Abstract
Background/Aim: Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. Materials and Methods: In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. Results: AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 μM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. Conclusion: AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.
- Ally lisothiocyanate (AITC)
- human breast cancer MCF-7 cells
- DNA damage
- DNA condensation and fragmentation
Breast cancer is the most common form of cancer and the most frequently diagnosed cancer in women. It is also the leading cause of cancer-related death among females, and it remains the most prevalent cancer worldwide. Breast cancer has been recognized to be a predominant disease with aging; however, in the developed world about 5-7% of patients are diagnosed with breast cancer below the age of 40 (1). Worldwide, breast cancer accounts for 25% of all cancer cases and 15% of all cancer deaths (2). The risk factors of breast cancer include genetic mutations, reproductive history, weight gain, alcohol consumption, lack of physical exercise, and exposure to common chemicals and radiation (3, 4). The current treatment for patients with breast cancer includes surgery, radiotherapy, chemotherapy, target therapy; still presenting with increased adverse events (drug resistance and side effects). Therefore, numerous studies have focused on natural products for the treatment of human breast cancer.
One of the feature of cancer cells is genomic instability, which can be combined with both DNA damage and tumor-specific DNA repair defects that are involved in tumorigenesis (5). DNA damage response has been recognized to be a vital cell network for maintaining genome stability. Agents inducing DNA damage may trigger a cascade of molecular changes such as DNA damage signaling (DDS), DNA double-strand breaks (DSBs), or DNA damage response (DDR) for causing cell death or survival (6, 7). In particular, DSBs are recognized to be the most serious factors which may induce cell death (8). DSBs may be triggered via cells directly interacting with a damaging agent, reactive oxygen species (ROS), metabolic processes, impaired DNA repair processes, and telomere erosion (9-13). Some chemotherapy agents have been designed to target DNA repair to induce cancer cell apoptosis. Studies should focus on measuring if natural products cause cancer cell DNA damage and/or alter or halt the cellular DNA repair systems.
Epidemiological studies have demonstrated that people with a high dietary consumption of cruciferous vegetables such as broccoli, Brussels sprouts, cabbage, cauliflower, and watercress will have lower disease occurrence and elicited chemopreventive effects (14, 15). These vegetables, rich in isothiocyanates (ITCs), have been recognized as nutraceutical agents against cancer development (16-19). AITC, a constituent of naturally occurring ITCs, is derived from the glucosinolate sinigrin, and it presents multiple biological functions, including anti-cancer activities (20-24). It was reported that in human lung cancer NSCLC cells, combination therapy involving AITC followed by radiation treatment leads to increased DNA damage responses and cell killing when compared to single-agent therapy (25). Our earlier studies showed that AITC induced cell apoptosis in human breast cancer MCF-7 cells via AIF and Endo G signaling pathways (20). AITC induced apoptotic cell death in many human cancer cells in vitro and in vivo; however, no report shows if AITC induces DNA damage and impairs DNA repair in human breast cancer cells. Therefore, in the present study, we investigated the effects and mechanisms of AITC on DNA damage and repair in human breast cancer MCF-7 cells in vitro.
Materials and Methods
Chemicals and reagents. Allyl isothiocyanate (AITC), dimethyl sulfoxide (DMSO), propidium iodide (PI), and trypsin-EDTA were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Minimum Essential Medium Eagle (MEM) medium, fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Anti-p-ATMSer1981 and -p-H2A.XSer139 were obtained from GeneTex Inc. (Irvine, CA, USA); anti-p-ATRSer428, -PARP, -BRCA1 and -p-p53Ser15 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA); anti-MGMT, -p53 and -β-actin were obtained from Sigma-Aldrich Corp.; anti-DNA-PK was got from Calbiochem (San Diego, CA, USA) The goat anti-mouse IgG (secondary antibody) was obtained from Sigma-Aldrich Corp.. In the entire experiment, the AITC was dissolved in DMSO.
Cell culture. MCF-7 human breast cancer cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). Cells were cultured in 90% Minimum Essential Medium Eagle (MEM) medium contained 10% FBS, 2 mM of L-glutamine, and penicillin-streptomycin (100 Units/ml penicillin and 100 μg/ml streptomycin) onto 75 cm2 flasks at 37°C and a 5% CO2 atmosphere under 90% humidified incubator as previously described (20).
Determination of cell viability by flow cytometric assay. MCF-7 cells (1×105 cells/well) were maintained in 12-well culture plates for 24 h and incubated with 0, 10, 20, and 30 μM of AITC for 48 h. After incubation, cells were harvested from each well (each treatment), washed with PBS, and re-suspended in PBS containing 5 μg/ml of PI to measure viable cell number (cell viability) using flow cytometer as cited previously (20).
DNA condensation was measured by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. MCF-7 cells (1×105 cells/well) were maintained in 12-well culture plates for 24 h and incubated with AITC at the final concentrations (0, 10, 20, and 30 μM) for 24 and 48 h. After incubation, cells were washed with PBS and fixed with 4% paraformaldehyde (v/v) in PBS for 15 min. Cells were followed by adding 0.1% Triton X-100 in PBS for 5 min to permeabilize cells. All cells in each treatment were stained with DAPI (2 μg/ml) for 10 min, examined, and photographed using a fluorescence microscope at 200× as described previously (20).
DNA fragmentation was measured by DNA gel electrophoresis. MCF-7 cells (1.5×106 cells/dish) were plated in 10-cm dishes and incubated with 0, 10, 20, and 30 μM of AITC for 24 and 48 h. After incubation, cells were harvested, washed with PBS, and lysed in ice-cold lysis buffer. DNA from each treated cells was extracted, quantitated, and electrophoresed on a 1.5% agarose gel. The gel was stained by ethidium bromide and then observed and photographed under UV-box as previously described (20, 26).
DNA damage was measured by comet assay. MCF-7 cells (1×105 cells/well) were plated in 12-well culture plates and treated with 0, 10, 20, and 30 μM of AITC for 24 and 48 h. After incubation, cells from each well were performed single-cell electrophoresis (comet assay), examined, and photographed under microscopy. The comets of cells were randomly caltured at a constant depth of the gel from each treatment. The comet tail length was measured and quantified using the Tri Tek Comet ScoreTM software image analysis system (TriTek Corp, Sumerduck, VA, USA) as described previously (20).
DNA damage and repair associated protein expression were examined by western blotting. MCF-7 cells (1.5×106 cells/dish) were plated in 10-cm dishes and treated with 0, 10, 20, and 30 μM of AITC for 24 and 48 h. At the end of incubation, cells were collected for protein extraction by RIPA buffer [50 mM Tris-HCI (pH7.4), 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.) as described previously (26). Proteins from each treatment were quantitated using the Bradford method as described previously (26). Proteins were further performed by western blotting and probed by antibodies anti-p-ATMSer1981, -p-ATRSer428, -p53, -p-p53Ser15, -p-H2A.XSer139, - BRCA1, -DNA-PK, -MGMT, -PARP, and -β-actin, followed with goat anti-mouse IgG coupled to HRP. The membranes that were bound antibodies were detected by chemiluminescence kits (Amersham Biosciences ECLTM, Buckinghamshire, UK) and quantified by Image J as described previously (20, 26).
Statistical analysis. The results (data) were performed at least three independent assays and expressed as the mean values with a standard deviation (SD) (mean±SD) in the figures and were analyzed using one-way ANOVA. **p<0.01 or ***p<0.001 is considered as significant differences between the AITC-treated and untreated (control) groups.
Results
AITC reduced cell viability of MCF-7 cells. To confirm whether or not AITC induced DNA damage in MCF-7 cells, we examined the cytotoxic effects of AITC on total cell viability. MCF-7 cells were incubated with or without AITC (0, 10, 20, and 30 μM) for 48 h. After treatment, cells were harvested and the percentage of viable cell numbers was measured by flow cytometric assay, and results were presented in Figure 1. Results indicated that increased doses of AITC (10-30 μM) decreased the viable cell number (cell viability) from 77% to 39% compared to the untreated (control) group. These effects are dose-dependent (Figure 1).
AITC induced DNA condensation (apoptotic cell death) in MCF-7 cells. To confirm whether AITC decreased cell viability in MCF-7 cells was mediated by induction of DNA condensation (one of the marks of apoptotic cell death), MCF-7 cells were incubated with AITC (0, 10, 20, and 30 μM) for 24 and 48 h. After treatment, cells were stained with DAPI solution to monitor nucleus morphology under fluorescence microscopy, and results are presented in Figure 2. Results indicated that increased doses of AITC (10-30 μM) led to increases in DAPI staining intensity when compared to the control group in MCF-7 cells for 24 and 48 h.
AITC induced DNA fragmentation in MCF-7 cells. DNA fragmentation (laddered DNA) was examined by DNA agarose gel electrophoresis in MCF-7 cells. Cells were incubated with AITC (0, 10, 20, and 30 μM) for 24 and 48 h and then harvested to extract DNA for agarose gel electrophoresis. Results are presented in Figure 3. The typical ladder pattern of oligonucleosomal fragments (DNA ladders) was shown after AITC (10, 20, and 30 μM) treatments in MCF-7 cells, which indicated the development of apoptotic cell death (DNA fragmentation is one of the markers of cell apoptosis) occurring in MCF-7 cells.
AITC induced DNA damage in MCF-7 cells. To further confirm whether or not AITC decreased viable cell number was mediated by induction of DNA damage in MCF-7 cells, cells were incubated with AITC (0, 10, 20, and 30 μM) for 24 and 48 h. After treatment, cellular DNA damage was measured by the comet assay and the results are presented in Figure 4. Results indicated that increased doses of AITC (10-30 μM) led to a significantly increased comet tail length than that of control groups in MCF-7 cells (Figure 4). The observations also showed AITC induced DNA damage (the higher of comet tail, the higher of DNA damage) at 24 and 48 h treatment and these effects are dose-dependent.
AITC affected the expression of DNA damage and repair associated proteins in MCF-7 cells. For further investigating the molecular mechanism involved in AITC-induced DNA damage in MCF-7 cells, cells were harvested after exposure to AITC for protein extraction. Then total proteins were quantitated and their expression was determined by western blotting. Results were shown in Figure 5. AITC increased the levels of p-ATMSer1981, p-ATRSer428, p53, and p-p53Ser15 at 10-30 μM (Figure 5A) and p-H2A.XSer139, BRCA1, and active PARP (Figure 5B) at 24 and 48 h treatment. However, AITC increased MGMT at 24 h treatment, but decreased it at 48 h treatment; furthermore, AITC decreased DNA-PK at 24 and 48 h treatment (Figure 5B). Based on these observations, AITC induced DNA damage for cell death, therefore affecting associated protein expression in MCF-7 cells.
Discussion
Considerable evidence has shown that dietary fruits and phytochemicals can prevent cancer development. One of the effects of dietary fruits is their antioxidant activities on cervical cancer (27). Overexpression of ROS leads to cell death when cells are exposed to chemicals or carcinogens. ROS have high reactivity to cause lipid peroxidation and oxidative damage to DNA and proteins (28). However, some anti-cancer drugs can induce DNA damage in cancer cells. Damage to cellular DNA may trigger a cascade of multifaceted molecular events for cells to undergo either apoptosis or senescence or enable accessibility of the DNA repair machinery to the damage site (29, 30). Cells involve several systems that monitor their DNA integrity, detect DNA lesions, cell-cycle checkpoints, and regulate DNA repair pathways for cell survival after exposure to chemical agents (31). Activation of DNA damage response has been applied for achieving anti-tumor effects of chemotherapy and radiotherapy. Therefore, measuring DNA damage may be the way for examining chemicals or agents and whether or not they induce cytotoxic effects on cancer or normal cells.
In this study, we investigated the cytotoxic effects of AITC in MCF-7 cells. Cells were treated with AITC for 48 h at final concentrations of 0, 10, 20, and 30 μM, and the total viable cell numbers (cell viability) were measured and results are presented in Figure 1. Data indicated that AITC induced cytotoxic effects in MCF-7 cells and these effects are dose-dependent. Similar results were found in other human cancer cell lines such as prostate cancer cells (32) and bladder cancer cells (33). However, AITC did have an inhibitory effect on breast cancer MCF-7 cells, but it did not inhibit human breast cancer MDA-MB-231 cells (34). Furthermore, there is no report showing that AITC induced DNA damage in MCF-7 cells. Thus, we further investigated whether or not AITC decreased viable MCF-7 cells via induction of DNA damage.
For further investigation, AITC decreased the viable cell number of MCF-7 cells by inducing DNA damage. Cells were treated with 0, 10, 20, and 30 μM of AITC and assayed by DAPI staining and results are presented in Figure 2. Results showed that AITC induced chromatin condensation and these effects are dose-dependent. Similar results were also reported in human brain cancer cells (35). We also used DNA gel electrophoresis to confirm that AITC induced DNA fragmentation (laddered DNA) (Figure 3), which is one of the characters of apoptotic cell death (cell apoptosis). Some of the anti-cancer drugs induced cancer cell death via induced DNA fragmentation (36). Thus, these findings are in agreement with our earlier report noted that AITC induced cell apoptosis in MCF-7 cells (20).
There is no report showing that AITC induced DNA damage in MCF-7 cells; thus, we used the comet assay to determine DNA damage in MCF-7 cells after exposure to AITC at 24 and 48 h and results indicated AITC induced DNA damage in MCF-7 cells (Figure 4). The protocol of the comet assay (single-cell gel electrophoresis) has been shown to allow the assessment of oxidation of purine and pyrimidine bases (37) and to evaluate cell DNA damage (38) due to it being a simple, sensitive, and low-cost protocol (39). The higher doses of AITC induced a longer comet tail than that of the untreated (control) group. The comet tail length has been recognized to be the most sensitive parameter responding to DNA damage.
We further investigated induction of DNA damage in MCF-7 cells after exposure to AITC which may affect the expression of DNA damage and repair associated proteins in vitro. Therefore, after MCF-7 cells were treated with AITC (0, 10, 20, and 30 μM) for 24 and 48 h, cells were collected. Total proteins were quantitated for western blotting and results are presented in Figure 5. Figure 5A indicates that AITC increased the levels of phosphorylated ataxia-telangiectasia-mutated (p-ATM), phosphorylated-Rad3-related (p-ATR), p53, and p-p53 in MCF-7 cells at both treatment times and these effects were dose-dependent. When DNA damage occurs, it will lead to an increase in p53 activation and p53 is linked to DNA-damage-response signaling pathway (40). After DNA double-strand breaks were induced in the cells, they would respond to this damage through ATM, ATR, and DNA-dependent protein kinase (DNA-PK) which would help maintain cell survival (41). DNA-PK, ATM, and ATR have been known to share specific substrates and have some overlapping functions. ATM, ATR, and DNA-PK must be tightly regulated to prevent aberrant activation. Tumor cells rely heavily on ATR for survival; thus, ATR has been focused on the target for anti-cancer therapy. Our results from Figure 5B indicated AITC decreased DNA-PK protein expression in MCF-7 cells in the present study.
AITC increased the expressions of p-H2A.XSer139, BRCA1, and PARP at 10-30 μM at 24 and 48 h treatment in MCF-7 cells (Figure 5B). BRCA1, p-H2A.XSer139, and PARP are involved in DNA damage repair systems (42). Moreover, phosphorylation of H2A.X is also a highly specific and sensitive molecular marker for DNA damage response (43). The p-H2A.XSer139 activates ATM, which may phosphorylate p53 and histone H2A.X (γH2AX) (43) to activate ATM kinase in a positive feedback loop (44). The loss of ATM exhibits a higher predisposition to breast cancer (45). BRCA1 is phosphorylated on multiple residues to respond to DNA damage (46, 47) and the absence of BRCA1 results in increased genomic alterations (48). DNA repair pathways involved in the reaction of DNA damage (49, 50). Results from Figure 5B also showed that AITC decreased DNA-PK at 24 and 48 h treatment and decreased MGMT at 48 h treatment in MCF-7 cells (Figure 5B). DNA-PK and p53 have been reported to be a sensor complex that detects DNA replication disruption (51). DNA-PK is activated by dsDNA breaks (41, 52); thus, it may repair other proteins involved in DNA damage repair systems, such as PARP, which was increased in our case (Figure 5B). PARP plays an impart role in DNA damage and repair responses and genome stability (53), which act as DNA damage sensors and then the activated PARP contributes to cell survival during DNA damage (45).
In conclusion, our findings suggest that AITC decreased the viable cell number (cell viability) of MCF-7 cells via DNA damage induction. Western blotting results indicated that AITC altered DNA repair associated protein expression, such as p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, BRCA1, p-H2A.XSer139, DNA-PK, MGMT, and PARP in MCF-7 cells in vitro (Figure 6). Thus, AITC induced DNA damage and altered repair associated protein expression for relevant signaling pathways in MCF-7 cells.
Acknowledgements
This study was supported by the Research Funds of China Medical University (CMU104-TC-04) and Chang Bing Show Chwan Memorial Hospital (RD106068), Changhua, Taiwan. 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
Study conception and design: CLL, PPW, and KIL. Acquisition of data: CLL, JCC, PYC, and ACH. Analysis and interpretation of data: CLL, JCL, FSC, and TAC. Drafting of article: CLL, PPW, and KIL. Critical revision: CLL and KIL. All Authors discussed the results and commented on the article.
Conflicts of Interest
The Authors confirm that there are no conflicts of interest in regard to this study.
- Received April 16, 2021.
- Revision received June 24, 2021.
- Accepted July 16, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.