Research ArticleExperimental Studies

Casticin Induces DNA Damage and Impairs DNA Repair in Human Bladder Cancer TSGH-8301 Cells

AN-CHENG HUANG, YIH-DIH CHENG, LI-HSUEH HUANG, YUNG-TING HSIAO, SHU-FEN PENG, KUNG-WEN LU, JIN-CHERNG LIEN, JONG-LONG YANG, TZU-SHUN LIN and JING-GUNG CHUNG
Anticancer Research April 2019, 39 (4) 1839-1847; DOI: https://doi.org/10.21873/anticanres.13291

Abstract

Background/Aim: Casticin shows anti-cancer effects in many types of cancer. However, there is no information regarding its role in DNA damage in human bladder cancer. The aim of this study was to investigate the effects of casticin on TSGH-8301 cells in vitro. Materials and Methods: Viability of cells was assayed by flow cytometry. DNA damage was assayed by DAPI staining, comet assay, and gel electrophoresis. Protein levels were examined by western blotting and confocal laser microscopy. Results: Casticin decreased viability of cells and induced DNA damage. Furthermore, casticin decreased expression of p-ATM, p-ATR, MDC1 and MGMT levels after 48 h of treatment, however, it increased p-ATR and MGMT levels after 12 h. In contrast, casticin increased the levels of p-p53, p-H2A.X, and PARP after 48 h of treatment. As shown by confocal microscopy, casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. Conclusion: Casticin decreased viability of human bladder cancer cells through DNA damage.

Bladder cancer is the ninth most common malignancy worldwide and the fourth most common cancer in the United States (1). Worldwide, it is estimated that about 386,000 people are diagnosed with bladder cancer each year and about 150,000 die from this disease (2). In Taiwan, bladder cancer was the 10th most common cancer in 2014 based on the 2017 Taiwan Health and Welfare Report and its incidence rate was about 9.3 per 100,000 individuals (3). Currently, the incidence rate of bladder cancer shows a continuously increasing trend in the world (4) and numerous studies have focused on finding an effective treatment from natural products for bladder cancer patients.

In cells, unrepaired DNA double-strand breaks (DSBs) may lead to genomic instability resulting in reduced cell survival and the development of cancer (5-7). DNA-damage response (DDR), a complex signal transduction pathway, can sense DNA damage and induce the mechanisms involved in the repair of damaged DNA (8, 9). Numerous studies have shown that chemicals or radiation can induce DNA damage and accompany DNA damage with repair in vitro and in vivo (10-13). Furthermore, some of the anticancer drugs have also been shown to induce DNA damage (14-16). Thus, anticancer chemicals derived from natural products can also be investigated with regard to their ability to induce DNA damage in cancer cells in vitro.

Casticin (3’, 5-dihydroxy-3, 4’, 6, 7-tetramethoxyflavone), is a flavonoid isolated from Vitex Fructusand (17). It has been shown to have anti-inflammatory (18, 19), hepatoprotective (20) and anticancer (21, 22) activities. In our earlier studies, we have shown that casticin induced G2/M arrest and mitochondria-dependent apoptosis in human melanoma A375.S2 cells in vitro and suppressed tumor volume in a xenotransplantation model in vivo (23). Furthermore, numerous studies have shown that casticin has anti-invasive properties via suppressing NF-κB and MAPK signaling (24) as well as antimetastatic effects (25). Recently, it was reported that casticin induced DNA damage and inhibited the expression of DNA repair associated proteins such as Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) in mouse melanoma cancer B16F10 cells in vitro (26). Casticin has also been shown to suppress proliferation and to induce apoptosis in many human cancer cells. Furthermore, it has been shown to induce DNA damage in mouse melanoma cancer cells. However, there are no reports showing the effect of casticin on DNA damage and the expression of DNA repair-associated proteins in human bladder cancer cells in vitro. Therefore, this study aimed to investigate the effect of casticin on DNA damage in TSGH-8301 human bladder cancer cells and its mechanism of action. Casticin was found to induce DNA damage and to suppress expression of DNA repair associated proteins in vitro.

Figure 1.
Figure 1.

Casticin decreased the percentage of viable TSGH-8301 cells. Cells were incubated with 0, 0.5, 1, 2.5, 5 and 10 μM of casticin (A) for 48 h, or were treated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h (B) and then harvested to measure the percentage of viable cells by flow cytometry. Experiments were performed in triplicate as described in Materials and Methods. Data are presented as mean±S.D. *p<0.05 defined a significant difference between casticin treated and control groups.

Materials and Methods

Chemicals and reagents. Casticin, dimethyl sulfoxide (DMSO), ethidium bromide, phosphate-buffered saline (PBS), propidium iodide (PI), Triton X-100, trypan blue, RNase A and proteinase K were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). RPMI-1640 medium, L-glutamine, penicillin, streptomycin and trypsin-EDTA were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against O6-Methylguanine-DNA methyltransferase (MGMT), poly ADP-ribose polymerase (PARP), phospho-ataxia telangiectasia mutated (p-ATM), phospho-ataxia telangiectasia and rad3-related (p-ATR), and β-actin were purchased from Calbiochem (San Diego, CA, USA), antibodies against p-H2A.X, and breast cancer 1, early onset (BRCA1) from GeneTex Inc. (Irvine, CA, USA), p-p53 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and mediator of DNA damage checkpoint 1 (MDC1) from Millipore (Billerica, MA, USA). Anti-mouse IgG secondary antibody was obtained from Amersham Pharmacia Biotech, Inc (Piscataway, NJ, USA).

Cell culture. Human bladder cancer cell line (TSGH-8301) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were grown in RPMI-1640 medium (GIBCO®/Invitrogen Life Technologies; Carlsbad, CA, USA) containing 10% heat inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT, USA), 2 mM L-glutamine and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin) in 75 cm2 tissue culture flasks in a humidified atmosphere of 5% CO2 at 37°C (26).

Measurement of viability of TGSH-8301 cells treated by casticin. TSGH-8301 cells (1×105 cells/well) cultured in 12-well plates for 24 h were incubated with 0, 0.5, 1, 2.5, 5 and 10 μM of casticin for 48 h. Alternatively, cells were treated with 2.5 μM for 0, 6, 12, 24, and 48 h. After incubation, cells were washed twice with PBS, re-suspended in PBS containing 5 μg/ml of PI and the total number of viable cells was measured using flow cytometry as described previously (26).

4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining for DNA condensation in TSGH-8301 cells. TSGH-8301 cells (1×105 cells/well) cultured in 12-well plates for 24 h were incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h. After incubation, cells were fixed with 3.7% paraformaldehyde (wt/v) in PBS for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Nuclei were stained with 2 μg/ml of DAPI for 30 min. All samples were examined and photographed using a fluorescence microscope at 200× as described previously (26).

Figure 2.
Figure 2.

DAPI staining for DNA condensation in TSGH-8301 cells after exposure to casticin. Cells (×105 cells/well) cultured in 12-well plates for 24 h were incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h. Cells were then fixed with 3.7% paraformaldehyde (wt/v) in PBS for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Nuclei were stained with 2 μg/ml of DAPI for 30 min. All samples were examined and photographed using a fluorescence microscope at 200x and the intensity of fluorescence was measured as described in Materials and Methods. Data are presented as mean±S.D. *p<0.05 defined a significant difference between casticin treated and control groups.

Comet assay for measurement of DNA damage in TSGH-8301 cells. Comet assay (single-cell gel electrophoresis) was used to measure DNA damage in TSGH-8301 cells after exposure to casticin, as previously described (26). Briefly, TSGH-8301 cells (1×105 cells/well) were treated with 2.5 μM of casticin for 0, 24, and 48 h. Cells were examined for DNA damage and comets were randomly captured at a constant depth of the gel. The comet tail length was calculated and quantified by using the Tri Tek Comet Score™ software image analysis system (TriTek Corp, Sumerduck, VA, USA) as previously described (26).

Gel electrophoresis for DNA fragmentation in TSGH-8301 cells. TSGH-8301 cells (1×106 cells/dish) were incubated with 2.5 μM casticin for 0, 6, 12, 24 and 48 h. Cells were collected and lysed in ice-cold lysis buffer and DNA was extracted, electrophoresed on a 1.5% agarose gel and photographed as previously described (27).

Western blotting for examining the levels of DNA damage-associated proteins in TSGH-8301 cells. TSGH-8301 cells (1×106 cells/well) were placed onto 10-cm dishes, incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h and lysed in lysis buffer [50 mM Tris-HCI (pH=7.4), 125 mM NaCl, 0.1% Triton X-100, and 5 mM EDTA containing both 1% protease inhibitor and 1% phosphatase inhibitor mixture II] (Sigma-Aldrich Corp.). The lysates were centrifuged and total proteins were quantitated by using BioRad assay kit (BSA as a protein control). A total of 20 μg of extracted proteins were loaded onto SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins which were then transferred onto a PVDF membrane (Bio-Rad Laboratories, Richmond, CA, USA). The membrane was blocked with 1% BSA and probed with primary antibodies and the corresponding secondary antibodies as previously described (26, 27). Finally, the blots were detected and visualized using an enhanced chemiluminescence kit (NEN Life Science Products, Inc, Boston, MA, USA) as previously described (27, 28).

Confocal laser scanning microscopy for examining protein translocation in TSGH-8301 cells. Confocal laser scanning microscopy assay was used for examining protein translocation as described previously (26). Briefly, TSGH-8301 cells (5×104 cells/well) were placed on 4-well chamber slides and incubated with or without 2.5 μM casticin for 48 h. After incubation, cells were fixed with 4% formaldehyde in PBS and permeabilized using 0.3% Triton-X 100 in PBS, washed and incubated with anti-DNA-PKcs and -p-p53 antibodies followed by incubation with FITC-conjugated goat anti-mouse IgG (secondary antibody; green fluorescence). The nuclei of all cells were stained with PI (red fluorescence). All samples were examined and photographed under a Leica TCS SP2 Confocal Spectral Microscope as previously described (26, 27, 29).

Figure 3.
Figure 3.

Casticin induced DNA damage in TSGH-8301 cells. Cells were incubated with 2.5 μM of casticin for 0, 24 and 48 h and DNA damage was measured by the comet assay, as described in Materials and Methods. Data are presented as mean±S.D. *p<0.05 defined a significant difference between casticin treated and control groups.

Statistical analysis. All data are presented as mean±standard deviation. Statistical analysis was performed using one-way ANOVA. *p<0.05 was considered to indicate statistically significant difference between the casticin-treated and control groups.

Results

Casticin decreased the number of viable cells in TSGH-8301 cells. TSGH-8301 cells were treated with 0, 0.5, 1, 2.5, 5 and 10 μM of casticin for 48 h and the total number of viable cells was measured as described in Materials and Methods (Figure 1A). Cells were also treated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h and total number of viable cells was also measured (Figure 1B). Results indicated a dose-dependent decrease in the number of total viable cells after 48 h treatment with casticin (Figure 1A). Furthermore, there was a time-dependent decrease in the number of viable cells after treatment with casticin, with the longest duration of treatment leading to the smallest number of viable cells (Figure 1B).

Casticin induced DNA condensation in TSGH-8301 cells. In order to understand whether or not casticin decreased total viable cell number via the induction of DNA condensation, TSGH-8301 cells were incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h and stained with DAPI (Figure 2). Treatment with casticin for 12-24 h resulted in significant DNA condensation, as indicated by the brighter DAPI staining in TSGH-8301 cells compared to controls. Calculation of the intensity of DAPI fluorescence indicated that the effect of casticin on TSGH-8301 cells was time-dependent.

Figure 4.
Figure 4.

Casticin induced DNA fragmentation in TSGH-8301 cells. Cells were incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h. Extracted DNA was electrophoresed and DNA fragmentation was assayed as described in Materials and Methods.

Casticin induced DNA damage in TSGH-8301 cells. In order to investigate whether casticin decreased total viable cell number via the induction of DNA damage, TSGH-8301 cells were incubated with 2.5 μM of casticin for 24 and 48 h and analyzed by the comet assay (Figure 3). Treatment with casticin for 24 and 48 h resulted in significant induction of DNA damage in TSGH-8301 cells as indicated by the comet tail development. To further confirm this finding, cells were incubated with 2.5 μM of casticin for 0, 6, 12, 24 and 48 h. Cells were lysed and DNA was extracted for gel electrophoresis (Figure 4). Results indicated that casticin induced DNA damage (smeared DNA) after 12-48 h treatment.

Casticin affected the levels of proteins associated with DNA damage in TSGH-8301 cells. In order to investigate whether casticin-induced DNA damage is associated with changes in protein expression, western blotting was used. TSGH-8301 cells were incubated with 2.5 μM casticin for 0, 6, 12, 24 and 48 h and then cells were harvested for western blotting analysis (Figure 5). Casticin decreased expression of p-ATM after 6-48 h and p-ATR, MDC1 and MGMT after 48 h of treatment. However, casticin increased the levels of p-ATR after 12 h treatment, and those of MGMT after 12-24 h. In contrast, casticin increased the levels of p-p53 and p-H2A.X after 24-48 h and the levels of PARP after 12-48 h of treatment in TSGH-8301 cells.

Figure 5.
Figure 5.

Casticin altered the expression of DNA damage and repair-associated proteins in TSGH-8301 cells. Extracts of cells incubated with 2.5 μM casticin for 0, 6, 12, 24 and 48 h, were analyzed by western blotting. Membranes were probed with primary antibodies against p-ATM, p-ATR, BRCA1, p-p53, PARA, DNA-PKcs, p-H2A.X, MDC1, and MGMT. β-actin was used as an internal control.

Casticin affected the translocation of DNA-PKcs and p-p53 on TSGH-8301 cells. It was also tested whether casticin affected the nuclear translocation of DNA-PKcs and p-p53. TSGH-8301 cells were incubated with 2.5 μM of casticin for 48 h and examined and photographed by confocal laser microscopy (Figure 6A and B). Results indicated that casticin promoted the expression of DNA-PKcs and p-p53 and the nuclear translocation of p-p53 in TSGH-8301cells.

Figure 6.
Figure 6.

Casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. Cells (5×104 cells/well) cultured on 4-well chamber slides were incubated with 0 and 2.5 μM of casticin for 48 h. Following fixation and permeabilization cells were incubated with anti-DNA-PKcs and -p-p53 antibodies and a secondary antibody (FITC-conjugated goat anti-mouse IgG; green fluorescence). All cells were stained with PI (red fluorescence) for nuclei examination and photographed using a Leica TCS SP2 Confocal Spectral Microscope as described in Materials and Methods.

Discussion

Numerous studies have demonstrated that compounds that derive from natural products decrease the viability of many human cancer cells in vitro and in vivo, by inducing apoptosis. The majority of these compounds have been known to also induce DNA damage. In order to further increase our understanding of the mechanisms of action of these compounds, in the present study, we investigated whether casticin induced DNA damage in human bladder cancer TSGH-8301 cells in vitro.

Casticin has been shown to reduce viability of many human cancer cells through the induction of cell-cycle arrest and apoptosis (30, 31). Herein, our results also confirmed that casticin decreased viability of TSGH-8301 cells. Therefore, we further investigated whether DNA condensation and damage were involved in this effect. DAPI staining and comet assay were used for examining DNA condensation and damage, respectively, and results indicated that casticin induced DNA condensation and damage in a time-dependent manner (Figures 2 and 3). DNA gel electrophoresis was also used to confirm that casticin induced DNA damage in TSGH-8301 cells (Figure 4) (32, 33).

In order to confirm whether or not casticin induced cell death via the induction of DNA damage, protein extracts of TSGH-8301 cells treated with casticin were analyzed by western blotting. Results indicated that casticin decreased the levels of p-ATM and p-ATR after 6-48 h and 48 h of treatment, respectively, and decreased the levels of MDC1 and MGMT after 48 h of treatment (Figure 5). It was reported that different DNA breaks will rapidly activate the members of the PIKK (phosphatidylinositol 3-kinase-like kinase) family of proteins, such as protein kinases ATM, ATR, and DNA-PK, that phosphorylate a multitude of substrates, allowing them to jointly orchestrate DNA repair and cell recovery (34). It has also been reported that ATM is responsive to DNA double strand breaks (DSBs) and that DNA-PK promotes DNA re-ligation via non-homologous end joining (NHEJ) (35). Furthermore, it has been reported that the lack of ATM in both human and mice is involved in lymphoid malignancies (36-38).

It was also found that casticin increased the levels of p-ATR at 12 h, BRCA1 at 6-48 h, MGMT at 12-24 h, p-p53 at 24-48 h, PARP at 12-48 h, and also significantly increased p-H2A.X levels at 24-48 h of treatment in TSGH-8301 cells. ATM, ATR, and DNA-PKcs directly sense DNA damage and activate other mediators that control cell-cycle progression, DNA repair, and apoptosis (8, 39). ATR is mainly involved in the response to single strand DNA breaks (SSBs) (40). The Mre11-Rad50-Nbs1 (MRN)/ATM interaction at DNA breaks further enhances the activity of ATM kinase, which phosphorylates the histone H2A.X at Ser 139 (γH2A.X) and regulates the recruitment of downstream mediators such as MDC1, 53BP1, and BRCA1 (34) to facilitate DNA repair (41). Herein, our results (Figure 5) indicated that casticin decreased MDC1 and MGMT levels at 48 h of treatment. It is well documented that if irreparable, DNA damage activates a p53-dependent apoptotic process to avoid the propagation of genomically compromised cells (41, 42). Furthermore, activation of p53 through ATM may also trigger the G1/S checkpoint to inhibit cells with unrepaired DSBs from entering the S-phase (43, 44).

Figure 7.
Figure 7.

The possible pathway of casticin-induced DNA damage and cell death in TSGH-8301 cells.

Based on these observations, we conclude that casticin decreased viability of cells through DNA damage, which was confirmed by DAPI staining and comet assay, and by inhibiting the expression of DNA repair-associated proteins, such as p-ATM, p-ATR, BRCA1, MDC1 and MGMT in TSGH-8301 cells in vitro (Figure 7).

Acknowledgements

This work was supported by a grant from Saint Mary's Hospital Luodong (SMH106010) sponsored by An-Cheng Huang. 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.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Authors' Contributions

    A.C. Huang, T.S. Lin and J.G. Chung conceived and designed the experiments; A.C. Huang, Y.D. Cheng, L.H. Huan, Y.T. Hsiao and S.F. Peng performed the experiments; Y.T. Hsiao, S.F. Peng and K.W. Lu analyzed the data; J.C. Lien and J.L. Yang contributed reagents/materials/analysis tools; A.C. Huang, T.S. Lin and J.G. Chung wrote the paper.

  • Conflicts of Interest

    The Authors confirm that there are no conflicts of interest.

  • Received February 21, 2019.
  • Revision received March 14, 2019.
  • Accepted March 15, 2019.

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Casticin Induces DNA Damage and Impairs DNA Repair in Human Bladder Cancer TSGH-8301 Cells
AN-CHENG HUANG, YIH-DIH CHENG, LI-HSUEH HUANG, YUNG-TING HSIAO, SHU-FEN PENG, KUNG-WEN LU, JIN-CHERNG LIEN, JONG-LONG YANG, TZU-SHUN LIN, JING-GUNG CHUNG
Anticancer Research Apr 2019, 39 (4) 1839-1847; DOI: 10.21873/anticanres.13291
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