ANTICANCER RESEARCH 35: 795-804 (2015)

Cantharidin Induces DNA Damage and Inhibits DNA Repair-associated Protein Expressions in TSGH8301 Human Bladder Cancer Cell

JEHN-HWA KUO1,2, TING-YING SHIH3, JING-PIN LIN4, KUANG-CHI LAI5,6, MENG-LIANG LIN7, MEI-DUE YANG8* and JING-GUNG CHUNG3,9*

1Special Class of Healthcare, Industry Management, Central Taiwan University of Science and Technology, Taichung, Taiwan, R.O.C.; 2Department of Urology, Jen-Ai Hospital, Taichung, Taiwan, R.O.C.; 3Department of Biological Science and Technology, 4School of Chinese Medicine, and 5School of Medicine, China Medical University, Taichung, Taiwan, R.O.C.; 6Department of Surgery, China Medical University Beigang Hospital, Yunlin, Taiwan, R.O.C.; 7Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, R.O.C.; 8Department of Surgery, China Medical University Hospital, Taichung, Taiwan, R.O.C.; 9Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan, R.O.C.

Abstract. Cantharidin is an active component of mylabris, protein kinase, poly-ADP ribose polymerase, phosphate- which has been used as a traditional Chinese medicine. ataxia-telangiectasia and RAD3-related, O-6-methylguanine- Cantharidin has been shown to have antitumor activity against DNA methyltransferase, breast cancer susceptibility protein 1, several types of human cancers in vitro and in animal models mediator of DNA damage checkpoint protein 1, phospho- in vivo. We investigated whether cantharidin induces DNA histone H2A.X, but increased that of phosphorylated p53 damage and affects DNA damage repair-associated protein following 6 and 24 h treatment. Confocal laser microscopy levels in TSGH8301 human bladder cancer cells. Using flow was used to examine the protein translocation; cantharidin cytometry to measure viable cells, cantharidin was found to suppressed the levels of p-H2A.X and MDC1 but increased the reduce the number of viable cells in a dose-dependent manner. levels of p-p53 in TSGH8301 cells. In conclusion, we found Comet assay, 4’,6-diamidino-2-phenylindole (DAPI) staining that cantharidin-induced cell death may occur through the and DNA gel electrophoresis were used to measure DNA induction of DNA damage and suppression of DNA repair- damage and condensation; the results indicated that associated protein expression in TSGH8301 cells. cantharidin induced DNA damage (comet tail), DNA condensation (white DAPI staining) and DNA damage (DNA DNA damage can cause genomic instability if not repaired, smear). Results from western blotting showed that cantharidin possibly leading to the development of diseases including inhibited the expression of DNA-dependent serine/threonine cancer (1, 2). It is well-known that exogenous factors (ultraviolet light, ionizing radiation, and heavy metals are environmental agents) and endogenous sources (reactive oxygen species and reactive nitrogen species) can induce *These Authors contributed equally to this study. DNA damage (3). Many anticancer drugs also cause DNA Correspondence to: Jing-Gung Chung, Ph.D., Department of damage (4, 5) and inhibit DNA damage-repair systems (6, Biological Science and Technology, China Medical University. No 7). In cells, DNA damage leads to DNA damage response, 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 422053366 recognizing DNA lesions in order to promote DNA repair ext. 8000, Fax: +886 422053764, e-mail: [email protected] systems. Suppressing the DNA repair system in cancer cells and Mei-Due Yang, Department of Surgery, China Medical can lead to an increased efficiency of DNA damage-inducing University Hospital, Taichung 404, Taiwan No 91, Hsueh-Shih drugs (8). DNA damage repair, therefore, plays an important Road, Taichung 404, Taiwan. Tel: +886 422053366 ext. 3113, Fax: role in both carcinogenesis and cancer treatment (9). +886 422053764, e-mail: [email protected] Mylabris is the dried body of the Chinese blister beetle Key Words: Cantharidin, DNA damage, Comet assay, DNA repair, (10), which has long been used for treating cancer in the TSGH8301 human bladder cancer cells. Chinese population. Cantharidin is an active constituent of

0250-7005/2015 $2.00+.40 795 ANTICANCER RESEARCH 35: 795-804 (2015) mylabris (10). Cantharidin has been used for dermatological diseases to remove warts and molluscum contagiosum for over 50 years (11). Cantharidin has various biological activities including induction of cell apoptosis in many human cancer cell types (12-17). It has been reported that cantharidin induced cell apoptosis through the Nuclear Factor-KappaB (NF-ĸB) pathway (18) and inhibited the migration of breast cancer cells (19). Cantharidin has been shown to be a potent and selective inhibitor of protein phosphatases 1 and 2a (20). It has also shown anti-metastatic potential against TSGH-8301 human bladder cancer cells by suppressing the expression of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9), which might be mediated by targeting the p38/c-Jun N-terminal protein kinase (JNK1/2)/Mitogen-activated protein kinases (MAPK) pathways (21). Furthermore, earlier studies of ours showed that cantharidin induced DNA damage and inhibited Figure 1. Cantharidin reduced the percentage of viable TSGH8301 cells. Cells (2×105 cells/well) were placed in 12-well plates and were expression of DNA repair-associated proteins in H460 incubated with cantharidin (0, 1.25, 2.5, 5 and 10 μM) for 48 h. All human lung cancer cells in vitro (22). samples were stained with PI (5 μg/ml) and analyzed by flow cytometry Many studies have focused on the effects of cantharidin for total viable cells as described in the Materials and Methods in many human cancer cell lines and few have reported that section. *Significant at p<0.05 compared to the control. Data are cantharidin induces DNA damage and inhibits DNA means±S.D. (n=3). damage repair systems. In the present study we measured the effects of cantharidin on TSGH8301 human bladder cancer cells in vitro. Cellular viability. TSGH8301 cells were seeded at a density of 5×105 cells/well in a 12-well plate for 24 h and were then treated Materials and Methods with cantharidin ranging from 0 to 10 μM for 48 h. Cells were collected, washed and stained with PI (5 μg/ml) in phosphate- Chemicals and reagents. RPM-1640 medium, L-glutamine and buffered saline (PBS) and then underwent flow cytometry (Becton- penicillin-streptomycin were purchased from GIBCO®BRL Dickinson, San Jose, CA, USA) in order to measure the total /Invitrogen Life Technologies (Carlsbad, CA, USA). Cantharidin, percentage of viable cells as described previously (21). dimethyl sulfoxide (DMSO), propidium iodide (PI), trypsin-EDTA, anti-ataxia-telangiectasia mutated (ATM), anti-MDC1 (mediator of Comet assay (single-cell gel electrophoresis). TSGH8301 cells DNA-damage checkpoint 1), anti-BRCA-1 (breast cancer 1), anti- (2×105 cells/well) were maintained in 12-well plates and were PARP (Poly (ADP-ribose) polymerase), anti-p53, anti-Phospho-p53 treated with IC50 (7.5 μM) of cantharidin for 6, 24 and 48 h. At the (p-p53), anti-DNA-dependent protein kinase (DNA-PK), anti-O-6- end of each treatment, aliquots of 105 cells were collected to methylguanine-DNA methyltransferase (MGMT) antibodies and measure cell DNA damage by using the comet assay as described anti-p-H2A.X (H2A histone family, member X) were purchased previously (23). Comets of cells on slides acquired DNA damage from Sigma Chemical (St. Louis, MO, USA). Anti-p-alamin by the CometScore™ Freeware analysis (TriTek Corporation, adenosyltransferase (ATR) was purchased from Cell Signaling Sumerduck, VA, USA). Comet tail lengths were calculated, (Danvers, MA, USA) and anti-14-3-3σ was purchased from quantified, and expressed as mean±SD (23). (Merck, Darmstadt, Germany). Nitrocellulose membrane was obtained from Amersham Pharmacia Biotech (Buckinghamshire, 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining. UK). Bio-Rad Protein assay kit was obtained from Bio-Rad, TSGH8301 cells were seeded at a density of 5×105 cells/well in a Hercules (CA, USA). Horseradish peroxidase-conjugated anti- 12-well plate for 24 h then were treated with cantharidin (7.5 μM) mouse secondary antibody was obtained from Santa Cruz for 6, 24 and 48 h. At the end of incubation, cells were fixed with Biotechnology Inc. (Santa Cruz, CA, USA) 3.7% formaldehyde in PBS for 10 min at room temperature followed by DAPI staining and were examined and photographed Cell culture. The TSGH8301 human bladder carcinoma cell line was using a fluorescence microscope at ×200 as previously described obtained from the Food Industry Research and Development Institute (23). (Hsinchu, Taiwan, ROC). TSGH8301 cells were cultured in RPMI- 1640 medium supplemented with 10% fetal bovine serum (Gibco DNA gel electrophoresis for DNA damage. TSGH8301 cells (1×106 BRL, Grand Island, NY, USA) with 1.5 mM L-glutamine and 1% cells/well) were treated with cantharidin (7.5 μM) for 0, 6, 12 and penicillin-streptomycin (100 Units/ml penicillin and 100 μg/ml 24 h and were collected. Cells were lysed in 400 μl of ice-cold lysis streptomycin) at 37˚C under a humidified atmosphere with 5% CO2. buffer (containing 50 mM Tris–HCl, pH 7.5, 10 mM EDTA and Cells were cultured in 75 cm2 tissue culture flasks. 0.3% Triton X-100) for 30 min and were centrifuged for 1,500 rpm.

796 Kuo et al: Cantharidin-induced DNA Damage and Inhibition of DNA Damage Repair Protein in Human Bladder Cancer Cells

Figure 2. Cantharidin-induced DNA damage in TSGH8301 cells was measured by the comet assay. Cells (2×105 cells/well) were placed in 12-well plates and were incubated with 7.5 μM of cantharidin for 6, 24 and 48 h and DNA damage was determined by the comet assay as described in the Materials and Methods section. A: Representative image of comet assay; B: Comet length. Arrow showing the comet tail (DNA damage). *Significant at p<0.05 compared to the control. Data are means±S.D. (n=3).

Lysates were incubated with RNase A (100 μg/ml) for 30 min at Western blotting for examining protein expression. TSGH8301 cells 50˚C and then 200 μg/ml proteinase K was added and the mixtures (1×106 cells/well) were placed in a 10 cm dish and treated with 0, were incubated for 1 h at 50˚C. DNA was extracted with and 7.5 μM of cantharidin for 6, 24 and 48 h. Cells were harvested, phenol/chloroform and then precipitated with ethanol/sodium lysed and the total proteins were measured by Bio-Rad Protein assay acetate at −20˚C as described previously (23). DNA from each kit, as described previously (23). In brief, isolated proteins were sample was electrophoresed on a 1.5% agarose gel and electrophoresed by 10% sodium dodecyl sulfate–polyacrylamide gel photographed as previously described (23). (SDS-PAGE) and were transferred to a nitrocellulose membrane. The

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Figure 3. Cantharidin-induced DNA condensation in TSGH8301 cells, as measured by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. Cells (1×105 cells/well) were placed in 12-well plates for 24 h and were treated with 7.5 μM of cantharidin for 6, 24 and 48 h. Cells were then stained with DAPI and examined and photographed using a fluorescence microscope at ×200 magnification. Significant at *p<0.05, **p<0.01, ***p<0.001, compared to the control. Data are means±S.D. (n=3).

membrane was then incubated with primary antibodies to DNA-PK, Confocal laser microscopy. TSGH8301 cells (5×104 cells/well) MDC1, BRCA1, MGMT, p-p53, p-ATR, PARP and p-H2A.X at 4˚C were placed on 4-well chamber slides and were incubated with 7.5 then washed and stained by secondary antibody for 1 h. All μM of cantharidin for 48 h, followed by rinsing and then fixed in membranes were visualized with a chemiluminescent detection 4% formaldehyde in PBS for 15 min. Cells were made permeable system and the protein expressions were measured as described by by using 0.3% Triton-X 100 in PBS for 1 h at room temperature. the manufacturer (23). Cells were then washed with PBS and blocked with 5% bovine

798 Kuo et al: Cantharidin-induced DNA Damage and Inhibition of DNA Damage Repair Protein in Human Bladder Cancer Cells

Figure 4. Cantharidin-induced DNA fragmentation in TSGH8301 cells. Cells (5×105 cells/well) were placed in 12-well plates for 24 h and were treated with 7.5 μM of cantharidin (CTD) for 6, 12 and 24 h. DNA was extracted and DNA gel electrophoresis was performed for examining inter-nucleosomal DNA cleavage (DNA smear).

serum albumin (BSA) in PBS for 20 min and were stained with green fluorescent anti-MDC1, anti-p-H2A.X and anti-p-p53 overnight. Cells were then washed and stained with secondary antibody (Fluorescein isothiocyanate-conjugated goat anti-mouse IgG). Nuclei were stained by using PI (red fluorescence). All samples were mounted, examined, viewed and photomicrographed using a Leica TCS SP2 Confocal Spectral Microscope (Leica, Figure 5. Cantharidin affected the protein expression associated with DNA damage and repair in TSGH8301 cells. Cells (5×105 cells/well) Mannheim, Germany) as described previously (21-23). were placed in 12-well plates and then were incubated with 7.5 μM of cantharidin (CTD) for 6, 24 and 48 h. Then cells were collected and Statistical analysis. All data are presented as the means±standard protein levels from each treatment were measured by SDS-PAGE and deviation (S.D.) of three independent experiments. The comparisons immunoblotting as described in Materials and methods. A: DNA-PK, between cantharidin-treated and untreated groups were performed PARP and p-ATR; B: MGMT, BRCA1, MDC1, p-H2A.X and p-p53. by Student’s t-test. Differences were considered statistically significant at p<0.05.

Results

Cantharidin reduces the percentage of viable TSGH8301 Cantharidin-induced DNA damage in TSGH8301 cells. cells. To investigate the cytotoxic effects of cantharidin on Cells were treated with 7.5 μM of cantharidin for 6, 24 and TSGH8301 cells, cells were treated with 0, 1.25, 2.5, 5 and 48 h, and DNA damage was measured by the comet assay 10 μM of cantharidin for 48 h, and the total number of viable (results are shown in Figure 2). Cantharidin induced comet cells was counted (Figure 1). Cantharidin exhibited cell tail production in TSGH8301 cells at 24 and 48 h treatment cytotoxicity at concentrations of 1.25 μM or more (p<0.05). (Figures 2A and B). However, 6-h treatment did not show Cantharidin reduced the number of viable TSGH8301 cells significant DNA damage when compared to the control in a dose-dependent manner. groups.

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Figure 6. Continued

800 Kuo et al: Cantharidin-induced DNA Damage and Inhibition of DNA Damage Repair Protein in Human Bladder Cancer Cells

Figure 6. Cantharidin affected protein expression and translocation in TSGH8301 cells, as examined by confocal laser microscopy. Cells (5×104 cells/well) were placed on 4-well chamber slides and were incubated with or without 7.5 μM of cantharidin for 48 h. Cells were fixed in 4% formaldehyde in PBS, washed and immunostained as described in the Materials and Methods section. Cells were then examined and photo- micrographed under a Leica TCS SP2 Confocal Spectral Microscope. A: Mediator of DNA damage checkpoint protein 1 (MDC1); B: phospho- Histone H2A.X (p-H2A.X); C: phosphorylated p53 (p-p53).

Cantharidin-induced DNA damage and condensation in Cantharidin affects the expression of DNA damage and TSGH8301 cells. To confirm the fact cantharidin induced repair-associated proteins in TSGH8301 cells. In order to DNA damage in TSGH8301 cells, as indicated by the comet confirm that cantharidin induced cytotoxic effects on assay, cells were exposed to 0 and 7.5 μM of cantharidin for TSGH8301 cells, the expression of certain DNA damage and 6, 24 and 48 h, and were then stained using the DNA- repair-associated proteins was measured by western blotting, binding fluorescent dye DAPI, followed by fluorescent following treatment with 7.5 μM of cantharidin for 6, 24 and microscopy examination. Figure 3 shows that cantharidin 48 h. The results shown in Figure 5 indicated that cantharidin induced DNA condensation in TSGH8301 cells in a time- suppressed the expressions of DNA-PK, PARP, p-ATR, dependent manner. Figure 3A shows that cantharidin induced MGMT, BRAC1, MDC1, p-H2A.X but increased that of p- greater DAPI staining following longer treatments, and p53 (Figure 5B) at 6 and 24 h treatment. reduced cell number (Figure 3B) when compared to cantharidin-untreated (control) cells; these effects were time- Cantharidin affects MDC1, p-H2A.X and p-p53 expression dependent. and translocation in TSGH8301 cells. In order to examine whether the effects of cantharidin on the expression of Cantharidin-induced DNA damage and fragmentation in MDC1, p-H2A.X and p-p53 expressions are involved in the TSGH8301 cells. DNA smearing in DNA gel electrophoresis translocation of these proteins,TSGH8301 cells were treated is characteristic of DNA damage in cells. TSGH8301 cells with 7.5 μM of cantharidin for 48 h and were then viewed were incubated with 7.5 μM of cantharidin for 6, 24 and 48 and photographed under confocal laser microscopy. h. Results of the DNA gel electrophoresis are presented in Cantharidin was shown to increase the expression of p-p53 Figure 4. DNA smearing was observed in TSGH8301 cells (Figure 6C), MDC1 (Figure 6A) and p-H2A.X (Figure 6B) treated with 7.5 μM of cantharidin at 12 and 24 h. in TSGH8301 cells when compared to control groups.

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smearing (Figure 4). DNA strand breaks have also been documented in leukemia cells after cantharidin treatment in vivo (24). The comet assay is considered a significant method for measuring DNA damage (25, 27) in eukaryotic cells from a single-cell basis. Data from this technique came in agreement with our earlier report showing cantharidin- induced DNA damage in TSGH8301 cells (22). It is also well documented that DAPI staining can be useful for measuring DNA condensation and our results revealed that cantharidin induced DNA condensation and this effect was time-dependent (Figure 3). It was reported that deficits in DNA repair capacity can reduce cellular resistance to spontaneous and exogenous DNA damage (28). The DNA repair system then eliminates DNA damage to maintain cell survival (29, 30). Figure 5B indicates that cantharidin suppressed the protein levels of DNA-PK, PARP, and p-ATR in TSGH8301 cells. Damage such as double-stranded DNA breaks in cells can activate ATM and ATR to maintain genomic integrity (31, 32). The suppression of DNA damage-induced poly(ADP- ribosyl)ation by PARP inhibitors impairs early DNA damage-response events (33). Figure 5B also shows that Figure 7. The possible flow chart for cantharidin-induced DNA damage and inhibition of DNA repair-associated protein expression in TSGH8301 cantharidin increased p-p53 expression but inhibited that of cells. Abbreviations: p-Ataxia Telangiectasia Mutated and Rad3 Related MGMT, BRCA1 and MDC1 in TSGH8301 cells. It has been Protein Kinase (p-ATR), breast cancer 1 (BRCA1), DNA-dependent reported that p53 can be phosphorylated by ATM, ATR and protein kinase (DNA-PK), phospho-Histone H2A.X (p-H2A.X), mediator DNA-PKCs, and then induced p53 in response to DNA of DNA damage checkpoint protein 1 (MDC1), phosphorylated p53 (p- damage (34), and that such damage can induce p-p53 p53) and O-6-methylguanine-DNA methyltransferase (MGMT). expression (35). DNA-PK can be activated by DNA damage (36) and it was also reported that DNA-PK was involved in DNA damage repair system (37). BRCA-1 was reported to be involved in DNA repair and Discussion to maintain genomic stability (38). It is known that H2A.X is critical for the efficient accumulation of DNA repair factors Previous research has shown that exposure to cantharidin in at sites of DNA breakage (39). When mice are H2A.X- vitro causes DNA damage in different human cancer cells deficient they exhibit higher radiosensitivity when compared (22-24). Our previous studies showed that cantharidin can to normal mice (39). In human cells, phosphorylated H2A.X induce DNA damage in TSGH8301 cells, but this was only (γH2A.X) is recognized by MDC1 to accumulate numerous shown by the comet assay. Herein, TSGH8301 cells were DNA damage response factors on chromatin regions selected for further investigation regarding DNA damage surrounding DNA lesions (40). Furthermore, it was reported and repair-associated proteins involved in the action of that among the mammalian DNA damage response proteins cantharidin in causing DNA damage. It is well-documented that rely on γH2A.X for ionizing radiation-induced foci that tumor cells can repair DNA damage caused by formation, MDC1 is the main direct binder of γH2A.X (41). anticancer drugs via DNA repair responses. DNA repair In conclusion, we suggest that cantharidin induced cell pathways have been suggested to be of prognostic or death through the induction of DNA damage. Figure 7 predictive value (25). provides a possible mechanism of cantharidin function on Numerous studies have shown that there is a connection anticancer activity. between DNA damage and apoptosis (26). Herein, we showed that cantharidin treatment of TSGH8301 cells Acknowledgements caused DNA damage based on the observations that i) This study was supported by the Jen-Ai Hospital, Dali, Taichung, cantharidin induced comet tail production (Figure 2), ii) Taiwan, R.O.C. Experiments and data analysis were performed in part DAPI staining showed cantharidin-induced DNA damage through the use of the Medical Research Core Facilities Center, Office and condensation (Figure 3); and iii) DNA gel of Research and Development at China medical University, Taichung, electrophoresis showed that cantharidin induced DNA Taiwan, R.O.C.

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