Research ArticleExperimental Studies

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

JEHN-HWA KUO, TING-YING SHIH, JING-PIN LIN, KUANG-CHI LAI, MENG-LIANG LIN, MEI-DUE YANG and JING-GUNG CHUNG
Anticancer Research February 2015, 35 (2) 795-804;

Abstract

Cantharidin is an active component of mylabris, which has been used as a traditional Chinese medicine. Cantharidin has been shown to have antitumor activity against several types of human cancers in vitro and in animal models in vivo. We investigated whether cantharidin induces DNA damage and affects DNA damage repair-associated protein levels in TSGH8301 human bladder cancer cells. Using flow cytometry to measure viable cells, cantharidin was found to reduce the number of viable cells in a dose-dependent manner. Comet assay, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA gel electrophoresis were used to measure DNA damage and condensation; the results indicated that cantharidin induced DNA damage (comet tail), DNA condensation (white DAPI staining) and DNA damage (DNA smear). Results from western blotting showed that cantharidin inhibited the expression of DNA-dependent serine/threonine protein kinase, poly-ADP ribose polymerase, phosphate-ataxia-telangiectasia and RAD3-related, O-6-methylguanine-DNA methyltransferase, breast cancer susceptibility protein 1, mediator of DNA damage checkpoint protein 1, phospho-histone H2A.X, but increased that of phosphorylated p53 following 6 and 24 h treatment. Confocal laser microscopy was used to examine the protein translocation; cantharidin suppressed the levels of p-H2A.X and MDC1 but increased the levels of p-p53 in TSGH8301 cells. In conclusion, we found that cantharidin-induced cell death may occur through the induction of DNA damage and suppression of DNA repair-associated protein expression in TSGH8301 cells.

DNA damage can cause genomic instability if not repaired, possibly leading to the development of diseases including 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 DNA damage (3). Many anticancer drugs also cause DNA damage (4, 5) and inhibit DNA damage-repair systems (6, 7). In cells, DNA damage leads to DNA damage response, recognizing DNA lesions in order to promote DNA repair systems. Suppressing the DNA repair system in cancer cells can lead to an increased efficiency of DNA damage-inducing drugs (8). DNA damage repair, therefore, plays an important role in both carcinogenesis and cancer treatment (9).

Mylabris is the dried body of the Chinese blister beetle (10), which has long been used for treating cancer in the Chinese population. Cantharidin is an active constituent of 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-κB) 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 expression of DNA repair-associated proteins in H460 human lung cancer cells in vitro (22).

Many studies have focused on the effects of cantharidin in many human cancer cell lines and few have reported that cantharidin induces DNA damage and inhibits DNA damage repair systems. In the present study we measured the effects of cantharidin on TSGH8301 human bladder cancer cells in vitro.

Materials and Methods

Chemicals and reagents. RPM-1640 medium, L-glutamine and penicillin-streptomycin were purchased from GIBCO®BRL/Invitrogen Life Technologies (Carlsbad, CA, USA). Cantharidin, dimethyl sulfoxide (DMSO), propidium iodide (PI), trypsin-EDTA, anti-ataxia-telangiectasia mutated (ATM), anti-MDC1 (mediator of DNA-damage checkpoint 1), anti-BRCA-1 (breast cancer 1), anti-PARP (Poly (ADP-ribose) polymerase), anti-p53, anti-Phospho-p53 (p-p53), anti-DNA-dependent protein kinase (DNA-PK), anti-O-6-methylguanine-DNA methyltransferase (MGMT) antibodies and anti-p-H2A.X (H2A histone family, member X) were purchased from Sigma Chemical (St. Louis, MO, USA). Anti-p-alamin adenosyltransferase (ATR) was purchased from Cell Signaling (Danvers, MA, USA) and anti-14-3-3σ was purchased from (Merck, Darmstadt, Germany). Nitrocellulose membrane was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bio-Rad Protein assay kit was obtained from Bio-Rad, Hercules (CA, USA). Horseradish peroxidase-conjugated anti-mouse secondary antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)

Cell culture. The TSGH8301 human bladder carcinoma cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). TSGH8301 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) with 1.5 mM L-glutamine and 1% penicillin-streptomycin (100 Units/ml penicillin and 100 μg/ml streptomycin) at 37°C under a humidified atmosphere with 5% CO2. Cells were cultured in 75 cm2 tissue culture flasks.

Figure 1.
Figure 1.

Cantharidin reduced the percentage of viable TSGH8301 cells. Cells (2×105 cells/well) were placed in 12-well plates and were incubated with cantharidin (0, 1.25, 2.5, 5 and 10 μM) for 48 h. All samples were stained with PI (5 μg/ml) and analyzed by flow cytometry for total viable cells as described in the Materials and Methods section. *Significant at p<0.05 compared to the control. Data are means±S.D. (n=3).

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 with cantharidin ranging from 0 to 10 μM for 48 h. Cells were collected, washed and stained with PI (5 μg/ml) in phosphate-buffered saline (PBS) and then underwent flow cytometry (Becton-Dickinson, San Jose, CA, USA) in order to measure the total percentage of viable cells as described previously (21).

Comet assay (single-cell gel electrophoresis). TSGH8301 cells (2×105 cells/well) were maintained in 12-well plates and were treated with IC50 (7.5 μM) of cantharidin for 6, 24 and 48 h. At the end of each treatment, aliquots of 105 cells were collected to measure cell DNA damage by using the comet assay as described previously (23). Comets of cells on slides acquired DNA damage by the CometScore™ Freeware analysis (TriTek Corporation, Sumerduck, VA, USA). Comet tail lengths were calculated, quantified, and expressed as mean±SD (23).

4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining. TSGH8301 cells were seeded at a density of 5×105 cells/well in a 12-well plate for 24 h then were treated with cantharidin (7.5 μM) for 6, 24 and 48 h. At the end of incubation, cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature followed by DAPI staining and were examined and photographed using a fluorescence microscope at ×200 as previously described (23).

DNA gel electrophoresis for DNA damage. TSGH8301 cells (1×106 cells/well) were treated with cantharidin (7.5 μM) for 0, 6, 12 and 24 h and were collected. Cells were lysed in 400 μl of ice-cold lysis buffer (containing 50 mM Tris–HCl, pH 7.5, 10 mM EDTA and 0.3% Triton X-100) for 30 min and were centrifuged for 1,500 rpm. Lysates were incubated with RNase A (100 μg/ml) for 30 min at 50°C and then 200 μg/ml proteinase K was added and the mixtures were incubated for 1 h at 50°C. DNA was extracted with phenol/chloroform and then precipitated with ethanol/sodium acetate at −20°C as described previously (23). DNA from each sample was electrophoresed on a 1.5% agarose gel and photographed as previously described (23).

Figure 2.
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).

Western blotting for examining protein expression. TSGH8301 cells (1×106 cells/well) were placed in a 10 cm dish and treated with 0, and 7.5 μM of cantharidin for 6, 24 and 48 h. Cells were harvested, lysed and the total proteins were measured by Bio-Rad Protein assay kit, as described previously (23). In brief, isolated proteins were electrophoresed by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and were transferred to a nitrocellulose membrane. The membrane was then incubated with primary antibodies to DNA-PK, MDC1, BRCA1, MGMT, p-p53, p-ATR, PARP and p-H2A.X at 4°C then washed and stained by secondary antibody for 1 h. All membranes were visualized with a chemiluminescent detection system and the protein expressions were measured as described by the manufacturer (23).

Figure 3.
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).

Confocal laser microscopy. TSGH8301 cells (5×104 cells/well) were placed on 4-well chamber slides and were incubated with 7.5 μM of cantharidin for 48 h, followed by rinsing and then fixed in 4% formaldehyde in PBS for 15 min. Cells were made permeable by using 0.3% Triton-X 100 in PBS for 1 h at room temperature. Cells were then washed with PBS and blocked with 5% bovine 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, Mannheim, Germany) as described previously (21-23).

Figure 4.
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).

Statistical analysis. All data are presented as the means±standard deviation (S.D.) of three independent experiments. The comparisons between cantharidin-treated and untreated groups were performed by Student's t-test. Differences were considered statistically significant at p<0.05.

Results

Cantharidin reduces the percentage of viable TSGH8301 cells. To investigate the cytotoxic effects of cantharidin on TSGH8301 cells, cells were treated with 0, 1.25, 2.5, 5 and 10 μM of cantharidin for 48 h, and the total number of viable cells was counted (Figure 1). Cantharidin exhibited cell cytotoxicity at concentrations of 1.25 μM or more (p<0.05). Cantharidin reduced the number of viable TSGH8301 cells in a dose-dependent manner.

Figure 5.
Figure 5.

Cantharidin affected the protein expression associated with DNA damage and repair in TSGH8301 cells. Cells (5×105 cells/well) 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 protein levels from each treatment were measured by SDS-PAGE and immunoblotting as described in Materials and methods. A: DNA-PK, PARP and p-ATR; B: MGMT, BRCA1, MDC1, p-H2A.X and p-p53.

Cantharidin-induced DNA damage in TSGH8301 cells. Cells were treated with 7.5 μM of cantharidin for 6, 24 and 48 h, and DNA damage was measured by the comet assay (results are shown in Figure 2). Cantharidin induced comet tail production in TSGH8301 cells at 24 and 48 h treatment (Figures 2A and B). However, 6-h treatment did not show significant DNA damage when compared to the control groups.

Figure 6.
Figure 6.
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 TSGH8301 cells. To confirm the fact cantharidin induced DNA damage in TSGH8301 cells, as indicated by the comet assay, cells were exposed to 0 and 7.5 μM of cantharidin for 6, 24 and 48 h, and were then stained using the DNA-binding fluorescent dye DAPI, followed by fluorescent microscopy examination. Figure 3 shows that cantharidin induced DNA condensation in TSGH8301 cells in a time-dependent manner. Figure 3A shows that cantharidin induced greater DAPI staining following longer treatments, and reduced cell number (Figure 3B) when compared to cantharidin-untreated (control) cells; these effects were time-dependent.

Cantharidin-induced DNA damage and fragmentation in TSGH8301 cells. DNA smearing in DNA gel electrophoresis is characteristic of DNA damage in cells. TSGH8301 cells were incubated with 7.5 μM of cantharidin for 6, 24 and 48 h. Results of the DNA gel electrophoresis are presented in Figure 4. DNA smearing was observed in TSGH8301 cells treated with 7.5 μM of cantharidin at 12 and 24 h.

Cantharidin affects the expression of DNA damage and repair-associated proteins in TSGH8301 cells. In order to confirm that cantharidin induced cytotoxic effects on TSGH8301 cells, the expression of certain DNA damage and repair-associated proteins was measured by western blotting, following treatment with 7.5 μM of cantharidin for 6, 24 and 48 h. The results shown in Figure 5 indicated that cantharidin suppressed the expressions of DNA-PK, PARP, p-ATR, MGMT, BRAC1, MDC1, p-H2A.X but increased that of p-p53 (Figure 5B) at 6 and 24 h treatment.

Cantharidin affects MDC1, p-H2A.X and p-p53 expression and translocation in TSGH8301 cells. In order to examine whether the effects of cantharidin on the expression of MDC1, p-H2A.X and p-p53 expressions are involved in the translocation of these proteins,TSGH8301 cells were treated with 7.5 μM of cantharidin for 48 h and were then viewed and photographed under confocal laser microscopy. Cantharidin was shown to increase the expression of p-p53 (Figure 6C), MDC1 (Figure 6A) and p-H2A.X (Figure 6B) in TSGH8301 cells when compared to control groups.

Figure 7.
Figure 7.

The possible flow chart for cantharidin-induced DNA damage and inhibition of DNA repair-associated protein expression in TSGH8301 cells. Abbreviations: p-Ataxia Telangiectasia Mutated and Rad3 Related Protein Kinase (p-ATR), breast cancer 1 (BRCA1), DNA-dependent protein kinase (DNA-PK), phospho-Histone H2A.X (p-H2A.X), mediator of DNA damage checkpoint protein 1 (MDC1), phosphorylated p53 (p-p53) and O-6-methylguanine-DNA methyltransferase (MGMT).

Discussion

Previous research has shown that exposure to cantharidin in vitro causes DNA damage in different human cancer cells (22-24). Our previous studies showed that cantharidin can induce DNA damage in TSGH8301 cells, but this was only shown by the comet assay. Herein, TSGH8301 cells were selected for further investigation regarding DNA damage and repair-associated proteins involved in the action of cantharidin in causing DNA damage. It is well-documented that tumor cells can repair DNA damage caused by anticancer drugs via DNA repair responses. DNA repair pathways have been suggested to be of prognostic or predictive value (25).

Numerous studies have shown that there is a connection between DNA damage and apoptosis (26). Herein, we showed that cantharidin treatment of TSGH8301 cells caused DNA damage based on the observations that i) cantharidin induced comet tail production (Figure 2), ii) DAPI staining showed cantharidin-induced DNA damage and condensation (Figure 3); and iii) DNA gel electrophoresis showed that cantharidin induced DNA 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 cantharidin increased p-p53 expression but inhibited that of MGMT, BRCA1 and MDC1 in TSGH8301 cells. It has been reported that p53 can be phosphorylated by ATM, ATR and DNA-PKCs, and then induced p53 in response to DNA damage (34), and that such damage can induce p-p53 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 to maintain genomic stability (38). It is known that H2A.X is critical for the efficient accumulation of DNA repair factors at sites of DNA breakage (39). When mice are H2A.X-deficient they exhibit higher radiosensitivity when compared to normal mice (39). In human cells, phosphorylated H2A.X (γH2A.X) is recognized by MDC1 to accumulate numerous DNA damage response factors on chromatin regions surrounding DNA lesions (40). Furthermore, it was reported that among the mammalian DNA damage response proteins that rely on γH2A.X for ionizing radiation-induced foci formation, MDC1 is the main direct binder of γH2A.X (41).

In conclusion, we suggest that cantharidin induced cell death through the induction of DNA damage. Figure 7 provides a possible mechanism of cantharidin function on anticancer activity.

Acknowledgements

This study was supported by the Jen-Ai Hospital, Dali, Taichung, Taiwan, R.O.C. Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research and Development at China medical University, Taichung, Taiwan, R.O.C.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Received November 4, 2014.
  • Revision received December 4, 2014.
  • Accepted December 8, 2014.

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Cantharidin Induces DNA Damage and Inhibits DNA Repair-associated Protein Expressions in TSGH8301 Human Bladder Cancer Cell
JEHN-HWA KUO, TING-YING SHIH, JING-PIN LIN, KUANG-CHI LAI, MENG-LIANG LIN, MEI-DUE YANG, JING-GUNG CHUNG
Anticancer Research Feb 2015, 35 (2) 795-804;
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