1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] plays an anticancer role in multiple types of cancer and potentiates the cytotoxic effects of several common chemotherapeutic agents. The hypercalcemia caused by 1,25(OH)2D3 alone or resistance to cisplatin weaken the anticancer effects of vitamin D. Thus, in this study, we aimed to investigate the synergistic effects of 1,25(OH)2D3 and cisplatin on the apoptosis and cell cycle progression of gastric cancer cells. BGC-823 human gastric cancer cells were treated with 1,25(OH)2D3 or cisplatin alone, or a combination of both agents. Cell apoptosis was assessed by TUNEL assay and flow cytometry. The expression of the apoptosis-related proteins, poly(ADP-ribose) polymerase (PARP), Bax, Bcl-2, caspase-3 and caspase-8, was examined using immunoblot analysis. ERK and AKT phosphorylation were examined by immunoblot analysis. The cell cycle distribution was determined by propidium iodide staining and flow cytometric analysis. p21 and p27 protein expression was also examined using immunoblot analysis. Our results revealed that co-treatment with 1,25(OH)2D3 enhanced cisplatin-induced apoptosis and upregulated the expression of Bax, and promoted the cleavage of PARP and caspase-3. The phosphorylation levels of ERK and AKT were reduced following combined treatment with 1,25(OH)2D3 and cisplatin. The percentage of cells in the G0/G1 phase was greater in the cells treated with the combined treatment than in those treated with either 1,25(OH)2D3 or cisplatin alone. p21 and p27 expression was upregulated following co-treatment with both agents. The results of this study suggest that 1,25(OH)2D3 potentiates cisplatin-mediated cell growth inhibition and cell apoptosis, which involves the upregulation of Bax, a decrease in ERK and AKT phosphorylation levels, and increased p21 and p27 levels.
It has been previously demonstrated that vitamin D3 affects cell proliferation, differentiation and apoptosis (
The role of vitamin D in inhibiting cancer cell growth, inducing cell differentiation and promoting cell apoptosis has been a research hotspot for the prevention and therapy of certain types of cancer, including gastric cancer (
Cisplatin is a major chemotherapeutic agent used in the treatment of gastric cancer. The National Comprehensive Cancer Network (NCCN) guideline suggested that cisplatin should be used as a first-line anticancer drug in the treatment of gastric cancer. Cisplatin exerts anticancer effects through various mechanisms; however, its most prominent mode of action is through the generation of DNA lesions followed by the activation of DNA damage response and the induction of cell apoptosis (
1,25(OH)2D3 has been shown to synergistically or additively enhance the antitumor activities of a number of chemotherapeutic agents, including carboplatin, cisplatin, docetaxel and paclitaxel in prostate cancer (
1,25(OH)2D3 was purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,25(OH)2D3 was dissolved in absolute ethanol (ETOH) to the concentration of 10−3 M and stored in solution at −20°C. 1,25(OH)2D3 was freshly diluted in culture medium to reach the required concentrations prior to each experiment. The ethanol concentration in each test condition never exceeded 0.1%.
Cisplatin was purchased from Shanghai Haoran Bio-Technology Co., Ltd. (Shanghai, China). Cisplatin was dissolved in sterile 0.9% NaCl to the concentration of 50 μg/ml and stored in solution at 4°C. Cisplatin was freshly diluted in culture medium to reach the required concentrations prior to each experiment.
The BGC-823 gastric cancer cell line was purchased from the Central Laboratory of Xiangya Medical College of Central South University, Changsha, China. The cells were cultured according to standard conditions. In brief, the BGC-823 gastric cancer cells were grown in RPMI-1640, 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humid environment with 5% CO2. Cell media and reagents were obtained from Gibco-Invitrogen (Carlsbad, CA, USA). The culture media were changed every 48 h, and the cells were passaged every 2–3 days to produce new generations. The cells were plated in 25-cm2 flasks (Costar Life Sciences, Tewksbury MA, USA), and split every 48 h by washing with D-Hank’s solution and detached using 0.05% trypsin-EDTA. Half of the cells were plated in new flasks with fresh culture medium and the remaining cells were used for the experiments. The cells which had undergone 3 passages were selected for the experiments.
The cells in the 1,25(OH)2D3 treatment group were cultured in RPMI-1640 culture medium with 10 nM 1,25(OH)2D3 for 72 h. The cells in the cisplatin treatment group were treated with 0.2 μg/ml cisplatin solution for 2 h following normal culture for 24 h; the cells were then cultured with fresh medium, followed by washing twice with D-Hank’s solution. The cells in the group co-treated with 1,25(OH)2D3 and cisplatin were treated with 0.2 μg/ml cisplatin for 2 h following culture for 24 h with 1,25(OH)2D3 alone, and were then cultured with fresh culture medium with 10 nM 1,25(OH)2D3. The control group was treated with ETOH in RPMI-1640 culture medium. The total culture time was 72 h for each group.
The BGC-823 cells were seeded in 6-well plates (5×104 cells/well) and left overnight to attach. The cells were then treated with 1,25(OH)2D3 or cisplatin alone, or a combination of both. The cells treated with an equivalent amount of ETOH were used as the vehicle control. The cells were washed with D-Hank’s solution and replenished with fresh medium every 24 h. Following treatment for 72 h, the cells were harvested for immunoblot analysis using RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitors.
The cell protein concentration was measured using a BCA protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. An immunoblot analysis was performed using a Bio-Rad wet electroblotting system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The results from immunoblot analysis were quantified by measuring the optical density of the immunoreactive bands using ImageJ software.
Mouse and rabbit antibodies against Bax, Bcl-2, caspase-3, caspase-8, poly(ADP-ribose) polymerase (PARP), cleaved PARP, phosphorylated (p-)ERK1/2, ERK1/2, p-AKT, AKT, p21, p27 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin was used as the loading control to ensure equal protein loading among all wells in immunoblot analysis.
The In Situ Cell Death Detection Fluorescein kit (Roche Applied Science, Indianapolis, IN, USA) was used to detect cell apoptosis. Cell apoptosis was analyzed by TUNEL assay. In brief, the procedure was as follows: BGC-823 gastric cancer cell suspension was prepared following scheduled experiment treatment, the test sample was washed 3 times in phosphate-baffered saline (PBS) and adjusted to 2×107 cells/ml. Subsequently, 100 μl/tube suspension were transferred into a V-bottomed EP tube, and then 100 μl/tube of a freshly prepared fixation solution were added (4% paraformaldehyde in PBS, pH 7.4) to the cell suspension. The cells were then resuspended and incubated for 60 min at 22°C. The EP tubes were centrifuged at 300 × g for 10 min and the fixative was removed by flicking off or suction. The cells were then washed once with PBS, the EP tubes were centrifuged at 300 × g for 10 min again, and finally, the cells were resuspended in 100 μl/tube permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 5 min on ice. The TUNEL reaction mixture was prepared immediately according to the kit’s instructions prior to use in the experiments and kept on ice until use. The cells were washed twice with PBS, then resuspended in 50 μl/tube TUNEL reaction mixture and incubated for 60 min at 37°C in a humidified atmosphere in the dark. The cells were then transferred to a tube to a final volume of 500 μl in PBS. The well-prepared samples were tested in a Beckman flow cytometry (Beckman Coulter, Miami, FL, USA) for the analysis of cell apoptosis.
The BGC-823 gastric cancer cells were treated with the vehicle control (ETOH), 1,25(OH)2D3 alone, cisplatin alone, or combined treatment with 1,25(OH)2D3 and cisplatin for 24 h. Following treatment, the BGC-823 cells (1×106/sample) were collected by trypsin digestion, then washed twice in cold PBS and fixed for 24 h in 70% ETOH at −20°C. The cells were then washed twice in PBS and incubated with RNAse (8 μg/ml; Fermentas, St. Leon-Rot, Germany) at 37°C for 1 h. The cells were stained with propidium iodide (0.5 mg/ml; Sigma-Aldrich) for 30 min at 37°C in the dark. The cellular DNA content was determined using a Beckman flow cytometry (Beckman Coulter) and ModFit LT 3.0 software (Verity Software House Inc., Topsham, ME, USA). The experiment was repeated 3 times.
Statistical analysis was performed by employing GraphPad Prism 5.0 software (GraphPad Software, CA, USA). All data are presented as the means ± standard error of the mean (SEM). Each experiment was repeated 3 times. The difference between the mean values of 2 groups was evaluated using the Student’s t-test. A value of P<0.05 was considered to indicate a statistically significant difference.
The apoptosis of BGC-823 gastric cancer cells was evaluated by TUNEL assay. The apoptosis of the treated cancer cells was expressed using a fluorescent signal determined by flow cytometry. The density plots obtained by flow cytometry are shown as
Treatment with 10 nM 1,25(OH)2D3 or 0.2 μg/ml cisplatin alone significantly enhanced cell apoptosis compared with the control, as indicated by the increased fluorescence intensity of the DNA fragments in apoptotic cells (P<0.05). Co-treatment with 10 nM 1,25(OH)2D3 and 0.2 μg/ml cisplatin led to a significantly (P<0.05) greater number of apoptotic BGC-823 cells compared to treatment with cisplatin or 1,25(OH)2D3 alone (
Following treatment with 1,25(OH)2D3 alone or in combination with cisplatin for 72 h, the cells were harvested for immunoblot analysis. The expression of a series of apoptosis-related proteins was then determined. The cleavage of PARP was significantly higher in the group co-treated with 1,25(OH)2D3 and cisplatin (P<0.01) compared with the group treated with cisplatin or 1,25(OH)2D3 alone (
Cell apoptosis is regulated by multiple pathways. AKT and ERK1/2 are two important kinases involved in cell proliferation and apoptosis in gastric cancer (
ERK1/2 phosphorylation was also observed following treatment with 1,25(OH)2D3 or cisplatin alone or the combined treatment. Both agents decreased the phosphorylation levels of ERK1/2 (P<0.01). Similarly, co-treatment with 1,25(OH)2D3 and cisplatin significantly reduced the phosphorylation levels of ERK1/2 compared to treatment with cisplatin alone (P<0.05). These results indicated that co-treatment with 1,25(OH)2D3 and cisplatin further enhanced the anti-proliferative effects of cisplatin on BGC-823 gastric cancer cells.
The evaluation of the cell cycle distribution was carried out following treatment with 1,25(OH)2D3 or cisplatin alone or the comined treatment. Co-treatment with 1,25(OH)2D3 and cisplatin significantly increased the percentage of cells in the G0/G1 phase when compared to the group treated with ETOH (vehicle control), 1,25(OH)2D3 or cisplatin alone (P<0.05) (
As the anti-proliferative effects of 1,25(OH)2D3 commonly involve the upregulation of p21 and/or p27 (
Gastric cancer remains the second most common cause of cancer-related mortality worldwide (
In the present study, we demonstrated the differential effects of treatment with vitamin D3 alone or in combination with cisplatin on the apoptosis of BGC-823 gastric cancer cells. Treatment with 1,25(OH)2D3 and cisplatin alone induced the apoptosis of BGC-823 cells, as shown by TUNEL assay. Furthermore, enhanced apoptosis was observed following co-treatment with both agents, and the fluorescence intensity of apoptotic cells markedly increased by 5-fold of the control, and by 1.6-fold that of cisplatin (
Having demonstrated the synergism between 1,25(OH)2D3 and cisplatin, we sought to explore the underlying mechanisms. Caspases play a crucial role in apoptotic cell death induced by vitamin D3 (
We also found that the pro-apoptotic protein, Bax, was upregulated following co-treatment with 1,25(OH)2D3 and cisplatin, while treatment with cisplatin alone did not increase the expression of Bax. The translocation of Bax to the mitochondria has been shown to be of particular importance for the induction of vitamin D-mediated apoptosis in certain cell types. The treatment of MCF-7 breast cancer cells with 1,25(OH)2D3 has been shown to result in the redistribution of Bax from the cytosol to the mitochondria (
In our previous study, we reported that 1,25(OH)2D3-mediated apoptosis is associated with the downregulation of the AKT and ERK survival signaling pathways (
It has been demonstrated that the MAPK-ERK pathway is one of the most significant signal transduction pathways (
The anti-proliferative effects of 1,25(OH)2D3 commonly involve the cell cycle arrest of different cancer cells. 1,25(OH)2D3 inhibits cell proliferation, induces cell cycle arrest and promotes the accumulation of cells in the G0/G1 phase in multipotent mesenchymal cells (MMCs) (
Although a number of 1,25(OH)2D3 responsive genes are known, the exact mechanisms of growth regulation by 1,25(OH)2D3 have not been completely defined. However, an increase in p21 and/or p27 expression is an almost universal feature (
In conclusion, to the best of our knowledge, the present study demonstrates for the first time that 1,25(OH)2D3 plays a synergistic role in cisplatin-mediated growth inhibition and apoptosis in gastric cancer cells. The combined use of 1,25(OH)2D3 and cisplatin may be used as a strategy to overcome resistance to cisplatin and dose limitations, and to improve the anticancer effects of chemotherapy.
This study was supported by grants from the National Clinical Key Specialty Construction Project.
Apoptosis of BGC-823 cells determined by TUNEL assay following co-treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and cisplatin. Apoptotic intensity of BGC-823 cells was determined by flow cytometry after TUNEL assay. The effects of treatment with (A) vehicle-treated control (ETOH), (B) 1,25(OH)2D3, (C) cisplatin, and (D) co-treatment with 1,25(OH)2D3 and cisplatin are indicated. (E) The overlapped peak demonstrates the effects as a whole. The experiments were repeated 3 times.
Effects of co-treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and cisplatin on the expression of apoptosis-related proteins in BGC-823 cells. BGC-823 gastric cancer cells were treated with 1,25(OH)2D3, or cisplatin alone or a combination of both agents. Each experiment was repeated independently 3 times, and representative blots are shown. The expression of poly(ADP-ribose) polymerase (PARP), cleaved PARP, caspase-3, caspase-8, Bcl-2 and Bax is demonstrated. β-actin was used as a loading control for total cellular proteins. Values represent the means ± standard error of the mean (SEM) of triplicate assays. *P<0.05 compared with ETOH treatment alone; **P<0.01 compared with ETOH, 1,25(OH)2D3, or cisplatin treatment alone.
Regulation of AKT and ERK1/2 phosphorylation by co-treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and cisplatin. BGC-823 gastric cancer cells were treated with 1,25(OH)2D3, cisplatin alone or a combination of both agents. Each experiment was repeated independently 3 times, and representative blots are shown. The expression of AKT, pAKT, ERK1/2, pERK1/2 is demonstrated. β-actin was used as a loading control for total cellular proteins. Values represent the means ± standard error of the mean (SEM) of triplicate assays. *P<0.05 compared with ETOH; **P<0.01 compared with ETOH; #P<0.05 compared with cisplatin treatment alone.
Effects of co-treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and cisplatin on the cell cycle distribution in BGC-823 gastric cancer cells. BGC-823 gastric cancer cells were treated with 1,25(OH)2D3, or cisplatin alone or a combination of both agents. (A) Flow cytometric analysis of BGC-823 cells with propidium iodide staining. (B) Cell cycle distribution of BGC-823 cells following treatment. The percentages of cells in different cell cycle phases are expressed as the means ± standard error of the mean (SEM). The experiment was repeated independently 3 times. *P<0.05 compared with ETOH, 1,25(OH)2D3, or cisplatin treatment alone.
Effects of co-treatment with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and cisplatin on p21 and p27 protein expression in BGC-823 gastric cancer cells. BGC-823 gastric cancer cells were treated with 1,25(OH)2D3, or cisplatin alone or a combination of both agents. Each experiment was repeated independently 3 times, and representative blots are shown. β-actin was used as a loading control for total cellular proteins. Values represent the means ± standard error of the mean (SEM) of triplicate assays. *P<0.05 compared with ETOH; **P<0.01 compared with ETOH or 1,25(OH)2D3 alone; #P<0.05 compared with cisplatin treatment alone.
Effects of treatment with 1,25(OH)2D3 alone or in combination with cisplatin on the apoptosis of BGC-823 cells.
Group | Fluorescence intensity (means ± SEM) |
---|---|
Control (ETOH) | 4.73±0.55 |
1,25(OH)2D3 | 9.2±1.14 |
Cisplatin | 14.17±4.01 |
1,25(OH)2D3 + cisplatin | 23.07±3.00 |
P<0.05 compared to the control;
P<0.05 compared to treatment with cisplatin or 1,25(OH)2D3 alone.
1,25(OH)2D3, 1,25-dihydroxyvitamin D3; SEM, standard error of the mean.
Effects of treatment with 1,25(OH)2D3 alone or in combination with cisplatin on cell cycle distribution of BGC-823 cells.
Percentage of cells in each cell cycle phase (means ± SEM) | |||
---|---|---|---|
| |||
G0/G1 | S | G2/M | |
Control (ETOH) | 33.17±1.27 | 51.37±1.82 | 14.27±0.73 |
1,25(OH)2D3 | 30.77±0.73 | 56.03±0.58 | 13.17±0.59 |
Cisplatin | 33.57±1.17 | 59.14±1.21 | 7.60±0.44 |
1,25(OH)2D3 + cisplatin | 38.87±1.14 |
55.83±0.79 | 5.28±0.35 |
P<0.05 compared to control, or treatment with 1,25(OH)2D3 or cisplatin alone.
1,25(OH)2D3, 1,25-dihydroxyvitamin D3; SEM, standard error of the mean.