A novel coordination complex of platinum (PT) induces cell death in colorectal cancer by altering redox balance and modulating MAPK pathway

Abstract

Background: Colorectal cancer (CRC) is a heterogeneous tumor having various genetic alterations. The current treatment options had limited impact on disease free survival due to therapeutic resistance. Novel anticancer agents are needed to treat CRC specifically metastatic colorectal cancer. A novel coordination complex of platinum, (salicylaldiminato)Pt(II) complex with dimethylpropylene linkage (PT) exhibited potential anti-cancer activity. In this study, we explored the molecular mechanism of PT-induced cell death in colorectal cancer.

Methods: Colony formation was evaluated using the clonogenic assay. Apoptosis, cell cycle analysis, reactive oxygen species, mitochondrial membrane potential and caspase-3/- 7 were assessed by flow cytometry. Glutathione level was detected by colorimetric assay. PT-induced alteration in pro-apoptotic/ anti-apoptotic proteins and other signaling pathways were investigated using western blotting. P38 downregulation was performed using siRNA.

Results: In the present study, we explored the molecular mechanism of PT-mediated inhibition of cell proliferation in colorectal cancer cells. PT significantly inhibited the colony formation in human colorectal cancer cell lines (HT-29, SW480 and SW620) by inducing apoptosis and necrosis. This platinum complex was shown to significantly increase the reactive oxygen species (ROS) generation, depletion of glutathione and reduced mitochondrial membrane potential in colorectal cancer cells. Exposure to PT resulted in the downregulation of anti-apoptotic proteins (Bcl2, BclxL, XIAP) and alteration in Cyclins expression. Furthermore, PT increased cytochrome c release into cytosol and enhanced PARP cleavage leading to activation of intrinsic apoptotic pathway. Moreover, pre-treatment with ROS scavenger N-acetylcysteine (NAC) attenuated apoptosis suggesting that PT-induced apoptosis was driven by oxidative stress. Additionally, we show that PT-induced apoptosis was mediated by activating p38 MAPK and inhibiting AKT pathways. This was demonstrated by using chemical inhibitor and siRNA against p38 kinase which blocked the cytochrome c release and apoptosis in colorectal cancer cells.

Conclusion: Collectively, our data demonstrates that the platinum complex (PT) exerts its anti-proliferative effect on CRC by ROS-mediated apoptosis and activating p38 MAPK pathway. Thus, our findings reveal a novel mechanism of action for PT on colorectal cancer cells and may have therapeutic implication.

Keywords: Apoptosis; Colorectal Cancer; MAPK; Platinum; Redox balance.

Fig. 1

PT inhibits colony formation. a-b HT-29 c-d SW480 e-f SW620 cells were seeded as single cell at 500 cells/well in 6-well plate. After 4–6 h, PT (5 and 10 μM) was added for 24 h and incubated at 37 °C. After 24 h media containing PT was replaced with fresh complete media and cells were further incubated for 10–12 days for colony at 37 °C. Crystal violet staining was done and colonies were quantified using light microscope and images were captured by Bio-Rad Gel-Doc system. Results are shown as representative of three independent experiment (n = 3). ***p < 0.001 PT (5) vs control; ***p < 0.001 PT (10) vs control

Fig. 2

PT induces apoptosis and cell cycle arrest. a HT-29 b SW480 c SW620 cells were treated with 5 and 10 μM of PT for 24 h. Total cell death including apoptosis and necrosis was analyzed by Annexin V/PI staining using flow cytometry. d HT-29 and e SW620 cell cycle distribution was measured by PI staining using flow cytometry and the percentage of cell population was determined in the G0/G1, S and G2/M phases. Results shown are representative of three independent experiment (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs control

Fig. 3

PT alters redox balance. a HT-29 b SW480 c SW620 cells were exposed to PT for 24 h. Cells were incubated with 2′,7′–dichlorofluorescein diacetate (DCFDA) for 15 min and the fluorescence of the oxidized 2′,7′–dichlorofluorescein was detected by flow cytometry. c HT-29 d SW480 e SW620 were treated with PT for 24 h. The absorbance was detected at 405 nm using plate reader. The bar graphs are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control

Fig. 4

PT inhibits mitochondrial membrane potential. a HT-29 b SW480 c SW620 cells were treated PT for 24 h. Cells were washed with PBS and incubated with rhodamine 123 (25 ng/ml) at 37 °C for 20 min. Positive stained cells for rhodamine 123 were analyzed by flow cytometry. The bar graphs are presented as mean ± SD of three independent experiments. ***p < 0.001 vs control

Fig. 5

PT blocks anti-apoptotic protein expression and activates cytochrome c and PARP cleavage. a HT-29 c SW480 e SW620 cells were exposed to different concentration of PT for 24 h at 37 °C. Soluble fraction of total cell lysates were immunoblotted with indicated antibodies. Cytosolic fraction of HT-29, SW480 and SW620 were prepared and immunoblotted for cytochrome c. b, d, f Density of the protein bands of three independent experiments were quantified and expressed as relative protein expression to actin. The bar graphs are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Full Length blots were presented in Supplementary Figure S1

Fig. 6

PT activates intrinsic apoptotic pathway. a HT-29 b SW480 c SW620 cells treated with different concentration of PT for 24 h at 37 °C. Activation of caspase-3 and -7 was analyzed by flow cytometry. d HT-29 e SW620 cells were exposed to different concentration of PT for 24 h at 37 °C. Total cell lysates were immunoblotted with the indicated antibodies. Density of the protein bands of three independent experiments were quantified and expressed as relative protein expression to actin. The bar graphs are presented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 vs control. Full Length blots were presented in Supplementary Figure S2

Fig. 7

PT-induced apoptosis depends on ROS and p38 MAPK. a HT-29 c SW480 e SW620 cells were pre-treated with NAC (5 mM), SP600125 (10 μM), SB202190 (10 μM), LY294002 (10 μM) and U0126 (10 μM) for 1 h and then exposed to PT (10 μM) for 24 h at 37 °C. Total cell death was determined by Annexin V/PI staining using flow cytometry. The bar graph shows the percentage of total cell death and apoptotic/ necrotic cells and the results are presented as mean ± SD of three independent experiments. b %Total cell death ***p < 0.001 PT vs control and ****p < 0.001 PT + NAC vs PT signify a statistically significant difference. **p < 0.01 PT + SB202 vs PT; **p < 0.01 PT + LY vs PT statistically significant. d %Total cell death ***p < 0.001 PT vs control; ***p < 0.001 PT + NAC vs PT; ***p < 0.001 PT + SB202 vs PT; *p < 0.05 PT + LY vs PT. f % Total cell death ***p < 0.001 PT vs control; ***p < 0.001 PT + NAC vs PT; *p < 0.05 PT + SB202 vs PT

Fig. 8

PT activates p38 MAPK and inhibits AKT phosphorylation. a-c HT-29 b-d SW620 cells exposed to different concentration of PT for 24 h at 37 °C. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Density of the protein bands of three independent experiments were quantified and expressed as relative protein expression to actin. The bar graphs are presented as mean ± SD of three independent experiments. * < p < 0.05, **p < 0.01, ***p < 0.001 vs control. Full Length blots were presented in Supplementary Figure S3

Fig. 9

PT induces apoptosis by activating p38 MAPK. a HT-29 b SW620 cells were pre-treated with p38 MAPK inhibitor SB202190 (10 μM) for 1 h and then exposed to PT for 24 h at 37 °C. Cytosolic fraction were prepared and immunoblotted for the indicated antibodies. c SW620 cells were transfected with control and p38 siRNA duplex for 48 h then exposed to PT for 24 h at 37 °C. Total cell lysate were prepared and immunoblotted with indicated antibodies. Density of the protein bands of three independent experiments were quantified and expressed as relative protein expression to actin. The bar graphs are presented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 vs control. d SW620 cells were transfected with control and p38 siRNA duplex for 48 h then exposed to PT for 24 h at 37 °C. These cells were incubated with Annexin V/PI and %total cell death was detected by flow cytometry. e Results shown are representative of three independent experiments. ***p < 0.001 Control siRNA+PT vs Control siRNA; *** p < 0.001 p38siRNA + PT vs Control siRNA+PT. Full Length blots were presented in Supplementary Figure S4