control vs. ZnCl2 supplements were 6.1±0.6 µM vs. 5.5±0.1 µM. (n = 4, P > 0.05).

3.7 Discussion

Metronidazole, nifurtimox, nitrofurantoin, nitroxoline, ciprofloxacin and ofloxacin are all

FDA-approved antibiotics and have been used to treat numerous infections. Several studies have reported that these nitroheterocyclic agents exhibit anticancer activities in diverse cancer cell lines. However, the anticancer effects in many of them only occurred at high concentrations (> 50 or 100 μM). Some of these antibiotics even show pro- survival effects at high concentrations [146]. The anti-proliferative effects of these antibiotics in prostate cancer cells are determined by SRB assay in this study. Except for nitroxoline, the IC50 of other antibiotics were higher than 50 μM after 48 and 72-hour treatments. Nitroxoline is a potent and broad-spectrum antibiotic used to treat UTI, in which the MICs against different bacteria ranging from 2 to 64 μg/ml (10 to 326 μM)

[118,147,148]. Nitroxoline showed the inhibition of proliferation with IC50 of 5.6 to 16

μM in prostate cancer cells after a 24-hour treatment. The similar ranges between MIC

56 against bacteria and IC50 against prostate cancer cells suggest the therapeutic potential for the drug repurposing.

Recent studies have demonstrated that nitroxoline exhibits potential anticancer effects both in vitro and in vivo [124–126,149]. However, the mode of anticancer actions of nitroxoline has not been clearly identified. In the present study, nitroxoline demonstrated the anti-proliferative effects in both hormone-sensitive and hormone-refractory prostate cancer cells by induction of cell cycle arrest in G1 phase and sequential apoptosis. Cyclin

D and Rb are crucial regulators for G1-S transition. Up-regulation of Cyclin D1 has been reported in various cancer cells, including breast cancer and prostate cancer cells.

Evidence shows that the expression of Cyclin D1 is higher in androgen-independent bone metastases, suggesting the association between Cyclin D1 over-expression and metastatic progress [150]. Further, functional inactivation of Rb caused by chromosomal abnormalities also results in un-controlled cell proliferation in many types of human cancers [151]. In the present study, nitroxoline induced a significant down-regulation of

Cyclin D1 expression as well as Rb phosphorylation that were consistent with the G1 arrest of cell cycle. Moreover, the increase of apoptosis population and the cleavages of

PARP were observed at longer nitroxoline treatment, indicating growth inhibition followed by apoptotic cell death. mTOR pathway, the key coordinator of protein synthesis, is important for cell growth and division. During cell cycle progression, cells in G1 phase enlarge the size and synthesize mRNAs and proteins, which are essential for DNA synthesis before the entry into S phase. Aberrant regulation of mTOR signaling is associated with tumorigenesis.

Immunohistochemistry of prostate tissue array exhibits that the markers of mTOR pathway are elevated in advanced stage of cancers compared to normal cells [152]. The present study showed that nitroxoline treatment caused inhibition of mTOR and its downstream substrate, p70S6K in a concentration- and time-dependent manner. The data were correlated with the down-regulation of Cyclin D expression and growth inhibition.

AMPK, a well-known negative regulator of mTOR pathway, is involved in many physiological effects and is a potential target of drugs for modulating various diseases such as metabolic syndrome and cancers [127,128]. Metformin, a known AMPK activator, decreases Cyclin D1 expression and inhibits proliferation in prostate cancer cells via activation of AMPK pathway [153]. In this study, nitroxoline showed the similar anti-proliferative effects to metformin associated with AMPK activation. Silencing of

AMPK prevented nitroxoline-induced inhibition of mTOR-p70S6K pathway, the reduction of Cyclin D1 and the cleavage of PARP. These results indicate that AMPK plays a central role in nitroxoline-mediated anticancer effects in prostate cancer cells.

AMPK activation triggers autophagy via suppressing mTOR activity or directly activating the Ulk1-Atg13-Atg17 complex to initiate autophagic process. In this study, it showed that nitroxoline caused AMPK activation and concurrently induced autophagy in

PC-3 cells. However, instead of promoting cell death, the induction of autophagic activity by nitroxoline diminished the apoptotic activity of nitroxoline, which were evidenced by increases of apoptosis population and PARP cleavage while co-treated with autophagy inhibitor. Notably, nitroxoline-treated LNCaP cells demonstrated the apoptosis effect without autophagy induction. The data in both cell lines suggest that autophagy is not

58 essential in nitroxoline-related anti-cancer actions. Moreover, autophagy induces both cyto-protective and cytotoxic effects depending on the cellular context. The data in this study demonstrated a cyto-protective role of autophagy in nitroxoline-mediated effect.

Increasing evidence suggest that induction of protective autophagy results in the resistance to radiation therapy or chemotherapy in different cancer cell lines [141].

Understanding the cross-talk between apoptosis and autophagy, and development of a combination strategy of current anti-cancer drugs with autophagy inhibitors could be a novel approach for cancer therapy.

DNA damage is a common anticancer mechanism that can be induced from several different origins such as the stresses from alkylation agents, oxidative stimulation and topoisomerase poisons. In this study, comet assay showed an increase of DNA in comet tail, indicating nitroxoline caused DNA damage response. Our data showed that oxidative stress was not involved in nitroxoline-mediated DNA damage. Moreover, the structure of nitroxoline (hydroxyquinoline derivative) is related to quinolone antibiotics. Evidence show that quinolone antibiotics may not only target topoisomerases in bacteria but also in mammalian cells [112,113]. Two fluoroquinolone antibiotics, ciprofloxacin and ofloxacin, has demonstrated the inhibitory effect against topoisomerase I and II in cancer cells [154].

In this study, nitroxoline did not cause TOPO-complex formation in ICE assay, suggesting that nitroxoline-induced DNA damage was not mediated by the inhibition of topoisomerases.

Chk2, a multifunctional serine/threonine kinase, regulates the cellular response to DNA damage by phosphorylating a number of cellular substrates. Chk2 activation is always

59 associated with induction of DNA repair mechanism. Therefore, it has been considered that Chk2 inhibitors may improve cytotoxic effects through the inhibition of DNA repair when combined with DNA damage agents. However, since Chk2 also functions as a tumor repressor and it triggers apoptotic signaling against excessive DNA damage, activation of Chk2 may also be regarded as an anticancer strategy in some cases.

Evidence shows that direct activation of Chk2 via over-expression in the absence of DNA damage leads to apoptosis by causing mitochondrial membrane potential loss and activation of caspases cascade in colon cancer cells [155]. In the present study, we observed that Chk2 but not Chk1 was activated by nitroxoline treatment. Moreover, inhibition of Chk2 activation by Chk2 inhibitor and siRNA transfection partially but significantly rescued cell apoptosis. The data suggests that Chk2 serve as a pro-apoptotic effector in nitroxoline-mediated effects. H2AX plays a key role in DNA repair process. It is phosphorylated and accumulated in damaged site, forming γ-H2AX foci. Many DNA repair proteins, including MRE11/RAD50/NBS1 complex, BRCA1 and 53BP-1, co- localize in γ-H2AX foci by directly or indirectly binding to γ-H2AX [156]. H2AX-/- mouse embryonic fibroblasts demonstrate the defect of DNA repair mechanism and are more sensitized to radiation or DNA damage agents [157]. Therefore, γ-H2AX foci can be used as a biomarker for DNA repair. Our data indicate that nitroxoline treatment only induces Chk2 activation without triggering γ-H2AX-related DNA repair mechanism.

It has been reported that Chk2 phosphorylates Cdc25A, a CDK phosphatase, resulting in the proteasomal degradation of Cdc25A and the delay of S phase entrance after ionizing radiation (IR) exposure [158]. In the present study, it showed that nitroxoline treatment

60 caused the decrease of Cdc25A protein expression; however, using either Chk2 inhibitor or siRNA transfection did not prevent the decrease of Cdc25A or the cell cycle arrest caused by nitroxoline. It suggests that Chk2 activation did not participate in nitroxoline induced cell cycle arrest. Overall, the data indicate that Chk2 functions as a pro-apoptotic inducer but does not contribute to cell cycle arrest. AMPK is a critical mediator in response to metabolic and genomic stress signals. Increasing evidence demonstrates a complicated cross-talk between DNA damage and AMPK pathways, in which AMPK organizes the anti-proliferative effects of various DNA damage stimuli such as radiation and chemotherapy [159]. The association between AMPK and Chk2 signaling pathway has not been clearly identified. Recent studies show that AMPK also participates in checkpoints regulation in cancer cells by responding to ionizing radiation [160,161]. It has been reported that exposure to UV radiation results in increasing the phosphorylation of AMPK, ATM and Chk2 in WT-MEFs; however, the induction of Chk2 phosphorylation is diminished in AMPKα-/- -MEFs. It suggests that AMPK may regulate

Chk2 activity [162]. Furthermore, metformin induces DNA damage-like response by activating Chk2 in cancer cells [163]. In this study, it demonstrated that although AMPK knockdown slightly increased the basal phosphorylation of Chk2, there was no additional increase of the phosphorylation of Chk2 in the presence of nitroxoline. The data suggests that AMPK acts as an upstream regulator of nitroxoline-caused Chk2 activation.

Zinc is an essential component to maintain regular cellular function. However, the association between the concentration of cellular zinc and cell death is still poorly understood. Studies have shown that clioquinol, an analogue of nitroxoline, functions as a

61 zinc ionophore and causes apoptosis in various cancer cells [164,165]. Exposure to clioquinol results in accumulation of intracellular zinc, and the addition of ZnCl2 significantly augments the cytotoxicity of clioquinol in cancer cells [164,165]. In the present study, Zn supplements did not significantly affect nitroxoline-mediated anti- proliferative effect and related signaling pathways. It suggests that the anticancer effects of nitroxoline are not associated with the changes of cellular zinc concentrations.

Chapter 4

Conclusion

Prostate cancer is one of the most common cancers worldwide. According to American

Cancer Society, the number of estimated deaths due to prostate cancer is 29,480 in 2014 in the United States [1]. The unsuccessful therapy of prostate cancer is associated with the development of resistance and complicated regulation of cancer biology. In order to further understand the regulation of molecular biology in prostate cancer cells and to identify the potential agents for prostate cancer treatment, this thesis focuses on the basic mechanistic study of metalloproteinase -2 and -9 regulations and the drug discovery in prostate cancer cells.

Metastasis is the major cause of prostate cancer related death. Recently, it has received more and more attentions to target this critical step of tumor progress [3,19]. MMP-2 and

MMP-9 are the promising targets to suppress angiogenesis and cancer invasion. Tubulin- binding agents, such as docetaxel, demonstrate significant effects in the treatment of metastatic prostate cancer. Many reports have shown that tubulin-binding agents induce the decrease of MMP-2 and/or MMP-9 protein expression although the mechanism has not been fully clarified. In the first part, we used three different tubulin-binding agents to verify that the down-regulation of MMP-2 and -9 expression is a common effect of this

63 class of agents and to explore the underlying mechanism in HRPC cells. Our data suggest that the expression of MMP-2 and -9 can be regulated through cell cycle progression. In the mitotic phase, the expression of MMP-2 and -9 are decreased which may be related to

Cdk1 activity. Although the detailed mechanism remains to be elucidated, our study provides a new perspective to look into the regulation of MMP-2 and -9 expression and new possible approach to develop MMP inhibitors against prostrate cancers.

In the second study, we have demonstrated that nitroxoline, a widely used antibiotic, displays the potential for development against prostate cancers. Nitroxoline has been used for many years to treat UTI. The safety in human body and the urine excretion of its pharmacokinetic profile make nitroxoline a good candidate for prostate cancer treatment.

Our data have demonstrated that nitroxoline inhibits the proliferation in both hormone- sensitive and hormone-refractory prostate cancer cells by induction of cell cycle arrest in

G1 phase and sequential apoptosis. These effects are mediated by the activation of

AMPK, resulting in sequential suppression of mTOR signaling pathway and inhibition of cyclin D1-Rb-Cdc25A axis. Nitroxoline also induces AMPK-mediated Chk2 activation that cooperatively contributes to apoptosis. However, the induction of cyto-protective autophagy may attenuate nitroxoline-induced anticancer activity. Therefore, applying autophagy inhibitors with nitroxoline or agents of similar mechanism can be a practical strategy.

MMP-2 MMP-9

Cyclin B 0.79a 0.71a

MPM-2 0.85a 0.80a

p-PLK1Thr210 0.87a 0.84a

p-Cdk1Tyr15 0.84b 0.80b

Table 1. The correlation coefficient (r) between two variables in DU145 cells.

The correlation coefficient (r) was calculated in DU-145 cells. The tubulin-binding agents, including paclitaxel, vincristine and evodiamine, down-regulated the protein levels of

MMP-2 and MMP-9, but up-regulated several mitotic proteins in DU-145 cells. The protein expression were examined by western blot analysis. a, inverse correlation between two variables; b, correlation between two variables.

Time (hour) Compounds 48 72

Metronidazole >100 >100

Nifurtimox 91.7±11.5 74.0±5.3

Nitrofuratoin 63.6±4.1 53.9±3.7

Nitroxoline 5.6±0.1 4.6±0.4

Ofloxacin 91.1±18.1 91.2±14.1

Ciprofloxacin >100 82.5±19.9

Table 2. Effect of selected antibiotics on cell proliferation in human prostate cancer cells

PC-3 cells were treated with the increased concentrations of indicated antibiotics for 24,

48, and 72 hours. After treatment, the cells were fixed and stained for SRB assay. IC50 values (µM) are presented as mean±SEM of three to four independent determinations.

Time (hour) Cell lines 24 48 72

PC-3 8.3±0.5 5.6±0.1 5.0±0.3

DU145 16.6±1.0 7.4±0.3 5.5±0.1

LNCaP 6.6±0.3 5.0±0.3 4.2±0.1

Table 3. Effect of nitroxoline on cell proliferation in human prostate cancer cells

DU-145 and LNCaP cells were treated with the increased concentrations of nitroxoline for indicated time. After treatment, the cells were fixed and stained for SRB assay. IC50 values (µM) are presented as mean±SEM of three to four independent determinations.

Figure 1. Tubulin-binding agents cause down-regulation of MMP-2 and MMP-9 protein expression.

PC-3 cells were treated with DMSO or indicated agents for 24 hours. After treatment, the cells were collected and lysed for performing Western blotting and examining the expression of MMP-2 and MMP-9. The histogram represents four independent experiments. Quantitative data are presented as mean±SEM. *** P < 0.001 vs. respective control.

Figure 2. Tubulin-binding agents do not change the mRNA levels of MMP-2 and -9.

PC-3 cells were treated with DMSO or indicated tubulin-binding agents for 24 hours and then harvested to examine the mRNA expression of MMP-2 and MMP-9 by RT-PCR.

The data represents three independent experiments.

Figure 3. anti-tubulin agents do not alter the extracellular levels of MMP-2 and -9

PC-3 cells were seeded in 96-well and treated with DMSO or indicated tubulin-binding agents for 24 hours. Conditioned media was collected and the amounts of MMP-2 and

MMP-9 were detected by ELISA. Reconstituted human MMP-2 and MMP-9 were used as positive controls. Data are representative in triplicate.

Figure 4. The effects of tubulin-binding agents on cell apoptosis

PC-3 cells were treated by indicated agents with increased concentrations for 24 hours.

After treatments, cells were harvested, fixed, and stained with propidium iodide for flow cytometry analysis. The data were representative as three independent experiments.

Figure 5. The reduction of MMP-2 and -9 levels are not related to phosphorylation

PC-3 cells were collected after treated with DMSO or indicated agents for 24 hours and then lysed with presence or absence of phosphatase inhibitors (50 mM NaF and 100 mM sodium orthovanadate) for Western blotting.

Figure 6. Effects of anti-tubulin agents on the phosphorylations of NF-kB and c-Jun

PC-3 cells were incubated with DMSO or tubulin-binding agents for 24 hours. The cells were collected and lysed for testing the protein expression by Western blotting. The histogram represents four independent experiments. Quantitative data are presented as mean±SEM. *p < 0.05, **p < 0.01, *** P < 0.001 vs. respective control.

Figure 7. Anti-tubulin agents decrease the phosphorylations of mTOR, Akt and p38

PC-3 cells were treated with DMSO or tubulin-binding agents. After 24 hours, the cells were collected, lysed and detected the protein phosphorylation by Western blotting. The data represents three independent experiments. Quantitative data are presented as mean±SEM. *p < 0.05, **p < 0.01, *** P < 0.001 vs. respective control.

Figure 8. Effects of several inhibitors on the phosphorylations of mTOR, Akt and p38 and the expression of MMP-2 and -9

PC-3 cells were treated with indicated inhibitors. After 24 hours, the cells were collected, lysed, and detected the protein phosphorylations by Western blotting.

Figure 9. The expression or phosphorylation of mitotic proteins induced by anti- tubulin agents correlate to MMP-2 and -9 down-regulations

continued

Figure 9 continued

PC-3 cells were treated with DMSO or tubulin-binding agents. After 24 hours, the cells were collected, lysed and detected the protein expression or phosphorylations by Western blotting. The data represents three independent experiments. Quantitative data are presented as mean±SEM. The correlation coefficient was made between levels of the mitotic proteins and levels of MMP-2 and -9 expression. (r) represented as correlation coefficient.

Figure 10. Co-treatment with MG-132 blocks the mitotic entry and prevents the down-regulations of MMP-2 and MMP-9

continued

Figure 10 continued

PC-3 cells were exposed to indicated agents (paclitaxel, 100 nM; vincristine, 100 nM; evodiamine, 10 µM) in the absence or presence of 2 µM MG-132 for 24 hours. (A) The cells were collected, fixed and stained with propidium iodide for cell cycle analysis by flow cytometry. The data are representative of three independent experiments. (B)

Protein expression were detected by Western blotting. The histogram represents three independent experiments. Quantitative data are presented as mean±SEM. **p < 0.01,

*** P < 0.001 vs. respective control.

Figure 11. Mitotic exit rescues the reduction of the MMP-2 and -9 protein expression

PC-3 cells were synchronized using nacodazole for 24 hours. After washout, cells were collected at indicated time. The cell morphology was observed and photographed using microscopy (B), or the cells were fixed and stained with propidium iodide for cell cycle analysis by flow cytometry (A), or tested the protein expression by Western blotting (C).

The data represents three independent experiments. Quantitative data are presented as mean±SEM. *** Indicates that P < 0.001 vs. control cells. Bar, 50 µm.

Figure 12. The expression of MMP-2 and -9 are decreased when cell cycle progress into mitotic phase

Synchronization of PC-3 cells was performed by thymidine block for 24 hours. After washout, cells were collected at indicated time and tested the protein expression by

Western blotting.

Figure 13. The increase of phosphorylation at Thr161 and decrease of phosphorylation at Y15 of Cdk1 induced by anti-tubulin agents correlate to MMP-2 and -9 down regulations

PC-3 cells were treated with DMSO or tubulin-binding agents. After 24 hours, the cells were collected, lysed, and detected the protein expression or phosphorylations by western blotting. The data represents four independent experiments. Quantitative data are presented as mean±SEM. The correlation coefficients was made between the levels of p-

Y15-Cdk1 and the levels of MMP-2 and -9 expression. r represented as correlation coefficient.

Figure 14. Treatment with roscovitine or transfection with Cdk1 siRNA prevent the down-regulation of MMP-2 and MMP-9

PC-3 cells were exposed to indicated agents (paclitaxel, 0.1 µM; vincristine, 0.1 µM; evodiamine, 10 µM) in the absence or presence of roscovitine (A) or Cdk1 siRNA (B) for

24 hours. The protein expression was detected by Western blotting. The data represents three independent experiments. Quantitative data are presented as mean±SEM. *p < 0.05,

**p < 0.01, *** P < 0.001 vs. respective control.

Figure 15. Effects of selected antibiotics on cell viability in PC-3 cells

Cells were treated with increased dose of indicated antibiotics and the viability was assessed for 24, 48 and 72 hours using SRB assay. Data represent three independent experiments and the quantitative data are expressed as mean±SEM of three independent experiments.

Figure 16. Effects of selected antibiotics on cell cycle distribution in PC-3 cells

PC-3 cells were stained with propidium iodide after 48 hours exposure to DMSO, 10 or

30 μM of indicated antibiotics. The percentages of apoptotic cells were determined by sub-G1 content of histogram and the values are expressed as means±S.E.M. of three independent experiments.

Figure 17. Effects of nitroxoline on anti-proliferation in prostate cancer cells

LNCaP and DU-145 cells were incubated with nitroxoline (0.3 to 100 µM) for the indicated time (24 to 72 hours). After treatment, the viability of cells was determined by

SRB assay. Quantitative data are presented as mean±SEM of three independent experiments.

Figure 18. Effect of nitroxoline on anti-proliferation

PC-3 cells were stained with CFSE dye and incubate with 10 μM nitroxoline for indicated time (0 to 72 hours). After treatment, the cells were harvested and analysed by flow cytometry. Data are representative of three independent experiments. Quantitative data are presented as mean±SEM of three independent experiments. *** P < 0.001 compared with the control.

Figure 19. The long-term exposure effect of nitroxoline on cell proliferation

PC-3 cells were seeded in 6-well dishes and incubated with nitroxoline for 10 days. After exposure, cells were fixed and stained with crystal violate. For quantitative data, the colonies were dissolved by 50 mM sodium citrate/ 50% ethanol and detected at 595nm.

Figure 20. Effects of nitroxoline on cell cycle profile in prostate cancer cells

LNCaP, DU-145 and PC-3 cells were treated with nitroxoline (1 to 30 µM) for 24 hours.

After treatment, the cells were fixed and stained with propidium iodide for flow cytometric analysis. Data are representative of three independent experiments.

Quantitative data are expressed as mean±SEM of three independent experiments. * P <

0.05 and ** P < 0.01 compared with 100% control.

Figure 21. Effect of nitroxoline on the protein expression of cell cycle progression regulators in PC-3 cells

PC-3 cells were incubated in the absence or presence of nitroxoline (10 µM) for the indicated time (3 to 24 hours). The cells were harvested and lysed for examining the indicated proteins by Western blotting analysis. Data are representative of three independent experiments. The protein expression of PC-3 cells were quantified and presented as mean±SEM of three to four independent experiments. * P < 0.05, ** P <

0.01 and *** P < 0.001 compared with 100% control.

Figure 22. Effect of nitroxoline on the protein expression of cell cycle progression regulators in LNCaP cells

LNCaP cells were incubated in the absence or presence of nitroxoline (10 µM) for the indicated time (3 to 24 hours). The cells were harvested and lysed for examining the indicated proteins by Western blotting analysis.

Figure 23. Effects of nitroxoline on the protein expression of mTOR, AMPK, Chk2 and autophagy pathways in PC-3 cells

PC-3 cells were treated with nitroxoline (1 to 30 µM or 10 µM) for the indicated time (24 hours or 3 to 24 hours). Cells were collected and lysed for detecting the expression of the indicated proteins by Western blotting analysis. The protein expression of PC-3 cells are quantified and expressed as mean±SEM of three to four independent experiments. * P <

0.05, ** P < 0.01 and *** P < 0.001 compared with 100% control.

Figure 24. Effects of nitroxoline on the protein expression of mTOR, AMPK, Chk2, and autophagy pathways in LNCaP cells

LNCaP cells were treated with nitroxoline (1 to 30 µM or 10 µM) for the indicated time

(24 hours or 3 to 24 hours). Cells were collected and lysed for detecting the expression of the indicated proteins by Western blot analysis.

Figure 25. Examination the effects on protein expression of nitroxoline-mediated

AMPK activation

Both the control and AMPK-silenced PC-3 cells were incubated with or without nitroxoline (10 μM) for 24 hours. Cells were collected and lysed for the testing the expression of indicated proteins by Western blot analysis. The protein expression are quantified relative to nitroxoline-free group and presented as mean±SEM of three to four independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with

100% control.

Figure 26. Effects of nitroxoline on autophagy-mediated cyto-protection

PC-3 cells were incubated in the presence or absence of nitroxoline and co-treated with or without chloroquine. After 48-hour treatment, the cells were fixed by 70% enthanol and stained with propidium iodide. Hypodiploid DNA content (apoptotic sub-G1 population) were detected and analyzed by flow cytometry. The histogram represents as meanSEM of four independent experiments. Autophagy-related protein expression was detected by

Western blot analysis after 24-hour treatment. The protein expression is representative of three independent experiments.

Figure 27. Effect of nitroxoline on DNA damage response

PC-3 cells were incubated with DMSO, nitroxoline (10 µM) or etoposide (50 µM) for 12 hours. Comet assay was applied to examine the integrity of chromosome DNA. The percentage of tail DNA was quantified and the histogram represents as mean±SEM of three independent determinations. *** P < 0.001 vs. respective control.

Figure 28. Effect of nitroxoline on ROS production

PC-3 cells were treated with or without nitroxoline (10 µM) for 1 or 3 hours. Before harvesting, the cells were stained with CM-H2DCFDA for 15 minutes at 37°C. The collected cells were washed once by PBS and analyzed by flow cytometry. 10mM H2O2 was used as positive control.

Figure 29. Effects of nitroxoline on the formation of TOPO-DNA cleavable complexes

continued

Figure 29 continued

TOPO-DNA cleavable complexes were examined by using the ImmunoComplex of

Enzyme (ICE) assay in PC-3 cells. Camptothecin and etoposide (VP-16), which were

DNA damage inducers through the inhibition of topoisomerase I and II, caused TOPO-

DNA complexes formation and were used as positive controls. After CsCl2 gradient isolation, TOPO-free form was presence in fractions 1 to 4 and TOPO-DNA complex form was presence in fractions 5 to 8. TOP-DNA complexes were visualized by immunoblotting using the primary antibodies against topoisomerase I (A), topoisomerase

IIα (B) and topoisomerase IIβ (C).

Figure 30. Examination of the effects on protein expression of nitroxoline-mediated

Chk2 activation

continued

100

Figure 30 continued

FACS analysis (A) and western blotting (B and C) were used to evaluate the effects on cell apoptosis or protein expression in PC-3 cells with inhibition of Chk2. PC-3 cells were co-treated with or without Chk2 inhibitor (20 µM) or transfected with si-Chk2.

Then, the cells were treated with 10 μM nitroxoline for indicated time (A.48 hours, B and

C, 24 hours). After treatment, cells were collected and fixed for FACS analysis or lysed for testing the protein expression by Western blot analysis. The protein expression are quantified relative to nitroxoline-free group and expressed as mean±SEM of three to four independent experiments.

101

Figure 31. Effects of nitroxoline on protein expression of p-Chk2 and -H2AX

PC-3 cells were treated with or without nitroxoline (10 µM), camptothecin (10 µM) or etoposide (25 µM) for 24 hours. (A) The cells were fixed, and stained with anti-Chk2

(red fluorescence), anti--H2AX (green fluorescence) antibodies and counterstained with

DAPI (blue fluorescence, for nuclear detection). The images were acquired and photographed by immunofluorescence microscopy. Scale bar, 20 mm. (B) The cells were collected, fixed and stained with -H2AX antibody for flow cytometric analysis. Dashed line area is basal fluoroscence; blue area is vehicle control; pink area is the indicated drug.

102

Figure 32. Effect of ZnCl2 supplement on nitroxoline-related anti-cancer effects

PC-3 cells were incubated with or without of ZnCl2 (100 µM) and treated with or without nitroxoline (10 µM) for 24 hours. After treatment, cells were collected and lysed for examining the expression of the indicated proteins by Western blot analysis. The protein expression are quantified relative to nitroxoline-free group and expressed as mean±SEM of three independent experiments.

103

Figure 33. Summary of the anti-cancer effects of nitroxoline

Nitroxoline causes AMPK activation, induces cell cycle arrest and results in cancer cells apoptosis. AMPK activation also triggers cyto-protective autophagy in PC-3 cells. On the other hand, nitroxoline causes DNA damage and activates Chk2 without inducing classic

DNA repair mechanism. Chk2 activation, which is associated with AMPK activation, also contributes to nitroxoline-mediated cell apoptosis.

104

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