Abstract
Demethoxycurcumin (DMC) is a key component of Chinese medicine (Turmeric) and has been proven effective in killing various cancer cells. Its role in inducing cytotoxic effects in many cancer cells has been reported, but its role regarding DNA damage on lung cancer cells has not been studied in detail. In the present study, we demonstrated DMC-induced DNA damage and condensation in NCI-H460 cells by using the Comet assay and DAPI staining examinations, respectively. Western blotting indicated that DMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DNA damage response), DNA repair proteins breast cancer 1, early onset (BRCA1), O6-methylguanine-DNA methyltransferase (MGMT), mediator of DNA damage checkpoint 1 (MDC1), and p53 (tumor suppressor protein). DMC activated phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Furthermore, we used confocal laser systems microscopy to examine the protein translocation. The results showed that DMC promotes the translocation of p-p53 and p-H2A.X from the cytosol to the nuclei in NCI-H460 cells. Taken together, DMC induced DNA damage and affected DNA repair proteins in NCI-H460 cells in vitro.
Lung cancer has been recognized as the second most common cancer in both genders and the leading cause of cancer-related death. Non-small cell lung carcinoma (NSCLC) is responsible for 80% of lung cancer cases and is the leading cause of cancer-related death worldwide with a 5-year survival rate of only 16% (1). NSCLC is relatively insensitive to chemotherapy and the outcome of standard platinum-based chemotherapies remains disappointing (1, 2). Thus, it is important to develop and identify new strategies for NSCLC treatment.
Recently, natural herbals and phytochemicals have attracted great interest as complementary and/or alternative therapies in psychiatric medicine (3). Curcuminoids, the major natural phenolic compounds, are present in the rhizome of turmeric (Curcuma longa Linn.) (4), contain curcumin (Cur, 75-80%), demethoxycurcumin (DMC, 15-20%), and bisdemethoxycurcumin (BDMC, 3-5%), respectively (5, 6). It is well-documented that curcumin has anticancer activities and it also acts as an anti-oxidant and anti-inflammatory (7-9) agent. DMC has anti-oxidant (10), anti-inflammatory (11), anti-cancer (12), and anti-angiogenesis (13) properties. DMC has also been reported to induce cytotoxic effects in many cancer cells (14-16). Recently, curcumin has shown clinical benefits in human chronic diseases including osteoarthritis, rheumatoid arthritis, and type II diabetes (17).
Induction of DNA damage and inhibition of the DNA repair system by DMC in human lung cancer cells has not been reported, and currently there exist no available information to study the molecular mechanisms of DMC.
Materials and Methods
Chemicals and reagents. Demethoxycurcumin (DMC), 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 Chemicals. (St. Louis, MO, USA). Anti-DNA-PK was purchased from Calbiochem (San Diego, CA, USA). Anti-14-3-3σ was purchased from Merck and anti-p-ATR was purchased from Cell Signaling (Danvers, MA, USA). Fetal bovine serum (FBS), culture medium RPMI-1640, L-glutamine and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, California, USA).
Cell culture. The human non-small lung cancer cell line (NCI-H460) was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamines at 37°C in a 75 cm2 tissue culture flask in a 5% CO2 humidified incubator. Cells were passaged by 0.25% trypsin-EDTA when they reached 80% confluence.
Cell viability. Cell viability was measured by flow cytometry as described previously (18). Briefly, NCI-H460 cells (5×105 cells) were seeded on the 12-well culture plate overnight and then were treated with DMC (0, 15, 20, 25, 30 and 35 μM) for 48 h. Cells were collected, washed and stained with PI (5 μg/ml) in phosphate-buffered saline (PBS). The total percentage of viable cells was measured by flow cytometry (Becton-Dickinson, San Jose, CA, USA).
Comet assay (Single cell gel electrophoresis) for DNA damage measurement. NCI-H460 cells (2×105 cells/well) were seeded on 12-well plates for 24 h and were then incubated with 0, 15, 25 and 35 μM of DMC or 0.5% H2O2 as a positive control for 48 h. After incubation, aliquots of 105 cells were isolated to measure cell DNA damage by using Comet assay as described previously (19). Comet tail length was calculated and quantified using the CometScoreTM Freeware analysis (TriTek Corporation, Sumerduck, VA, USA) (19).
4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining for DNA condensation examination. NCI-H460 cells (2×105 cells/well) were seeded on a 12-well plate for 24 h and were then incubated with DMC (0, 15, 20, 25, 30 and 35 μM) for 48 h. After incubation, cells were fixed with 3.7% formaldehyde in PBS for 10 min, followed by DAPI staining. After staining, cells were washed, examined and photographed using a fluorescence microscope at 400×, as described previously (19).
Western blotting for examining protein expressions. NCI-H460 cells (1×106 cells/well) were seeded onto a 100 mm tissue culture dish containing 10% FBS. Cells were then treated with 0, and 35 μM of DMC for 6, 24 and 48 h. After treatment, cells were harvested and suspended in sodium dodecyl sulfate (SDS) sample buffer, sonicated, and boiled for 10 min, as described previously (19). After a brief centrifugation, equivalent amounts of isolated proteins from the soluble fractions of cell lysates from each treatment were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and were transferred to nitrocellulose membrane, and immunoblotted as described previously (19). The transfer membrane was stained by primary antibodies (anti-14-3-3σ, MGMT, BRCA1, MDC1, p53, p-p53, and p-H2A.X) at 4°C and were then washed and stained by secondary antibody. Membranes were subsequently visualized with a chemiluminescent detection system and the protein expressions were measured as described by the manufacturer (19).
Confocal laser microscopy for examining protein translocation. NCI-H460 cells (5×104 cells/well) were placed on 4-well chamber slides and were incubated with 35 μM of DMC for 48 h. Cells were rinsed and fixed in 4% formaldehyde in PBS for 15 min. A 0.3% Triton-X 100 in PBS was used to enhance the permeability of the cells. Cells were washed with PBS, blocked with 5% BSA in PBS for 20 min and were subsequently stained with primary anti-p-H2A.X and anti-p-p53 (green fluorescence) overnight. Cells were then washed and stained with secondary antibody (FITC-conjugated goat anti-mouse IgG). Nuclein was stained with PI (red fluorescence). All samples were mounted and photomicrographed with a Leica TCS SP2 Confocal Spectral Microscope as described previously (20, 21).
Statistical analysis. The results are expressed as the mean±standard deviation (S.D.) from 3 independent experiments. Student's t-test was used for comparisons between DMC-treated and untreated groups. p Values <0.05 were considered to be significant.
Results
Effects of DMC on the total viability of NCI-H460 cells. NCI-H460 cells were treated with DMC (0, 15, 20, 25, 30 and 35 μM) for 48 h. The percentage of viable cells was measured by flow cytometric assay and the results are shown in Figure 1. The results indicated that levels of viable cells decreased significantly after treatment with DMC (except the 15 μM DMC dose) and these effects were dose-dependent (Figure 1).
Effects of DMC on DNA damage of NCI-H460 cells. Cells were treated with DMC (0, 15, 25 and 35 μM) and 0.5% H2O2 for 48 h and were then harvested for the Comet assay and the results are presented in Figures 2A and B. DMC, at all 3 doses tested, induced increased comet lengths compared to control. Positive control (0.5% H2O2) treatment clearly induced comet tail production. These results indicated that DMC-induced DNA damage in NCI-H460 cells.
Effects of DMC on DNA condensation of NCI-H460 cells. In order to further confirm whether the induction of cell death was via DNA condensation by DMC in NCI-H460 cells, DAPI staining was used to examine the formation of DNA condensation (Figures 3A and B). Results indicated that DMC induced DNA condensation in NCI-H460 cells and these effects were dose-dependent.
Effects of DMC on DNA damage-associated proteins of NCI-H460 cells. Cells were treated with 35 μM of DMC for 0, 6, 24 and 48 h and then DNA damage associated protein levels (14-3-3σ, MGMT, BRCA1, MDC1, p53, p-p53 and p-H2A.X) were measured by western blotting and the results are shown in Figure 4. DMC decreased the protein levels of 14-3-3σ, MGMT, BRCA1, MDC1, and p53 but increased p-p53 and p-H2A.X in NCI-H460 cells. These effects were time-dependent.
DMC affects the translocation of p-p53 and p-H2A.X in NCI-H460 cells. In order to investigate whether or not DMC affected DNA damage-associated protein translocation in NCI-H460 cells, cells were exposed to 35 μM of DMC and were then examined by confocal microscopy; the results are shown in Figure 5. DMC promoted the p-p53 (Figure 5A) and p-H2A.X (Figure 5B) translocation to nuclei when compared to control groups. These observations indicated that DMC induced DNA damage and affected DNA repair-associated protein expressions that may also involve p-p53 (Figure 5A) and p-H2A.X (Figure 5B) translocation from the cytoplasm into the nuclei in NCI-H460 cells.
Discussion
Although DMC has been shown to induce cytotoxic effects through cell-cycle arrest and apoptosis in many human cancer cells (22-24), there exist no reports to show that DMC induces DNA damage and affects associated protein expression in human cancer cells. Therefore, we exposed NCI-H460 cells to various doses of DMC in order to investigate the effects of DMC on cell viability. We found that i) DMC decreased the percentage of viable cells in a concentration-dependent manner (Figure 1); ii) a concentration-dependent increase in DNA damage and condensation was observed in NCI-H460 cells after exposure to DMC, which was assayed by Comet assay and DAPI staining, respectively (Figures 2 and 3); iii) DMC decreased the protein levels of 14-3-3σ, MGMT, BRCA1, MDC1, and p53 but increased p-p53 and p-H2A.X in NCI-H460 cells. These effects were time-dependent; iv) DMC induced DNA damage and repair that may also involve p-p53 (Figure 5A) and p-H2A.X (Figure 5B) translocation from cytoplasm into nuclei in NCI-H460 cells.
Based on the results presented in Figures 2 and 3, DMC was found to induce DNA damage and condensation in NCI-H460 cells. In normal or cancer cells, after exposure to agents or chemicals that lead to DNA damage in cells, cells can use their own DNA repair system to survive by eliminating DNA lesions or by adding new DNA bases (20, 25). Recently, a new type of anti-neoplastic therapy that manipulates DNA damage response (DDR) has been developed (26-28). Thus, DDR inhibition has been proven as an effective treatment for cancer. In the present study, our finding is the first to provide information regarding the effects of DMC on DNA damage and repair in NCI-H460 cells (Figures 2 and 3). Figure 4 indicates that DMC suppressed DNA damage and repaired associated protein expression 14-3-3σ, O6-methylguanine-DNA methyltransferase (MGMT), breast cancer 1, and early onset (BRCA1), mediator of DNA damage checkpoint 1 (MDC1), and tumor suppressor protein p53 but promoted the levels of p-p53 and p-H2A.X in examined NCI-H460 cells.
Results from Figure 4 show that DMC decreased the protein levels of MDC1 and MGMT. It has been reported that oligomerized MDC1 accumulation can facilitate the activation of Ataxia telangiechtasia mutated (ATM) and other DDR-related proteins at that special location (29). If MDC1 is defective, the downstream DDR functions of ATM can be impaired (29). DNA damage can develop after cells have been exposed to agents (environmental pollutants, carcinogens and anticancer drugs) to form O6 methylguanine. MGMT is a DNA repair enzyme that can eliminate O6 methylguanines (30). Thus, it was suggested that MGMT inhibition can be used as a means to increase tumor susceptibility to chemotherapy (31).
DMC also suppressed the protein levels of 14-3-3σ and BRCA1 in NCI-H460 cells. One of the cell cycle checkpoints is controlled by 14-3-3σ, which is activated after DNA damage and may lead to G2 phase arrest (32). Several checkpoint inhibitors are currently under investigation for their anticancer potential (33) and 14-3-3σ has been suggested as an effective therapeutic target (34). BRCA-1 plays an important role in DNA repair, cell-cycle checkpoint control and maintenance of genomic stability in breast and ovarian cancer (35), furthermore, BRCA-1 promoter methylation was found to be positively associated with increased mortality in breast cancer patients (36). The results from western blotting (Figure 4) also showed that DMC increased p-p53 and p-H2A.X in NCI-H460 cells. This was confirmed by confocal laser microscopy, which showed that DMC promoted the translocation of p-p53 and p-H2A.X from cytoplasm to nuclei (Figure 5). H2A.X plays a critical role in the efficient accumulation of DNA repair factors at the DNA break site; H2A.X-deficient mice have been found to exhibit higher radiosensitivity (37, 38).
Based on these observations, in Figure 6 we propose some possible mechanisms underlying DMC-induced DNA damage and inhibition of DNA repair proteins in NCI-H460 cells. In the present study, our findings regarding the effects of DMC on DNA damage and associated protein expression were limited by the in vitro design. In the future, studies including animal models are required to confirm DMC's functions in vivo.
Acknowledgements
This work was supported by grant CMU102-ASIA-20 from China Medical University, Taichung, 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, R.O.C.
Footnotes
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Conflicts of Interest
The Authors declare no conflicts of interest.
- Received January 16, 2015.
- Revision received March 2, 2015.
- Accepted March 5, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved