Cantharidin induces DNA damage and inhibits DNA repair associated protein expressions in human gastric cancer TSGH8301 cells
Jehn-Hwa Kuo1, Ting-Ying Shih2, Jing-Pin Lin3, Kuang-Chi Lai 4,5, Mei-Due Yang6,*, and Jing-Gung Chung2, 7 *
1Special Class of Healthcare, Industry Management, Central Taiwan University of
Science & Technology, Taichung, Taiwan.
2Departments of Biological Science and Technology, China Medical University,
Taichung, Taiwan.
3Schools of Chinese Medicine, China Medical University, Taichung, Taiwan. 4Schools of Medicine, China Medical University, Taichung, Taiwan.
5Department of Surgery, China Medical University Beigang Hospital, Yunlin,
Taiwan.
6Department of Surgery, China Medical University Hospital, Taichung 404, Taiwan 7Department of Biotechnology, Asia University, Wufeng, Taichung 413, Taiwan,
R.O.C.
Running title: Cantharidin induced DNA damage and inhibit DNA damage repair protein expression in human gastric cancer cells
#,*Both authors contributed equally to this work
*Correspondence to:
Jing-Gung Chung, Ph.D., Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 4
2205 3366 ext. 2161, Fax: +886 4 2205 3764, e-mail: [email protected] Mei-Due Yang. Department of Surgery, China Medical University Hospital, Taichung 404, Taiwan No 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 4 2205 3366 ext. 3113, Fax: +886 4 2205 3764, e-mail: [email protected]
Abstract. Cantharidin (CTD) is one of active component of mylabris which has been
used as a traditional Chinese medicine. CTD has been shown to have antitumor activity against several types of human cancer in vitro and in animal model in vivo. We investigated whether CTD could induce DNA damage and affect DNA damage repair associated protein levels in human gastric cancer TSGH8301 cells in vitro. Using flow cytometry assay for measuring percentage of viable cells and results indicated CTD decreased the viable cells in a dose-dependently. Comet assay, DAPI staining and DNA gel electrophoresis were used to measure DNA damage and condension and results indicated that CTD induced DNA damage (Comet tail) and DNA condensation (whited DAPI staining) and DNA damage (smear DNA). Results from Western blotting showed that CTD inhibited the protein expressions of DNA-dependent serine/threonine protein kinase (DNA-PK), poly-ADP ribose polymerase (PARP), ataxia-telangiectasia and Rad3-related (p-ATR), phosphate-ataxia-telangiectasia and Rad3-related (MGMT), breast cancer susceptibility protein 1 (BRAC1), Mediator of DNA damage checkpoint protein 1 (MDC1, phospho-histone H2A.X (p-H2A.X) but increased phosphorylated p53 (p-p53) at 6 and 24 h treatment. The confocal laser microscopy was used to examine the protein translocation and results indicated that CTD suppressed the levels of p-H2A.X and MDC1 but increased the levels of p-p53 in TSGH8301 cells. In conclusion, we found that CTD induced cell death may through the induced DNA damage and suppressed DNA repair
associated protein expression in TSGH8301 cells.
Keywords: Cantharidin (CTD); DNA damage; Comet assay; DNA repair; human
gastric cancer TSGH8301 cells.
Introduction
It was reported that DNA damage can caused genomic instability if not repair then it could leading to diseases development 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). It is also reported that many anticancer drugs could cause DNA damage (4, 5) and inhibited DNA damage repair systems (6, 7). In cells, after DNA damage occur that could led to the DNA damage response (DDR) to recognize DNA lesions for promoted DNA repair systems to repair the damage DNA. To suppress the DNA repair system in cancer cells that can lead to increase efficiency of DNA damage-inducing drugs (8). It was reported that DNA damage repair play an important role in both carcinogenesis and cancer treatment (9). Cantharidin (CTD) is an active constituent of mylabris (10) which have been used for treating cancer since long ago in Chinese population and mylabris is the dried body of the Chinese blister beetle (11). CTD has been used for dermatologic diseases to remove warts and MC for over 50 years (12). CTD presented biological activities including induced cell apoptosis in many human cancer cells (13-18). It was reported that CTD induced cell apoptosis through NF-κB pathway (19) and inhibited cell migration in breast cancer cells (20). CTD has been shown to be a potent and selective inhibitor of protein phosphatases 1 and 2a (21). CTD was suggested to present antimetastatic potential in TSGH-8301 human bladder cancer cells via suppressing
MMP-2 and MMP-9 expression that might be mediated by targeting the p38 and JNK1/2 MAPKs pathway (22). Furthermore, in our earlier studies have shown that CTD induced DNA damage and inhibited levels of DNA repair-associated proteins in human lung cancer H460 cells in vitro (23).
Many studies have focused on the effects of CTD on anticancer function in many human cancer cell lines and few report to show CTD induced DNA damage and inhibited DNA damage repair systems. However, this study is to investigate whether or not CTD could induced DNA damage and affect associated repair protein expressions, thus, in this study, we measured the effects of CTD on human gastric cancer TSGH8301 cells in vitro. The results showed that CTD induced DNA damage and affect DNA repair associated protein expression in human gastric cancer TSGH8301 cells in vitro.
Materials and methods
Chemicals and reagents. RPM-1640 medium, fetal bovine serum (FBS), L-glutamine
and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life
Technologies (Carlsbad, California, USA). Cantharidin (CTD), dimethyl sulfoxide (DMSO), propidium iodide (PI), Trypsin-EDTA, anti-p-ATM, anti-p53, anti-p-p53, anti-DNA-PK and anti-MGMT were purchased from Sigma Chemical. (St. Louis, MO, USA). Anti-DNA-PK was purchased from Calbiochem (San Diego, CA, USA). Anti-p-ATR was purchased from Cell Signaling (Danvers, MA, USA) and anti-14-3-3σ was purchased from Merck. Nitrocellulose membrane was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bio-Rad assay Kit was obtained from Bio-Rad, Hercules (CA, USA). Horseradish peroxidase-conjugated anti-mouse secondary antibody was obtained from Santa Cruz Biotechnology Inc.
(CA, USA)
Cell culture. The human bladder carcinoma TSGH8301 cell line was obtained from
the Food Industry Research and Development Institute (Hsinchu, Taiwan). 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 5% CO2 atmosphere. Cells were cultured in 75 cm2 tissue
culture flasks.
Cellular viability. TSGH8301 cells were seeded at a concentration of 5x105 cells/well
in a 12-well plate for 24 h then were treated with CTD 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 followed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) performed for measuring the total percentage of viable cells as described previously (22).
Comet assay (Single cell gel electrophoresis). TSGH8301 cells (2x105 cells/well)
were maintained in 12-well plates and were treated with 7.5 μM of CTD for 6, 24 and 48 h. At the end of treatment, aliquots of 105 cells were collected to measure cell
DNA damage by using Comet assay as described previously (24).
4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. TSGH 8301 cells were
seeded at a concentration of 5x105 cells/well in a 12-well plate for 24 h then were treated with CTD (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
using DAPI staining and were examined and photographed using a fluorescence microscope at 200X as previously described (24).
DNA gel electrophoresis for DNA damage. TSGH8301 cells (1x106 cells/well) were
treated with CTD (7.5 μM) for 0, 6, 12 and 24 h then 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. Then were incubated with RNase A (100 μg/ml) for 30 min at 50°C and then 200 μg/ml proteinase K was added and then 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 (24). Quantitated DNA from each sample and then were electrophoresed on a 1.5% agarose gel and were by photographed as previously described (24).
Western blotting for examining protein expressions. TSGH8301 cells (1x106
cells/well) were placed in 10 cm dish and treated with 0, and 7.5 μM of CTD for 6, 24 and 48 h. Cells were harvested, lysed and the total proteins were measured by Bio-Rad assay Kit as described previously (24). In briefly, isolated proteins were electrophoresed by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and were transferred to nitrocellulose membrane. Then membrane was incubated with primary antibodies (anti-DNA-PK, MDC1, BRCA1, MGMT, p-p53, p-ATR, PARP and p-H2A.X) at 4 °C then were 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 (24).
Confocal laser microscopy. TSGH8301 cells (5×104 cells/well) were placed on
rinsed and fixed in 4% formaldehyde in PBS for 15 min. To permeable cells by using 0.3% Triton-X 100 in PBS for 1 h at room temperature. Cells were washed with PBS and blocking with 5% BSA in PBS for 20 min and were stained with green
fluorescence anti-MDC1, anti-p-H2A.X and anti-p-p53 overnight then washed and
followed by staining with secondary antibody (FITC-conjugated goat anti-mouse IgG). And then nuclein was stained by using PI (red fluorescence). All samples were mounted, examined, viewed and photomicrograph by using a Leica TCS SP2 Confocal Spectral Microscope as described previously (22-24).
Statistical analysis. All data were presented as the means ± standard deviation (S.D.)
from 3 independent experiments. The comparisons between CTD-treated and untreated groups were performed by Student’s t-test. Differences were considered statistically significant at p<0.05.
Results
CTD decrease the percentage of viable TSGH8301 cells. To investigate the cytotoxic
effects of CTD on TSGH8301 cells, cells were treated with 0, 1.25, 2.5, 5 and 10 μM of CTD for 48 h, total number of viable cells was counted and results are shown in Figure 1. CTD showed cell cytotoxicity at >1.25 μM concentration was observed (p<0.05). CTD decreased the total viable cells in TSGH83901 cells are dose-dependently.
CTD-induced DNA damage in TSGH8301 cells. Cells were treated with 7.5 μM of
CTD for 6, 24 and 48 h then DNA damage was measured by Comet assay and the results are shown in Figure 2A and B. Results showed that CTD induced comet tail production in TSGH8301 cells at 24 and 48 h treatment (Fig. 2A and B). However, at
6 h treatment did not show significant DNA damage occur when compared to the control groups.
CTD-induced DNA damage and condensation in TSGH8301 cells. To confirm CTD
induced DNA damage in TSGH8301 cells which were evaluated by Comet assay, cells were exposed to 0 and 7,5 μM of CTD for 6, 24 and 48 h, then were stained by using the DNA-binding fluorescent dye DAPI followed by using fluorescent microscopy examination. Figure 3A and B showed that CTD induced DNA condensation in TSGH8301 cells in a time-dependent manner. Figure 3A showed that CTD induced higher DAPI staining at long time treatment and lower cell number (Fig. 3B) when compared to CTD-untreated (control) cells, these effects in a time-dependently.
CTD induced DNA damage and fragmentation in TSGH8301 cells. Smear DNA in
DNA gel electrophoresis has been known is the character of DNA damage in cells. TSGH8301 cells were incubated with 7.5 μM of CTD for 6, 24 and 48 h, DNA was isolated from each treatment cells, followed by DNA gel electrophoresis and results are showing in Figure 4. DNA smear was observed in TSGH8301 cells at 12 and 24 h treated with 7.5 μM of CTD.
CTD affect DNA damage and repair associated proteins expressions in TSGH8301 cells. In order to confirm CTD induced cytotoxic effects on TSGH8301 cells via
induced DNA damage and affect DNA damage and repair associated protein expression, cells were treated with 7.5 μM of CTD for 6, 24 and 48 h then protein expressions were examined by Western blotting and results are shown in Figure 5A and B. Results from Figures 5A and B indicated that CTD suppressed the expressions
of DNA-PK, PARP, p-ATR, MGMT, BRAC1, MDC1, p-H2A.X but increased p-p53
(Fig. 5B) at 6 and 24 h treatment.
CTD affect MDC1, p-H2A.X and p-p53 expression and translocation in TSGH8301
cells. For examining whether or not CTD inhibited the expression of MDC1 and
p-H2A.X and increased p-p53 expressions are involved the translocation of those
proteins in TSGH8301 cells. Cells were treated with 7.5 μM of CTD for 48 h then were viewed and photographed by confocal laser microscopy and the results shown in Figure 6A, B and C. Results indicated that CTD increased the expression of the p-p53 (Fig. 6C), MDC1 (Fig. 6A) and p-H2A.X (Fig. 6B) in TSGH8301 cells when
compared to control groups.
Discussion
Previous researches have shown that exposure to CTD in vitro causes DNA damage in different human cancer cells (23-25), although our previous studies have shown that CTD can induced DNA damage in TSGH8301 cells but only showed it by Comet assay, thus, there is not further investigation to examine whether or not CTD affect DNA damage and repair associated protein expression, therefore, herein, TSGH8301 cells were selected for further investigation regarding DNA damage and repair associated proteins involved in the action of CTD for causing DNA damage. It is well documented that after exposed to anticancer drugs tumor cells can via DNA repair responses to repair DNA damage which caused by anticancer drugs. DNA repair pathways have been suggested it could be for prognostic and/or predictive value (26).
Numerous studies have shown that a connection between DNA damage and apoptosis (27). Here, we show that CTD treatment in TSGH8301 cells caused DNA damage based on the observations 1) Comet assay showed that CTD induced comet
tail production (Fig. 2A and B); 2) DAPI staining showed CTD induced DAN damage and condension (Fig. 3A and B); 3) DNA gel electrophoresis showed that CTD induced DNA smear (Fig. 4). DNA strand breaks have also been documented in leukemia cells after CTD treatment in vivo (25). Results from Figures 2A and B have shown that CTD induced DNA damage by using Comet assay which has been recognized to be a significant method for measuring DNA damage (26, 28) in eukaryotic cells from a single-cell basis. This is in agreement with our earlier report shown that CTD induced DNA damage in TSGH8301 cells (23). It is also well documented that DAPI staining could be a usefully for measuring DNA condension and our results have shown that CTD induced DNA condension and these effects are time-dependent (Fig. 3A and B).
It was reported that deficits in DNA repair capacity can decreased the cellular resistant to spontaneous and exogenous DNA damage (29), furthermore, DNA damage happen in cells then cells could through the DNA repair system to eliminate DNA damage cell maintain survival (30, 31). Results from figure 5B indicated that CTD suppressed the protein levels of DNA-PK, PARP, and p-ATR in TSGH8301 cells. DNA damage such as double-stranded DNA breaks in cells can activate ATM and ATR to maintain genomic integrity (32, 33). It was reported that the suppression of DNA damage-induced poly (ADP-ribosyl) ation by PARP inhibitors impairs early DNA damage response events (34). Figure 5B also showed that CTD increased p-p53 but inhibit MGMT, BRCA1 and MDC1 in TSGH8301 cells. It has been reported that p53 can be phosphorylated by ATM, ATR and DNA-PKCs and let p53 in response to DNA damage (35) and DNA damage will induced p-p53 expression (36). DNA-PK can be activated by DNA damage (37) and DNA-PK is joined DNA damage repair system (38).
It was reported that BRCA-1 involved in DNA repair and to maintain genomic stability (39). It has been known that H2A.X plays critical factor for the efficient
accumulation of DNA repair factors in DNA break site (40). When mice have H2A.X
deficient they will have higher radiosensitivity when compared to the normal mice (40). In human cells, phosphorylated H2A.X (γH2A.X) is recognized by MDC1 to
accumulate a myriad of DNA damage responses (DDR) factors on chromatin regions surrounding DNA lesions (41). Furthermore, it was reported that among the mammalian DDR proteins that rely on γH2A.X for IRIF formation, MDC1 is the main
direct binder of γH2A.X (42).
In conclusion, CTD decreased the total viable cells via induction of cell death of TSGH8301 cells in vitro through induced DNA damage and affected DNA damage repair associated protein levels such as suppressed the expressions of DNA-PK,
PARP, p-ATR, MGMT, BRAC1, MDC1, p-H2A.X but increased p-p53 at 6 and 24 h
treatment (Fig. 7).
Acknowledgement
This study is supported in part by a research grant from China Medical University [CMU102-ASIA-20]. Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China medical University, Taichung, Taiwan, R.O.C.
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Figure legends
Figure 1. Cantharidin (CTD) decreased the percentage of viable human gastric TSGH8301 cells. Cells (2x105 cells/well) were placed in 12-well plates and then were
incubated with CTD (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 Materials and Methods. *P<0.05 was considered significant.
Figure 2. CTD-induced DNA damage in TSGH8301 cells was measured by Comet
assay. Cells (2x105 cells/well) were placed in 12-well plates and then were incubated
with 7.5 μM of CTD for 6, 24 and 48 h and DNA damage was determined by Comet assay as described in Materials and Methods. A: representative picture of Comet assay; B: Comet length (folds of control). Arrow showing the comet tail (DNA damage). *P<0.05 were considered significant.
Figure 3. CTD-induced DNA condension in TSGH8301 cells was 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 CTD for 6, 24 and 48 h. Then cells were DAPI stained as described in Materials and Methods. Cells were examined and photographed using a fluorescence microscope at 200X. *P<0.05 were considered significant.
Figure 4. CTD 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 CTD for 6, 12 and 24 h. DNA was extract and DNA gel electrophoresis was performed for examining internucleosomal DNA cleavage (DNA smear) was visualized.
Figure 5. CTD affected the protein expression associated with DNA damage and
repair in TSGH8301 cells. Cells (5x105 cells/well) were placed in 12-well plates and
then were incubated with 7.5 μM of CTD for 6, 24 and 48 h. Then cells were collected and proteins levels from each treatment were measured by SDS-PAGE and immunoblotting as in Materials and methods. A: DNA-PK, PARP and p-ATR; B:
MGMT, BRCA1, MDC1, p-H2A.X and p-p53.
Figure 6. CTD affected the protein expression and translocation in TSGH8301 cells
were 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 CTD for 48 h. Cells were fixed in 4% formaldehyde in PBS, washed and immunostained as described in Materials and Methods. A: MDC1; B: p-H2A.X; C: p-p53 were
Microscope.
Figure 7. The possible flow chart for CTD-induced DNA damage and inhibit DNA
