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ANTICANCER RESEARCH 33: 3807-3814 (2013)

Zoledronic Acid-induced Cytotoxicity Through Endoplasmic Reticulum Stress Triggered REDD1-mTOR Pathway in Cells

YA-CHUN LAN1*, CHIA-LING CHANG1*, MING-TA SUNG2*, PEN-HUI YIN2, CHIA-CHI HSU1, KUO-CHUNG WANG3, HSIN-CHEN LEE1, LING-MING TSENG3 and CHIN-WEN CHI1,2

1Department and Institute of , School of Medicine, National Yang-Ming University, Taipei, Taiwan, R.O.C.; Departments of 2Medical Research and Education, and 3Surgery, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C.

Abstract. Background: (ZOL) used for the related ER kinase-eukaryotic initiation factor 2 alpha- prevention/treatment of osteopathic complications has been CCAAT/enhancer binding protein homologous protein reported to have antitumor effects in breast cancer treatment. (PERK-eIF2α-CHOP) pathway. Conclusion: Taken together, However, little is known about the exact molecular these results indicated that ZOL-induced death was mechanisms for antitumor actions of ZOL. In this study, two caused by endoplasmic reticulum stress activating PERK- breast cancer cell lines were used to investigate the eIF2α-CHOP pathway to induce REDD1 expression and antitumor efficacy of ZOL and the underlying molecular inhibit the mTOR pathway. mechanisms. Results: The growth of two breast cancer cell lines was markedly decreased following treatment with ZOL. Breast cancer remains the second and fourth leading cause of Compared with MCF-7 cells, MDA-MB-231 cells were more cancer in women in America and Taiwan, respectively, despite sensitive to ZOL treatment. Western blot analysis showed that rapid treatment advances in recent years. Often associated with the inhibitory effect of zoledronic acid on growth was related breast cancer is high frequency of tumor to other to the extent of inhibition of phosphorylated-protein kinase organs, including the lungs, liver or , which is the major B (p-AKT), and phosphorylated-mammalian target of cause of death for patients with breast cancer (1). In bone rapamycin (p-mTOR). Moreover, the expression of the stress- metastasis, -mediated is stimulated responsive protein regulated in development and DNA by metastatic cells, resulting in osteolysis, bone fragility and damage response 1 (REDD1), an inhibitor of mTOR, was proneness to fracturing (2). Bisphosphonates, such as induced markedly to various degrees in different breast zoledronic acid (ZOL), are potent inhibitors of osteoclast- cancer cell lines after ZOL treatment. Interestingly, by mediated bone resorption (3-6) and are therefore widely used examining the upstream signaling pathway of REDD1, we in the treatment of and bone diseases with a found that ZOL can induce endoplasmic reticulum stress high bone turnover, such as Paget’s disease (7), tumor- responses through activating the protein kinase R (PKR)- associated osteolysis (8), and post-menopausal (9). Although the main target of bisphosphonates is thought to be the osteoclast, there is increasing evidence to indicate that bisphosphonates have direct antitumor activity (3, 10). Indeed, *These Authors contributed equally to this work. in vitro studies have shown that ZOL is the bisphosphonate Correspondence to: Dr. Ling-Ming Tseng, Department of Surgery, with the most potent antitumor effects, including induction of Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Road, Taipei tumor cell and inhibition of tumor cell proliferation 112, Taiwan. Tel: +886 228757535, e-mail: [email protected] (11-13). ZOL has also been reported to have antiangiogenesis and Dr. Chin-Wen Chi, Department of Medical Research and and anti-invasion effects in breast cancer (14, 15). Education, Taipei Veterans General Hospital, No. 322, Sec. 2, Shipai Combination of ZOL with other for the Road, Taipei 112, Taiwan. Tel: +886 228757627, Fax: +886 treatment of breast or has shown increased 228751562, e-mail: [email protected] antitumor efficacy than single-agent therapy (16-18). However, Key Words: Breast cancer, zoledronic acid, mTOR, REDD1, the exact biochemical mechanisms for antitumor action of endoplasmic reticulum stress, MCF-7, MDA-MB-231 cells. ZOL are still poorly understood.

0250-7005/2013 $2.00+.40 3807 ANTICANCER RESEARCH 33: 3807-3814 (2013)

It has been reported that ZOL inhibits growth and survival Materials and Methods of pancreatic cancer cells by functional down-regulation of the mitogen-activated protein kinases (MAPK) and Cell culture. The human breast cancer cell lines MCF-7 and MDA- phosphoinositol 3-kinase/protein kinase B (PI3K/AKT) MB-231 obtained from American Type Culture Collection (ATCC, pathways (19). Mammalian target of rapamycin (mTOR), Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s associated with tumorigenesis, is one important downstream medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented effector of the PI3K/AKT signaling pathway (20). Therefore, with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% L-glutamine (Invitrogen), 1% non-essential amino acid (Invitrogen) inhibition of the mTOR signaling pathway might be a possible and 1% penicillin-streptomycin-amphotericin B (Biological mechanism of ZOL-induced growth inhibition. On the other Industries, Kibbutz beit Haemek, Israel) at 37˚C in a humidified hand, regulated in development and DNA damage response 1 atmosphere containing 5% CO2. (REDD1) is another key factor inhibiting the phosphorylation of mTOR (21, 22), resulting in protein synthesis inhibition and Cytotoxicity assay (MTT assay). MTT assay was performed to tumor suppression (23). REDD1, identified as a cell stress- determine the effect of ZOL on cell growth of breast cancer cells. responsive protein, can be induced by stress signals including Briefly, cells were seeded in a 96-well culture plate at a density of 7×103 cells/ml and then treated with 0.1, 1, 10, 50, or 100 μM ZOL hypoxia, endoplasmic reticulum (ER) stress and reactive for 24, 48 and 72 h. After treatment, 3-[4,5-dimethylthiazol-2- oxygen species (ROS) that cause cell apoptosis and DNA yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St Louis, MO, damage (22, 24). Therefore, REDD1 might be a candidate USA) was added and cells were incubated for two hours at 37˚C in molecule involved in ZOL-induced antitumor effects. the dark. The resulting formazan product was dissolved in dimethyl It has been shown that ZOL inhibits farnesyl sulfoxide (DMSO; Sigma) and optical density (OD) values were pyrophosphate synthase (FPPS) activity in the mevalonate measured using a microplate reader (SpectraMax 250; Molecular pathway to induce ER stress (25). The ER is an organelle for Devices, Sunnyvale, CA, USA) at an absorption wavelength of 570 nm. Data are expressed as the fraction of cells inhibited by drug membrane protein synthesis, initial post-translational treatments compared with vehicle-treated cells. modification, folding and secretion (26). When cells are stimulated by nutrient or oxygen deficiency, of Measurement of apoptosis. To analyze cellular DNA contents, ZOL- the ER is disrupted, producing a stress known as ER stress, treated cells were collected, fixed in 70% ice-cold ethanol, and kept which then initiates a host of stress reactions, the general at –20˚C for at least 14 h. The fixed cells were washed twice with directions of which are stress amelioration and restoration of phosphate buffered saline (PBS), stained with propidium iodide (PI; homeostasis (27). Among these is the stimulation of 50 μg/ml) in PBS containing 0.2% Tween 20 and 2.5 μg/ml RNase at 4˚C overnight, and analyzed by flow cytometry. The profiles of chaperone proteins which in turn triggers multiple protective cellular DNA contents indicated the distribution of the cells in cellular responses to reduce unfolded protein, a process different phases of the cell cycle. The cells with DNA content less known as the unfolded protein response (UPR) (28). than that of the G0/G1 cells were classified as the sub-G0/G1 However, the presence of excessive unfolded proteins population and considered as apoptotic cells. induces caspases to trigger apoptosis and results in cell death (29). There are three ER proximal sensors to regulate UPR, Western blot analysis. Cells were scraped away from 10-cm dishes including protein kinase R (PKR)-related ER kinase (PERK), and suspended in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.25% sodium deoxycholate, 1% Triton X-100, pH 7.4) on ice. After inositol-requiring enzyme 1 (IRE1) and activating centrifugation (13,000 ×g) at 4˚C for 15 min, the protein transcription factor 6 (ATF6). PERK is a serine threonine concentration was determined by Bio-Rad protein assay (Bio-Rad, kinase. Under stress, PERK promotes eukaryotic initiation Hercules, CA, USA). Protein lysates (30 μg) were separated in 8-15% factor 2 alpha (eIF2α) phosphorylation to reduce protein sodium dodecyl sulfate (SDS)-polyacrylamide gels and then translation and induce CCAAT/enhancer binding protein transferred to a nitrocellulose membrane. The membrane was blocked homologous protein (CHOP) to lead to cell apoptosis (30). with 5% milk in 1× TBST buffer (10 mM Tri-base, 150 mM NaCl, Moreover, previous studies indicated that ER stress increased 0.05% Tween 20, pH 7.4) for one hour at room temperature. Primary antibodies including those against REDD1 (Proteintech, Chicago, IL, REDD1 expression in many cell types (31, 32). In eIF2α USA), mTOR, p-mTOR, PERK, IRE1α, CHOP (Cell Signaling, kinase PERK-deficient mouse embryonic fibroblasts, ER Danvers, MA, USA), ATF6 (Abcam, Cambridge, MA, USA), p- stress cannot promote REDD1 expression, suggesting that PERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), eIF2α, p- eIF2α phosphorylation is necessary for the induction of eIF2α (Invitrogen) and β-actin (Sigma) were then used to probe these REDD1 in the ER stress response. blots at 4˚C overnight. Membranes were washed with TBST three In this study, the direct effect of ZOL on the growth and times and then incubated with horseradish peroxidase (HRP)- survival of metastatic breast cancer cells were examined. The conjugated secondary antibodies for two hours. Following TBST washing, protein visualization was carried out using an enhanced molecular mechanism of ZOL-induced differential growth chemiluminescence kit (ECL; Millipore, Bedford, MA, USA). inhibition in breast cancer cells was investigated. The AKT/mTOR signaling pathway, REDD1 expression and ER Reverse transcription and real-time quantitative polymerase chain stress response were evaluated after ZOL treatment. reaction (qPCR). Total RNA was extracted and precipitated

3808 Lan et al: Zoledronic Acid-induced Cytotoxicity in Breast Cancer Cells according to the instructions of the manufacturer (TRIzol® Reagent; Invitrogen). RNA was dissolved in diethylpyrocarbonate (DEPC)- treated water (Invitrogen) and denatured at 65˚C for 10 min. After denaturation, the sample was put on ice immediately for one minute. For reverse transcription, RNA was incubated with ready-to-go you- prime first-strand beads (GE Healthcare Biosciences, Pittsburgh, PA, USA) and 2.5 μM random primer dN6 at 37˚C for one hour. cDNA samples were then mixed with forward and reverse primer (REDD1: forward 5’ GTT TGA CCT CTC CAC CAG CCT 3’, reverse 5’ GCA CAC AAG TGT TCA TCC TCA GG 3’; β-actin: forward 5’ TGG CAT TGC CGA CAG GAT 3’, reverse 5’ GCT CAG CAG CAG CAA TGA TCT 3’), 2× Fast SYBR PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) and 6 μl ddH2O. The PCR reaction was carried out with an initial denaturation at 95˚C for 10 minutes, followed by 50 cycles at 95˚C for 3 s and 60˚C for 30 s for real-time fluorescence quantitation of products.

Statistical analysis. The means and standard deviation (SD) were calculated from three independent experiments. Two-way ANOVA with post-hoc test was used for multi-group comparisons. Differences were considered statistically significant when p<0.05.

Results

The effect of zoledronic acid on cell growth. The MTT assay was used to examine the anti-proliferation effect and determine the inhibitory dose of ZOL in MCF-7 and MDA- MB-231 breast cancer cells. Comparing to untreated controls, growth of MCF-7 and MDA-MB-231 cells significantly decreased when treated with 50 and 100 μM ZOL for 48 and 72 h (Figure 1). The results also showed that the growth inhibition of ZOL-treated MCF-7 cells was cytostatic. However, the growth inhibition of ZOL-treated MDA-MB-231 cells occurred in a time- and dose-dependent manner, indicating that ZOL induced a cytotoxic effect on MDA-MB-231 cells. To further evaluate the antiproliferative effect of ZOL, we Figure 1. The effects of zoledronic acid (ZOL) on the growth of breast cancer cells. MCF-7 (A) and MDA-MB-231 (B) cells were treated with checked the cell-cycle phase distribution of ZOL-treated ZOL at the indicated concentrations for 24, 48 and 72 h and cell growth breast cancer cells by flow cytometry (Figure 2). The results was determined by the MTT assay. Data are expressed as the mean±S.D. showed that treatment of MCF-7 cells with 50 μM of ZOL of three replicates and are representative of three independent resulted in S-phase accumulation and an increased proportion experiments. *Significantly different as compared to the control at the same time point (p<0.001). of sub-G0/G1 cells (9.34%) in a time-dependent manner (p<0.001, Figure 2A and C). However, compared to 0 or 10 μM treatment, treatment with 50 μM of ZOL significantly increased the proportion of sub-G0/G1 MDA-MB-231 cells (31.46%) in a time-dependent manner (p<0.001, Figure 2B and D). These results together suggest that ZOL induced μM ZOL for 24, 48 and 72 h, the expression of more significant apoptosis in MDA-MB-231 cells than in phosphorylated-AKT (p-AKT), AKT, phosphorylated-mTOR MCF-7 cells. (p-mTOR) and mTOR in the MCF-7 cells were similar to those of the untreated controls (Figure 3A). However, in The effect of zoledronic acid on the AKT/mTOR signaling contrast to MCF-7 cells, the expressions of p-AKT, p-mTOR pathway. Since ZOL demonstrated an antiproliferative and mTOR in ZOL-treated MDA-MB-231 cells were activity towards breast cancer cells, we next examined the obviously inhibited as compared to those of the untreated underlying growth inhibitory effect of ZOL. We firstly controls (Figure 3). The results together suggest that evaluated the expression of AKT/mTOR signaling pathway inhibition of p-AKT, and p-mTOR by ZOL related well to proteins by western blot analysis. After treatment with 50 the ZOL-induced cytotoxic effects.

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Figure 2. The effects of zoledronic acid (ZOL) on the cell cycle of breast cancer cells. MCF-7 (A) and MDA-MB-231 (B) cells were treated with ZOL at the indicated concentrations for 24, 48 and 72 h and then the cell cycle distribution was determined by flow cytometry. The percentage of apoptotic sub-G0/G1 cells from three independent experiments are expressed as the mean±S.D. and shown for MCF-7 (C) and MDA-MB-231 (D) cells. *Significantly different as compared to the control at the same time point (p<0.001). Apop: apoptotic sub-G0/G1 cells.

The effect of zoledronic acid on REDD1 expression. In MB-231 cells, respectively. At 48 h after ZOL treatment, the addition to p-AKT, REDD1 is another key factor that fold of increase of REDD1 was 1.3 and 2.7 for MCF-7 and regulates cell growth through modulating the mTOR MDA-MB-231 cells, respectively. Interestingly, the fold of signaling pathway. Thus, we examined the mRNA level and increase of ZOL-induced REDD1 mRNA and protein were protein expression of REDD1 in ZOL-treated breast cancer higher in the MDA-MB-231 cells as compared to the MCF- cells by real-time qPCR and western blot analysis. The 7 cells. These results together showed that REDD1 is mRNA levels of REDD1 in MCF-7 and MDA-MB-231 cells involved in the molecular mechanism of ZOL-induced were significantly increased after 50 μM ZOL treatment for cytotoxicity and ZOL inhibited mTOR signaling pathway 24 and 48 h (Figure 4A and B). The protein expression of through inducing REDD1 expression. REDD1 in ZOL-treated MCF-7 and MDA-MB-231 cells was also significantly increased as compared to that in controls The effect of zoledronic acid on ER stress responses. It has (Figure 4C). After treatment with ZOL for 24 h, the fold of been reported that REDD1 can be up-regulated by the ER increase of REDD1 was 1.1 and 1.8 for MCF-7 and MDA- stress response. Thus, ER stress response-related proteins

3810 Lan et al: Zoledronic Acid-induced Cytotoxicity in Breast Cancer Cells

Figure 3. The effects of zoledronic acid (ZOL) on protein kinase B/mammalian target of rapamycin (AKT/mTOR) signaling pathways of breast cancer cells. MCF-7 (A) and MDA-MB-231 (B) cells were treated with 50 μM ZOL for 24, 48 and 72 h. Western blot analysis was used to detect the expression of p-AKT, AKT, p-mTOR and mTOR in breast cancer cells. β-Actin was used as a loading control.

including IRE1α, ATF6, PERK, eIF2α and CHOP were occurs not only through inhibiting the cell cycle, but also by evaluated in breast cancer cells after treatment with 50 μM affecting other intracellular signaling pathways. ZOL for 24 and 48 h. Comparing to those of the controls, It is interesting that the mechanism of cell growth the expressions of ATF6, PERK and CHOP were inhibition by ZOL differs in different types of breast cancer significantly up-regulated in MDA-MB-231 cells treated with cells. Previous studies showed that ZOL can down-regulate ZOL for 24 and 48 hours (Figure 5). ZOL also activated the PI3K/AKT and MAPK pathways to inhibit growth or induce expression of phosphorylated-PERK and -eIF2α (p-PERK apoptosis in prostate gland and pancreatic cancer cells (19, and p-eIF2α), but had no effect on the expression of IRE1α 33). Indeed, AKT can directly and indirectly regulate and eIF2α. These results reveal that ZOL treatment can inhibitor of nuclear factor kappa-B kinase (IKK) activity to induce ER stress responses through activating PERK-eIF2α- cause nuclear factor kappa-light-chain-enhancer of activated CHOP and ATF6 signaling pathways in MDA-MB-231 cells. B cells (NF-κB) to move into the nucleus, and activation and anti-apoptosis gene transcription (34). AKT can promote B- Discussion cell lymphoma 2 (BCL2) family expression to inhibit cell apoptosis and increase cell survival (35). Therefore, In this study, our results clearly showed that the efficacy of inhibition of AKT activity has usually been associated with ZOL was higher in MDA-MB-231 cells than MCF-7 cells, antiproliferative effects or apoptotic responses. These results including cell growth inhibition and apoptosis induction. We corresponded well with our findings that ZOL-induced also found a new pathway of ZOL-induced growth cytotoxicity may be mediated through inhibiting p-AKT inhibition. ZOL induced ER stress to activate the PERK- expression in MDA-MB-231 cells but not in MCF-7 cells eIF2α-CHOP pathway followed by increasing REDD1 (Figure 3). Moreover, significant inhibition of p-mTOR expression, then inhibited the mTOR signaling pathway to expression was observed in ZOL-treated MDA-MB-231 cells result in cell growth inhibition. but not in MCF-7 cells (Figure 3), which also explains why We found that MCF-7 and MDA-MB-231 cells treated ZOL had different inhibitory effects in these two breast with ZOL exhibited significant accumulation of cells in the S cancer cell lines. Together, these results suggest that ZOL phase (Figure 2). However, significantly more sub-G0/G1 inhibited growth and induced cell apoptosis in breast cancer cells were observed for MDA-MB-231 cells than MCF-7 cells through inhibiting the AKT/mTOR signaling pathway. cells. These results suggest that S phase accumulation is not It has been reported that REDD1 can promote tuberous a prerequisite for the increase of sub-G0/G1 cells. These sclerosis complex 1/2 (TSC1/2) activation and mTOR findings suggest that ZOL-induced cell growth inhibition Complex 1 (mTORC1) inhibition to cause tumor

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Figure 4. The effects of zoledronic acid (ZOL) on the regulated in development and DNA damage response 1 (REDD1) mRNA and protein expression of breast cancer cells. MCF-7 (A) and MDA-MB-231 (B) cells were treated with 50 μM ZOL (+) for 24 and 48 hours and relative mRNA expression was determined by quantitative polymerase chain reaction. Western blot analysis was used to detect the expression of REDD1 (arrow) in breast cancer cells (C). β-Actin was used as a loading control.

suppression (23). A previous study also reported that high CCN1 expression and are more sensitive to ZOL by REDD1 promoted the apoptotic death of differentiated down-regulation of CCN1 expression in a dose-dependent neuronal PC12 cells and led to their sensitization to manner (37). Indeed, CCN1 a survival factor, is hypoxic and oxidative stress (36). In this study, we overexpressed in 30% of patients with metastatic breast observed that ZOL enhanced the expression of REDD1 at cancer has the ability to induce tumorigenesis and both mRNA and protein levels in MCF-7 and MDA-MB- angiogenesis, thus down-regulation of CCN1 expression by 231 cells (Figure 4). Compared to MCF-7 cells, MDA-MB- ZOL could lead to cell growth inhibition and apoptosis (37, 231 cells are more sensitive to ZOL and showed higher 38). Briefly, it is worth further investigating the relation level of ZOL-induced REDD1. These results together between CCN1, REDD1 and mTOR pathways in ZOL- suggest that REDD1 may be centrally involved in the drug induced cell death. sensitivity of breast cancer cells to ZOL. ZOL has been reported to inhibit FPPS in the mevalonate Our results show that MDA-MB-231 cells but not MCF- pathway and then induce ER stress (25). ER stress was found 7 cells were more sensitive to ZOL through inducing to trigger the PERK/eIF2α/ATF4 pathway and up-regulate REDD1 and inhibiting mTOR signaling pathway (Figures REDD1 expression (32, 39). This suggests that ZOL may 3 and 4). However, a different mechanism of action of ZOL induce REDD1 expression through activation of the ER on breast cancer cells has also been suggested. Espinoza et stress response. Indeed, our results showed that p-PERK and al. found that differently from non-cysteine-rich protein 61 p-eIF2α were activated and REDD1 expression was (CCN1)-expressing MCF-7 cells, MDA-MB-231 cells have increased simultaneously in breast cancer cells on ZOL

3812 Lan et al: Zoledronic Acid-induced Cytotoxicity in Breast Cancer Cells

Acknowledgements

We thank Novartis for providing zoledronic acid. This work was supported by grant V100C-119 from the Taipei Veterans General Hospital, Taiwan.

References

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