Combined Parthenolide and Balsalazide Have Enhanced

Published OnlineFirst November 14, 2016; DOI: 10.1158/1541-7786.MCR-16-0101

Cell Death and Survival Molecular Cancer Research Combined Parthenolide and Balsalazide Have Enhanced Antitumor Efﬁcacy Through Blockade of NF-kB Activation Se-Lim Kim1, Seong Hun Kim1, Young Ran Park1, Yu-Chuan Liu1, Eun-Mi Kim2, Hwan-Jeong Jeong2, Yo Na Kim3, Seung Young Seo1, In Hee Kim1, Seung Ok Lee1, Soo Teik Lee1, and Sang-Wook Kim1

Abstract

Balsalazide is a colon-specific prodrug of 5-aminosalicylate balsalazide together resulted in significant recovery of body that is associated with a reduced risk of colon cancer in patients weight and improvement in histologic severity. Administration with ulcerative colitis. Parthenolide, a strong NF-kB inhibitor, of parthenolide and balsalazide to CAC mice also suppressed has recently been demonstrated to be a promising therapeutic carcinogenesis as demonstrated by uptake of 18F-fluoro-2- agent, promoting apoptosis of cancer cells. In the current study, deoxy-D-glucose (FDG) using micro-PET/CT scans. These the antitumor effect of balsalazide combined with parthenolide results demonstrate that parthenolide potentiates the efficacy in human colorectal cancer cells and colitis-associated colon of balsalazide through synergistic inhibition of NF-kB activa- cancers (CAC) was investigated. The results demonstrate that tion and the combination of dual agents prevents colon car- the combination of balsalazide and parthenolide markedly cinogenesis from chronic inflammation. suppress proliferation, nuclear translocation of NF-kB, IkB-a phosphorylation, NF-kB DNA binding, and expression of Implications: This study represents the first evidence that com- NF-kB targets. Apoptosis via NF-kB signaling was confirmed bination therapy with balsalazide and parthenolide could be a by detecting expression of caspases, p53 and PARP. Moreover, new regimen for colorectal cancer treatment. Mol Cancer Res; 15(2); treatment of a CAC murine model with parthenolide and 1–11. 2016 AACR.

Introduction intestine and does not reach the colon (2). Sulfasalazine, the first 5-ASA–containing drug, functions by releasing an active compo- Colorectal cancer is a serious complication of ulcerative colitis nent in the colon through the activity of azo reductase expressed and responsible for up to 15% of all deaths in patients with by colonic bacterial. Sulfasalazine has been approved for thera- inflammatory bowel disease (1). Early detection and prevention peutic usage because of its ability to improve intestinal mucosal strategies (such as colonoscopy, mucosal biopsies, and procto- permeability (3, 4). However, the high rate of adverse effects colectomy) for colorectal cancer patients with ulcerative colitis related to sulfapyridine limits its use for patients. Balsalazide is a have limitations, and therefore, there is increased interest in novel orally administered prodrug of 5-ASA in which an inert identifying chemopreventive agents to reduce the overall risk of carrier molecule, 4-aminobenzoilb-alanine, is bonded to 5-ASA. colitis-associated colorectal cancer (CAC). Balsalazide has been shown to be more effective than sulfasala- 5-Aminosalicylate (5-ASA) is used to treat ulcerative colitis zine in the treatment of active ulcerative colitis (5, 6) and to be as because of its ability to control and relieve inflammation. In most effective and well tolerated as delayed release 5-ASA in the chronic cases, 5-ASA is absorbed rapidly and extensively in the upper treatment of ulcerative colitis (7). A recent study has shown that 5- ASA use is associated with reduced risk of colorectal cancer in 1Department of Internal Medicine, Research Institute of Clinical Medicine of patients with ulcerative colitis (8). However, the molecular Chonbuk National University-Biomedical Research Institute of Chonbuk Nation- mechanisms of balsalazide in the ulcerative colitis to colorectal al University Hospital, Jeonju, Korea. 2Department of Nuclear Medicine, cancer development have not been fully elucidated and the Research Institute of Clinical Medicine of Chonbuk National University-Biomed- antitumor effect of balsalazide remains controversial. ical Research Institute of Chonbuk National University Hospital, Jeonju, Korea. NF-kB is one of the key regulators in inflammation and cancer 3 Department of Pathology, Research Institute of Clinical Medicine of Chonbuk progression (9). NF-kB activation is markedly enhanced in National University-Biomedical Research Institute of Chonbuk National Univer- fl sity Hospital, Jeonju, Korea. patients with in ammatory bowel disease and the level of activated NF-kB is significantly correlated with the severity of intes- S.-L. Kim and S.H. Kim contributed equally to this article. tinal inflammation (10, 11). Activated NF-kB has the ability to Corresponding Author: Sang-Wook Kim, Department of Internal Medicine, Chon- promote the expression of various proinflammatory genes, thus buk National University Hospital, 20 Geonji-ro, Deokjin-gu, Jeonju 561-712, Korea. strongly influencing the process of ulcerative colitis (12). Phone: 826-3250-2302; Fax: 826-3254-1609; E-mail: [email protected] Parthenolide, a natural product, has been used for the treat- doi: 10.1158/1541-7786.MCR-16-0101 ment of fever and inflammatory disease. It is well known to inhibit 2016 American Association for Cancer Research. IL-1 and TNFa-mediated NF-kB activation (13, 14). Recent

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studies have demonstrated that parthenolide induces apoptotic a 60-mm culture dish were incubated with the designated doses of cell death through inhibition of NF-kB activation in a number of parthenolide and/or balsalazide for 24 hours. Cells were washed human cancers (13, 15). Several studies have also shown that twice with cold PBS and then resuspended in 500 mL of a binding parthenolide is a potent inhibitor of NF-kB activation and sup- buffer (10 mmol/L HEPES/NaOH pH 7.4, 140 mmol/L NaCl, and 6 presses the expression of proinflammatory cytokines in experi- 2.5 mmol/L CaCl2) at a concentration of 1 10 cells/mL. mental murine models (13, 16). We have also demonstrated that Annexin V-FITC (5 mL) and PI (1 mg/mL) were then added and parthenolide inhibits phosphorylation of IkBa and NF-kB acti- the cells were analyzed with a FACStar flow cytometer. vation, resulting in initiation of apoptosis, suppression of colo- Cell cycle and sub-G1 cell analysis were determined by PI (Ex/ rectal cancer tumor growth, and CAC development (17, 18). On Em ¼ 488/617 nm) staining. In brief, 1 106 cells were incubated the basis of these observations, it can be assumed that the in a 60-mm culture dish with the designated doses of parthenolide combination of parthenolide and balsalazide can be a promising and/or balsalazide for 24 hours. Total cells including floating cells strategy to prevent the development of ulcerative colitis to colo- were then washed with PBS and fixed in 70% (v/v) ethanol. Cells rectal cancer, inhibiting NF-kB activation pathway. were washed again with PBS, then incubated with PI (10 mg/mL) The aim of our study was to evaluate the effects of combination with simultaneous RNase treatment at 37C for 30 minutes. therapy with parthenolide and balsalazide on the inhibition of Cellular DNA content was measured using a FACStar flow cyt- NF-kB activation in human colorectal cancer cells and to inves- ometer (Becton-Dickinson) and analyzed using lysis II and CellFit tigate whether parthenolide and balsalazide are effective in pre- software (Becton-Dickinson). venting carcinogenesis from chronic colitis. Parthenolide-induced apoptosis in colon cancer cells was assessed using Hoechst 33258. The cells were treated with various concentrations of parthenolide for 24 hours, and then stained Materials and Methods with Hoechst 33258 (1 mg/mL) at 37 C for 10 minutes. Nuclear Chemicals and reagents morphology was examined under a Confocal Laser Scanning Parthenolide and z-VAD-FMK were obtained from Calbio- Microscope (Carl Zeiss) to identify cells undergoing apoptosis. chem. Balsalazide was provided by Chong Kun Dang Pharm. Parthenolide was dissolved in dimethylsulfoxide (DMSO; Sigma) Cytoplasmic and nuclear extract preparation to a concentration of 100 mmol/L and stored at 20 C in the dark. Cells were harvested and resolved in a lysis buffer [20 mmol/L Balsalazide was dissolved in FBS-free media to a concentration of Tris-HCl (pH 7.5), 137 mmol/L NaCl, 10% glycerol (v/v), 1% fl 100 mmol/L at 4 C. Annexin V- uorescein isothiocyanate Triton X-100 (v/v), 1 mmol/L Na3VO4, 1 mmol/L phenylmethyl- (Annexin-V FITC) and propidium iodide (PI) were purchased sulphonylfluoride, and protease inhibitor cocktail]. After centri- from Invitrogen. Hoechst 33258 was from Sigma. fugation at 16,000 g for 15 minutes, the supernatants were used as cytoplasmic extracts. To extract the nuclear fraction, cells were Cell culture and cell line authentication resuspended in 150 mL of buffer A [10 mmol/L HEPES (pH 7.9), HCT 116, SW480 and HT-29 (ATCC) were employed as repre- 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiotreitol, sentative human colorectal cancer cells. The cells were cultured in 0.5 mmol/L phenylmethylsulphonylfluoride, 0.4% Nonidet P-40 RPMI1640 medium supplemented with 10% FBS, 100 U penicil- (v/v) and protease inhibitor cocktail] for 20 minutes on ice and lin, and 100 U streptomycin. The three cell lines listed above were then centrifuged at 2,300 g for 5 minutes. The resulting pellets validated by short-tandem repeat (STR) DNA fingerprinting using were resolved in 100 mL of buffer C [20 mmol/L HEPES (pH 7.9), the Promega PowerPlex 18D System and analyzed by GeneMapper 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 Software 5 at Cosmo Genetech Korea in January 2016. mmol/L dithiotreitol, 0.5 mmol/L phenylmethylsulphonylfluor- For treatment of cells with parthenolide, the cells were sub- ide, and protease inhibitor cocktail] for 30 minutes on ice. After cultured in RPMI1640 medium without FBS for 24 hours. Parthe- centrifugation at 16,000 g for 15 minutes, the supernatants were nolide and balsalazide were diluted in FBS-free medium to used as nuclear extracts. achieve designated concentrations, and the same concentration of DMSO was applied to the cells as a control. Electrophoretic mobility shift assay NF-kB activity was measured by EMSA. In brief, prior to Cell viability assay stimulation, cells were preincubated with the indicated concen- HCT 116, SW480, and HT-29 cells were plated at a density of trations of parthenolide and/or balsalazide at 37C for 24 hours. 1.0 104 cells per well in 96-well plates. Cells were treated with In following, cells were stimulated with TNFa (10 ng/mL), parthenolide and/or balsalazide for 24, 48, and 72 hours. After harvested by centrifugation, washed twice with ice-cold 1 PBS, then, the medium was removed, 200 mL of fresh medium plus and then nuclear extracts were prepared. A double-stranded 20 mL of 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium oligonucleotide for NF-kB (Promega) was end-labeled with bromide (MTT, 2.5 mg dissolved in 50 mL of DMSO, Sigma) were [g-32P] ATP and purified with a G-25 spin column (Boehringer added to each well. After incubation for 4 hours at 37 C, the Mannheim). Nuclear extracts were incubated for 20 minutes at culture medium containing MTT was removed and then 200 mLof room temperature with a gel shift binding buffer [5% glycerol, 1 DMSO was added, followed by shaking until the crystals were mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L dissolved. Viable cells were detected by measuring the absorbance NaCl, 10 mmol/L Tris–HCl, pH 7.5, 50 mg/mL poly(dI-dC) poly at 570 nm using a microplate reader (Molecular Devices). (dI-dC)] and 32P-labeled oligonucleotide. The DNA–protein complex formed was separated on 4% native polyacrylamide gel, Detection of apoptosis and the gel was transferred to Whatman 3 MM paper, dried, and Apoptotic cell death was determined by staining cells with exposed to X-ray film, captured and analyzed by the BAS-2500 Annexin V-FITC (Ex/Em ¼ 488/519 nm). In brief, 1 106 cells in Image Analyzer (Fuji Film).

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Western blotting phology; 1, focal inflammatory cell infiltrate around the crypt The protein concentration in cell lysates or tissue lysates was base; 2, diffuse infiltration of inflammatory cells around the crypts measured using a Protein Quantification kit from Bio-Rad. Fifty or erosion/destruction of the lower one-third of the glands; 3, micrograms of protein or 30 mg of nuclear extract protein per lane erosion/destruction of the lower two-thirds of the glands or loss of was loaded onto a SDS-polyacrylamide gel. After transferring and all the glands. Invasion depth was scored as follows (20): 0 ¼ no blocking, the polyvinylidene difluoride (PVDF) membrane was invasion, 1 ¼ invasion through mucosa, 2 ¼ invasion through probed with various antibodies (anti-p-IkB-a, anti-p65, anti- submucosa, 3 ¼ full invasion through muscularis and into serosa. phospho-p65, anti-caspase 8, anti-caspase-9, anti-caspase-3, All histologic analyses were performed by a pathologist who was anti-p53, anti-PARP, anti-Bcl-xL, anti-Bcl-2, anti-VEGF, anti- blinded to treatment status. MMP9, anti-cyclin D1 anti-cFLIP, anti-COX2 anti-GLUT1, anti-Hexokinase II, anti-actin and anti-Lamin B antibody). The Statistical analysis binding of antibody to antigen was detected using enhanced ECL The data are presented as the mean SE of at least three prime (GE Healthcare), captured and analyzed by the Las-3000 independent experiments performed in duplicate. Representative luminescent Image Analyzer (Fuji Film). blots are shown. All the data were entered into Microsoft Excel 5.0, and SPSS Software was used to perform two-tailed t tests or CAC murine model the ANOVA, where appropriate. A P value <0.05 was considered Sixty specific pathogen–free mice (Balb/C female mice, 6 week) significant. were purchased from Orient. Mice were given ad libitum access to water and standard rodent food until they reached the desired weight (18–20 g). Mice were maintained on a 12/12 hours light/ Results dark cycle under specific pathogen-free conditions. All procedures Effect of parthenolide on balsalazide-induced cell viability using the mice were reviewed and approved by Chonbuk National Human colorectal cancer cell lines, HCT116, SW480, and HT- University Animal Care and Use Committee (approval no: CBNU 29 cells were treated with various concentrations of balsalazide for 2015-0027). Twelve in each group were randomly assigned after 24 hours. Cell proliferation was significantly affected by treatment they were weighed. Mice were intraperitoneally injected with 7.4 with 20 mmol/L balsalazide in all cell lines (Fig. 1A). Next, to mg/kg body weight of azoxymethane (AOM) dissolved in phys- examine whether parthenolide promotes balsalazide-induced iologic saline. Seven days later, 3% dextran sulfate sodium (DSS) death of human colorectal cancer cells, the cells were treated with was given in the drinking water for 7 days, followed by 14 days of parthenolide and balsalazide for 24, 48, and 72 hours. After regular water. This cycle was repeated three times. Parthenolide (2 cotreatment with 20 mmol/L balsalazide and 5 or 10 mmol/L mg/kg) suspended in saline was administered by intraperitoneal parthenolide, the viability of cells was dramatically reduced in a injection three times a week at break time. Balsalazide (200 mg/ dose-dependent manner of parthenolide, showing 3.4-fold, 2.9- kg) suspended in saline was administered daily by oral gavage at fold, and 2.2-fold decrease for 24-hour treatment in HCT116, break time under anesthesia using isoflurane. SW480, and HT-29 cells with 10 mmol/L parthenolide, respectively (Fig. 1B). And the cotreatment with 20 mmol/L balsalazide Ex vivo micro-PET/CT imaging and 10 mmol/L parthenolide for 48 and 72 hours also showed After finishing CAC-related procedures, all mice were fasted for synergistic reduction of viability (4.5-fold for 48 hours and 12- 6 hours to minimize plasma glucose concentration before exam- fold for 72 hours in HCT 116 cells; 2.8-fold for 48 hours and 3.3- ination. The mice were anesthetized under 2% isoflurane and 18F- fold for 72 hours in HT-29 cells; 4.4-fold for 48 hours and 11-fold FDG (3.7 106 Bq) was injected via the tail vein with an insulin for 72 hours in SW480 cells). For the subsequent experiments, we syringe. After 30 minutes, mice were sacrificed by cervical dislo- used the concentration of 5 mmol/L parthenolide and 20 mmol/L cation and the entire colon was removed from the cecum to the balsalazide for the 24-hour treatment resulted in approximately anus. The colon was then opened longitudinally and washed 50% inhibition of viability. HCT 116 cells showed more remark- twice with saline. The colons of each group were placed on the able changes than other cells in viability with combined treat- scanner bed. Micro-PET/CT images were acquired using a FLEX X- ment. Moreover, population doubling time of HCT116 cells is PET/X-O small-animal imaging instrument (X-PET/CT System, shorter than HT-29 and SW480 cells (population doubling time: GE Healthcare), which combines a PET scanner and a multislice 21 hours for HCT116 cells, 23 hours for HT-29 cells, and 38 hours helical CT scanner. CT scans were performed with the following for SW480 cells), so it is much easier to handle for in vitro scanning parameters: 75 kVp and 0.25 mA. The CT images were experiment. For that reason, HCT116 cells were used as a repre- acquired with 256 projections over 2 minutes. After acquisition of sentative colorectal cancer cells for the subsequent experiments. PET/CT scans, for histologic analysis, the distal colons were fixed in 10% neutral buffered formalin for 24 hours and transferred to 70% ethanol for subsequent paraffin embedding. For Western Effect of parthenolide on balsalazide-induced apoptosis blotting, total protein was extracted using RIPA buffer (Invitro- To confirm the above observations, Annexin-V/PI analysis was gen) and homogenizer according to the manufacturer's instruc- performed using FACScan (Fig. 2A). We found that treatment of tions and quantified. HCT116 cells with a single agent induced low-level early and late apoptotic cell death [parthenolide alone (early apoptosis: 4.99%, Histologic analysis late apoptosis: 4.98%), balsalazide alone (early apoptosis: 2.28%, Five-micron tissue sections were stained with hematoxylin and late apoptosis: 3.56%)]. In agreement with cell growth inhibition, eosin, and histologic analysis was performed by a pathologist in a cotreatment with parthenolide and balsalazide dramatically double-blind manner. The inflammation scores of mucosal increased the apoptotic cell death (early apoptosis: 17.11%, late inflammation were determined as follows (19): 0, normal mor- apoptosis: 14.24%) by 5-fold compared with treatment with

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Figure 1. Inhibitory effect on cell growth induced by combination of parthenolide (PT) and balsalazide. A, Human colorectal cancer cells were treated with various concentrations of balsalazide for 24 hours. Cell viability was analyzed using MTT assay. Data represent the mean value SE of three independent experiments. , P < 0.05; , P < 0.01 versus control. B, Human colorectal cancer cells were treated with balsalazide (20 mmol/L) plus various concentrations of parthenolide for 24, 48, and 72 hours. The data represent the mean SE of three independent experiments , P < 0.01 versus control, #, P < 0.05 versus parthenolide only.

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Figure 2. Apoptotic effect induced by combination treatment with parthenolide (PT) and balsalazide. A, Apoptotic cell death induced by combination treatment. After treatment with parthenolide and/or balsalazide for 24 hours, HCT116 cells were harvested and stained with Annexin-V FITC and PI. B, Cell-cycle modification induced by combination treatment. After treatment with parthenolide and/or balsalazide for 24 hours, HCT116 cells were harvested and stained with PI. The percentage of sub-G1 population is shown in each histogram, and the total number of events analyzed for each condition was 10,000. C, DNA condensation and fragmentation induced by combination treatment. DNA condensation through apoptotic cell death was determined using Hoechst 33258 (1 mg/mL) in HCT116 cells. Apoptotic nuclei stained with Hoechst 33258 show intense fluorescence corresponding to chromatin condensation (arrow) and fragmentation. balsalazide alone, indicating that parthenolide promotes balsa- blotting. Neither phosphorylation of IkB-a nor inhibition of IkB- lazide-induced apoptosis in HCT116 cells. a phosphorylation was observed with a single-agent treatment. We also evaluated cell-cycle modifications induced by parthe- However, cells cotreated with parthenolide and balsalazide nolide and balsalazide in HCT116 cells (Fig. 2B). Twenty-four showed a significant inhibition in the TNFa-induced phosphor- hours after incubation with single or dual agents, cells were ylation of IkB-a (Fig. 3A). analyzed by PI staining and flow cytometric analysis. Treatment As the degradation of IkB-a is known to cause nuclear trans- with parthenolide and/or balsalazide resulted in the presence of a location of the p65 subunit of NF-kB, we next examined whether sub-G1 population, suggestive of apoptotic cell death. Peaks parthenolide and/or balsalazide modulates TNFa-induced nucle- accounting for 9.91% and 8.12% of the overall cell population ar translocation of p65. Nuclear and cytosolic extracts of HCT116 were detectable in HCT116 cells treated with parthenolide and cells treated with parthenolide and/or balsalazide were analyzed balsalazide, respectively. A much higher sub-G1 population by Western blotting. As shown in Fig. 3B, cotreatment of HCT116 (37.82%) was observed with cotreatment of parthenolide and cells with parthenolide and balsalazide resulted in a decrease in balsalazide than with single treatment, indicating that the com- nuclear p65 level, whereas the cytosolic p65 level was increased. bination of the two agents substantially promotes apoptosis of The results show that cotreatment with parthenolide and balsa- HCT116 cells. lazide also blocks TNFa-induced activation of p65. To understand the mechanism of cell death induced by com- To determine the effect of parthenolide and/or balsalazide on bination treatment, apoptotic nuclear morphology was evaluated the DNA-binding activities of NF-kB, we performed electropho- after Hoechst 33258 staining (Fig. 2C). In the control group, retic mobility shift assay. TNFa-induced DNA-binding activity of HCT116 cells were regular in morphology and grew fully in NF-kB was significantly suppressed by parthenolide treatment patches. In the parthenolide- or balsalazide-treated groups, only and the DNA-binding activity of NF-kB was almost completely a few cells exhibited nuclear condensation and fragmentation. blocked by cotreatment with parthenolide and balsalazide (Fig. However, cotreated cells showed apoptotic characteristics, such as 3C). These results demonstrate that parthenolide has a greater cell shrinkage, nuclear condensation, and fragmentation. inhibitory effect on TNFa-induced DNA-binding activity of NF- kB than balsalazide does. Inhibition of TNFa-induced NF-kB activation by cotreatment Phosphorylation of p65 has been shown to regulate its tran- with parthenolide and balsalazide scriptional activity (22), so we examined whether parthenolide Inflammatory cytokines are required to activate NF-kB; there- and/or balsalazide affected the post-translational modification of fore, TNFa was employed as a representative of NF-kB stimuli. p65. Cells were treated with parthenolide and/or balsalazide in The translocation of NF-kB to the nucleus is preceded by phos- the presence of TNFa, and phosphorylation of p65 at serine 536 phorylation, ubiquitination, and proteolytic degradation of IkB-a was evaluated in nuclear extracts using a 536 phosphoserine- (21). To determine whether inhibition of TNFa-induced NF-kB specific p65 antibody. As shown in Fig. 3D, parthenolide inhib- activation by parthenolide and/or balsalazide is caused by inhi- ited phosphorylation of p65 at serine 536 in a dose-dependent bition of IkB-a degradation, HCT116 cells were treated with manner. However, balsalazide did not prevent TNFa-mediated parthenolide and/or balsalazide for 24 hours and then with TNFa phosphorylation of p65 at serine 536. These results strongly (20 ng/mL) for 30 minutes. Phosphorylation and degradation of suggest that parthenolide enhances the effect of balsalazide on IkB-a in cytoplasmic extracts of the cells were analyzed by Western inhibition of NF-kB activation.

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Figure 3. Downregulation of NF-kB signaling by combination treatment with parthenolide (PT) and balsalazide. A, Cytosolic protein extracts were prepared from HCT116 cells treated with parthenolide and/or balsalazide for 24 hours and then treated with TNF-a for 30 minutes. The combination of parthenolide and balsalazide markedly suppressed IkB-a phosphorylation. Actin was used as a loading control for cytosolic protein. B, Cytosolic and nuclear extracts of HCT116 cells were prepared and used to determine the translocation of NF-kB subunit p65. The combination of parthenolide and balsalazide dramatically suppressed translocation of p65 to the nucleus from the cytosol. Actin and lamin B were used as loading controls of cytosolic and nuclear protein, respectively. C, Nuclear extracts were prepared and subjected to electrophoretic mobility shift analysis (EMSA) for NF-kB DNA binding using a 32P-labeled NF-kB probe. The cold probe lane contained the 1 hours poststimulation nuclear extract incubated with 32P-labeled NF-kB probe plus 10-fold excess unlabeled probe. The arrow indicates shifted bands. The combination of parthenolide and balsalazide dramatically blocked NF-kB DNA-binding activity. D, Nuclear protein extracts were prepared from cells treated with parthenolide or balsalazide or a combination with parthenolide and balsalazide for 24 hours. The combination of parthenolide and balsalazide dramatically suppressed p65 phosphorylation. Lamin B was used as loading control for nuclear protein.

Regulation of apoptotic proteins by cotreatment with treatment with parthenolide and cotreatment with balsalazide, parthenolide and balsalazide indicating that parthenolide potentiates the apoptotic effects of Most apoptotic cell death is initiated by activation of caspases balsalazide in HCT116 cells (Fig. 4B, second panel). (23). To delineate the mechanisms by which treatment with parthenolide potentiates balsalazide-induced apoptosis, we next Regulation of NF-kB target gene products by cotreatment with analyzed the protein expression levels of caspases by Western parthenolide and balsalazide blotting. Treatment of HCT116 cells with parthenolide decreased To investigate whether cotreatment with parthenolide and the levels of full-length caspases compared with no treatment, balsalazide inhibits the expression of NF-kB target gene products whereas balsalazide did not show any change in the levels of involved in aggressive cancers and inflammation, we analyzed caspases (Fig. 4A). However, Western blot analysis revealed that expression of Bcl-xL, Bcl-2, VEGF, MMP-9, Cyclin D1, cFLIP, and levels of full-length caspases in the cells cotreated with parthe- COX-2 by Western blotting (Fig. 5). The expression of antiapop- nolide and balsalazide were significantly decreased compared totic proteins, Bcl-2, Bcl-xL, and cFLIP, was markedly reduced by with the levels in the cells treated with parthenolide or balsa- cotreatment for 24 hours. Moreover, the proliferation and cell lazide alone. Furthermore, the decrease in caspase levels in the progression–related proteins, COX-2 and Cyclin D1, were sup- cotreated cells was significantly blocked by pretreatment with pressed. Invasion- and angiogenesis-related gene products, MMP- the general caspase inhibitor Z-VAD-FMK. These data indicate 9 and VEGF, were also dramatically inhibited by cotreatment with that parthenolide and balsalazide activate caspases and that parthenolide and balsalazide. cotreatment with parthenolide and balsalazide promotes activation of caspases. Amelioration of carcinogenesis in the CAC animal model by The activation of caspase-3 leads to the cleavage of its down- cotreatment with parthenolide and balsalazide stream molecular targets, PARP and p53, hallmarks of apoptosis Azoxymethane (AOM) is a procarcinogen that causes for- (24). The level of p53 was significantly increased by cotreatment mation of O6-methylguanine upon metabolic activation. with parthenolide and balsalazide compared with the level in cells AOM induces tumors in the distal colon of rodents and is treated with parthenolide or balsalazide alone (Fig. 4B, first commonly used to elicit colorectal cancer in experimental panel). The cleavage of PARP was significantly increased by animals (25). To test the physiologic relevance of the

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Parthenolide Enhances Antitumor Effect of Balsalazide

Figure 4. Regulation of caspases and apoptotic molecules by a combination treatment with parthenolide (PT) and balsalazide. A, Total cell lysates of HCT116 cells were prepared after treatment with parthenolide and/or balsalazide for 24 hours and then analyzed using caspase-3, -8, or -9 antibody by Western blotting. Protein levels of caspase-3, -8, and -9 were decreased by combination treatment. However, reduction of caspases was blocked by pretreatment with the pan- caspase inhibitor, Z-VAD-FMK. Actin was used as a loading control. B, Cytosolic protein extracts were prepared from HCT116 cells treated with parthenolide and/or balsalazide for 24 hours. The level of tumor suppressor protein, p53, was increased by cotreatment with parthenolide and balsalazide. Actin was used as a loading control of cytosolic protein. Nuclear protein extracts were prepared, and cleaved PARP was enhanced by the combination of parthenolide and balsalazide. Lamin B was used as a loading control for the nuclear fraction.

parthenolide/balsalazide–mediated suppression of carcino- thermore, we confirmed that 18F-FDG uptake in the colon was genesis from chronic inflammation in vivo, we conducted an significantly decreased by cotreatment with parthenolide and in vivo study using a murine model of AOM/DSS–induced balsalazide. colon cancer. First, we compared body weights between We further investigated whether protein levels induced by the AOM/DSS and administration of parthenolide- and/or balsa- NF-kB pathway in colonic tissues are affected after parthenolide lazide-treated mice (Fig. 6A). Cotreated mice significantly and balsalazide cotreatment (Fig. 6E). Consistent with our in vitro recovered the weight loss after the second break time. At the results, rapid decline of phosphorylation of IkB-a and p65 was start of the third break time, the body weight was 16.95 observed in cotreated mice colon. However, the level of p65 did 0.201 g for AOM/DSS mice and 20.304 0.359 g for parthe- not show any significant change after cotreatment with parthenolide/balsalazide cotreated mice, indicating that cotreatment nolide and/or balsalazide. The expression of the representative with parthenolide and balsalazide prevents the loss of body NF-kB target gene product, VEGF, was markedly suppressed by weight caused by carcinogenesis. cotreatment compared with the level observed after AOM/DSS Next, we evaluated colon length and the characteristics of colon treatment alone. Glucose transporters (GLUT) in the cell mem- carcinogenesis (Fig. 6B). Numerous nodular, polypoid, or cater- branes and the activity of intracellular enzymes such as hexoki- pillar-like tumors were observed in the middle and distal colon of nases (HK) mediate the intracellular accumulation of 18F-FDG AOM/DSS mice. Shortening of the colon, which is another char- (26). Therefore, levels of GLUT1 and HKII in the colonic tissues acteristic of colon carcinogenesis, was significantly improved in were determined by Western blotting. High expression of GLUT1 the cotreated mice. and HKyy was observed in the colon of AOM/DSS–treated mice We also evaluated the severity of acute murine colitis by compared with in the colon of normal control mice. The increased blinded histologic injury scoring from the cecum to the distal levels of both proteins in the colon of mice were dramatically colon (Fig. 6C). The histologic examination of colons from AOM/ suppressed by treatment with parthenolide and/or balsalazide, DSS–induced colon cancer mice showed inflammatory lesions and the expression of the proteins was well correlated with the that included total impairment of the glandular structure, muco- 18F-FDG PET/CT image. sal ulceration, crypt damage, and the infiltration of inflammatory cells. In contrast, cotreatment with parthenolide and balsalazide markedly reduced the impairment of the glandular architecture Discussion and infiltration of inflammatory cells. Histologic grading (inflam- In contrast to sporadic colorectal cancers that are mainly caused mation score and invasion depth) showed that the cotreatment by genetic factors, CAC is mostly associated with inflammatory significantly attenuated the overall score compared with the bowel disease (IBD), and, therefore, it is understood that chronic AOM/DSS mice. inflammation of the colonic epithelium can induce CAC. Spo- We then assessed the metabolic activity of carcinogenesis by radic colorectal cancers, accounting for 65%–85% of all colorectal 18F-FDG PET/CT imaging. As shown Fig. 6D, analysis of the PET/ cancers, develop through the "adenoma-cancer" progression, CT images revealed clearly elevated 18F-FDG uptake in the colon while CAC develops through "inflammation-dysplasia-cancer" of AOM/DSS–treated mice with the most intensive area of 18F- progression (27). Therefore, we suggest that modulation of FDG uptake in the distal colon. Compared with AOM/DSS– inflammation might be an effective approach for preventing CAC treated mice, 18F-FDG uptake in the colon of parthenolide- or cancers. In 1994, the risk of CAC among patients with ulcerative balsalazide-treated mice exhibited somewhat less uptake. Fur- colitis was reduced through treatment with sulfasalazine, a 5-ASA

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In this study, we used human colorectal cancer epithelial cells for in vitro study. However, ulcerative colitis is one of the T-cell– mediated IBD and excessive TNFa production in macrophages by T cells (32). Therefore, you may argue that human colorectal cancer cells are not reasonable to determine the effect of this therapeutic strategy in CAC. However, intestinal epithelial cells (IEC) exert their immunologic functions by processing and pre- senting antigens to T cells, so they can function as sensors of mucosal injury and actively participate in the mucosal response to intestinal inflammation. Chronic damage of intestinal epithelial barrier leads to development of CAC (33). By the reason, we selected commercial human colorectal cancer epithelial cells for in vitro experiments. Moreover, CAC in mice develops tumorigenesis from intestinal epithelium because of drinking water included DSS. Therefore, for this study, we consider that epithelial colorectal cancer cells for in vitro is partly correlated with in vivo observations. A central molecule in inflammation, NF-kB, has been shown to be activated in all cells, where it regulates expression of diverse target genes that promote cell proliferation, regulate immune and inflammatory response, and contribute to pathogenesis of various diseases, including cancers (34). In chronic inflammation, NF-kB has a specific role connecting inflammation to cancer. Over 15% fl Figure 5. of malignancies are initiated by chronic in ammatory disease; for fl Downregulation of NF-kB–regulated gene products by combination of example, squamous cell carcinoma by skin in ammation, viral parthenolide (PT) and balsalazide. Total cell lysates of HCT116 cells were hepatitis by liver cancer, and IBD by colorectal cancer (35). prepared after treatment with parthenolide and/or balsalazide for 24 hours and Studies have suggested that constitutive NF-kB activation in IBDs then analyzed using Bcl-xL, Bcl-2, VEGF, MMP9, Cyclin D1, cFLIP, COX2, and actin increases the risk of colorectal cancer (10, 12, 36). A number of – antibody by Western blotting. The protein levels of NF-kB regulated gene studies have demonstrated that parthenolide is a strong inhibitor products were significantly decreased by combination treatment with parthenolide and balsalazide. Actin was used as a loading control. of NF-kB activation and can effectively inhibit the expression of proinflammatory cytokines in cultured cells and experimental animal models (13, 37–42). In 1994, Zao and colleagues found derivative (28). It was also demonstrated that 5-ASA and its that administration of parthenolide significantly reduces the derivatives (i) inhibit Wnt/b-catenin signaling pathways; (ii) severity of DSS-induced colitis through inhibition of phosphor- activate PPAR-g; (iii) suppress NF-kB activity; and (iv) inhibit ylation of IkB-a and p65 proteins, resulting in reduction in oxidative damage and DNA mutations. However, recent studies expression of inflammatory mediators (16). Interestingly, our have demonstrated that there is not a statistically significant current study showed that treatment with parthenolide alone inhibitory effect of 5-ASA in colorectal cancer, raising controversy suppresses TNFa-induced phosphorylation of IkB-a, transloca- over the potency of 5-ASA and its derivatives for prevention of tion of p65 and phosphorylation of p65 at serine 536. Balsalazide colonic carcinogenesis from ulcerative colitis. To our knowledge, similarly downregulated phosphorylation of IkB-a and translo- there has been no investigation on the efficacy and underlying cation of p65; however, it did not show regulation of p65 molecular mechanisms of balsalazide and parthenolide combi- phosphorylation at serine 536, even at the maximum concentra- nation chemotherapy in vivo or in vitro. tion. Moreover, the combination of parthenolide and balsalazide In our previous studies, we found that parthenolide can be a significantly suppressed TNFa-induced DNA-binding activity of potential chemopreventive and therapeutic agent for colorectal NF-kB compared with treatment with balsalazide alone. Notably, cancer and CAC (17, 18). We have also shown that parthenolide as compared with simple translocation of p65 without phosphor- has potential to be applied in combination therapy for colorectal ylation, phosphorylation of p65 triggers strong transcriptional cancer treatment: combination with 5-fluorouracil (5-FU) activity of NF-kB producing significant modulation of target gene can overcome 5-FU resistance in human colorectal cancer, and transcription (43). To summarize the molecular mechanism of parthenolide sensitizes cancer cells to the TNF-related apoptosis- combined treatment, parthenolide potentiates balsalazide- inducing ligand (TRAIL) in TRAIL-resistant colorectal cancer cells induced inhibition of transcriptional activity of NF-kB by inhibit- (29–31). These findings indicate that parthenolide may increase ing of phosphorylation of p65 at serine 536. the efficacy of balsalazide in preventing carcinogenesis caused by The relation between NF-kB and apoptosis was elucidated chronic inflammation. The results in the current study showed recently by studies demonstrating that inhibition of NF-kB acti- that a combination treatment with parthenolide and balsalazide vation, either by the IkB super repressor or in Rel A (p65) significantly inhibited NF-kB activation and was linked to regu- knockout cells, results in increased apoptosis (43, 44). In partic- lation of apoptosis-related molecules and NF-kB target genes. ular, NF-8B is responsible for the expression of genes involved in Moreover, the combination of parthenolide and balsalazide proliferation, tumor survival, mitotic cell cycle, tumor cell inva- improved AOM/DSS–induced CAC in mice clinically and histo- sion, and angiogenesis. Our results revealed that the combination logically. These findings suggest that combination of parthenolide treatment with parthenolide and balsalazide significantly down- and balsalazide can be a useful therapeutic approach to treat CAC. regulated NF-kB–regulated gene products such as Bcl-2, Bcl-xL,

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Parthenolide Enhances Antitumor Effect of Balsalazide

Figure 6. Reduction of carcinogenesis in a mouse model of CAC by combination treatment with parthenolide (PT) and balsalazide. Mice treated with parthenolide and/or balsalazide were given AOM and DSS in their drinking water. Parthenolide was injected via the intraperitoneal route three times a week and balsalazidewas administrated via the intragastric route using oral zonde once a day at break time. A, The body weight of CAC mice was measured three times a week, and the means of the body weight of each group are presented. Data shown are presented as the mean SE of three independent experiments, and derived from 12 mice per group. , P < 0.05 versus AOM/DSS–treated group and combined treatment group. B, Length of inflamed colon on the final day of the study period. , P < 0.05 versus AOM/DSS-treated group. C, Hematoxylin and eosin staining (magnification 10) of colonic mucosal tissue section from mice. Inflammation scores and invasion depth of tissue specimens obtained from mice. Histologic score of H&E-stained specimens of the colon was determined by two pathologists in a blinded fashion. D,18F-FDG PET/CT imaging from representative colonic tissue in CAC mice of each group is presented. Radioactivity uptake represents the biological and metabolic activities of cancer and inflammation. E, Total tissue extracts were prepared after sacrifice and analyzed using phosphor-IkB-a, phosphor-p65, p65, VEGF, GLUT1, HKyy, and actin antibody by Western blotting. Actin was used as a loading control.

VEGF, MMP9, Cyclin D1, cFLIP, and COX2 in human colorectal AOM is a procarcinogen and is metabolically activated to a cancer cells through the inhibition of transcriptional activity of potent alkylating agent forming O6-methyl-guanine (25). Its NF-kB. oncogenic potential is markedly augmented in the setting of

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Kim et al.

chronic inflammation, such as that induced by repeated cycles of In conclusion, we have shown that combination of parthe- DSS treatment (45). The power of this model has recently been nolide and balsalazide exhibits synergistic suppression of NF- demonstrated in deciphering the epithelial versus myeloid cell kBandNF-kB–regulated gene products that are associated with contribution of IKKb to polyp formation in the setting of inflam- apoptosis, proliferation, invasion, angiogenesis, and inflammation (46). NF-kB activation is normally triggered in response to mation. Moreover, our data suggest a molecular mechanism by microbial and viral infections and proinflammatory cytokines, all which parthenolide enhances the effect of balsalazide on inhi- of which activate the IkB kinase (IKK) complex (21). IKK phos- bition of phosphorylation of p65 at serine 536, which is critical phorylates NF-kB–bound IkBs and targets them for ubiquitin- for the transcriptional activity of NF-kB. Therefore, adminis- dependent degradation, allowing liberated NF-kB dimers to enter tration of parthenolide and balsalazide significantly inhibits the nucleus (47). Our results in the current study showed that the inflammation–carcinoma sequence and can be a crucial phosphorylation of IkB-a and p65 in colonic tissues was dramat- regulator of CAC. ically reduced by cotreatment with parthenolide and/or balsalazide compared with AOM/DSS-induced CAC mice. However, p65 Disclosure of Potential Conflicts of Interest protein level did not change after treatment with parthenolide No potential conflicts of interest were disclosed. and/or balsalazide in colonic tissues, suggesting that NF-kB activation leads only to translocation of p65 without an increase Authors' Contributions in total protein level. Conception and design: S.L. Kim, S.Y. Seo, S.T. Lee, S.W. Kim A potential limitation of our study is that we used histologic Development of methodology: Y.R. Park, Y.N. Kim observation related to inflammation rather than carcinogenesis. Acquisition of data (provided animals, acquired and managed patients, Histologic scoring system such as epithelial damage and infiltra- provided facilities, etc.): S.H. Kim, E.-M. Kim, H.-J. Jeong, S.Y. Seo, S.T. Lee tion of immune cells in the mucosa/submucosa/muscularis/sero- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, sa are typical methods which evaluate severity of ulcerative colitis computational analysis): Y.N. Kim, S.Y. Seo, S.W. Kim Writing, review, and/or revision of the manuscript: S.L. Kim, Y.N. Kim, in animal models. However, Cooper and colleagues reported that S.Y. Seo, S.T. Lee histologic level of inflammation is significantly related to dyspla- Administrative, technical, or material support (i.e., reporting or organizing sia and/or cancer showing that CAC has higher inflammation data, constructing databases): Y.-C. Liu score than those of without cancer (48). Thus, our histologic Study supervision: I.H. Kim, S.O. Lee analysis partially supports that combined treatment with parthe- Other (specify): S.H. Kim nolide and balsalazide prevents carcinogenesis from chronic colitis. Acknowledgments Clinically, 18F-FDG PET/CT is a noninvasive imaging modality The authors thank Professor Mie-Jae Im from Chonbuk National University widely used for detection, staging, and follow-up of tumors, Medical School for proofreading and contributions. infections and inflammation (49). Spier and colleagues have also presented promising results for the use of 18F-FDG PET/CT in the Grant Support assessment of inflammation in patients with IBD (50). Moreover, This work was supported by Fund of Biomedical Research Institute, Chonbuk Hindryckx and colleagues have demonstrated that 18F-FDG PET/ National University Hospital. The costs of publication of this article were defrayed in part by the payment of CT can detect DSS-induced intestinal inflammation and imaging fi page charges. This article must therefore be hereby marked advertisement in ndings are correlated strongly with histologic examination (51). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In accordance with these observations, we were able to correlate 18 F-FDG PET/CT images with histologic alterations in an exper- Received March 22, 2016; revised October 8, 2016; accepted October 10, imental CAC model. 2016; published OnlineFirst November 14, 2016.

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Combined Parthenolide and Balsalazide Have Enhanced Antitumor Efficacy Through Blockade of NF- κB Activation

Se-Lim Kim, Seong Hun Kim, Young Ran Park, et al.

Mol Cancer Res Published OnlineFirst November 14, 2016.

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