Inhibitory Effect And Mechanisms Of Smac Mimetics LCL161 On The Proliferation And Apoptosis Of Colon Cancer Cells

Objectives: 1 To observe the effect of Smac mimetics LCL161 on the proliferation of colon cancer cells. 2 To investigate whether LCL161 induced apoptosis to induce the inhibition of proliferation in colon cancer cells. 3 To explore the mechanisms of LCL161-induced apoptosis in colon cancer cells.Methods: 1. MTT assay was performed to detect the inhibition of the proliferation induced by LCL161 in humans colon cancer cell lines. 2. Colony formation assay was used to observe the the effect of LCL161 on the clone ability of colon cancer cells. 3. Mitochondrial membrane potential was measured by the JC-1 staining on SW480 and HT-29 cells treated with LCL161. 4. Apoptosis ratio was assessed by flow cytometry with propidium iodide(PI)staining after the treatment of LCL161 on SW480 and HT-29 cells. 5. The ultrastructural details of two colon cancer cell lines with LCL161 were detected by transmission electron microscope(TEM). 6. Western blot was used to analyze the expressions of inhibitor of apoptosis proteins and TNF-a in the colon cancer cells with LCL161.Results: 1 Different concentrations of Smac mimetics LCL161 inhibited the cell proliferationin human colon cancer SW480 cell line and HT-24 cell line.1.1 In order to examine the effect of LCL161-induced inhibition of cellular proliferation in colon cancer cells SW480 and HT-29 cells were treated with LCL161(0,0.2,1,5,10 mmol/L). LCL161 at high concentrations inhibited both cell lines, cell viability, and the inhibitions were relative to the dose of LCL161 and time of action. 1.2 In an assay to estimate malignant cell colony formation, LCL161 decreased the number of cell colony in both cells, indicating LCL161 had the inhibition on cell colony formation. 2 LCL161 induced apoptosis in human colon cancer cells. 2.1 To confirm that the reduction in the cell numbers was reflective of apoptosis, 3. Mitochondrial membrane potential performed using JC-1 staining. The change of increasing red fluorescence and decreasing green one demonstrated mitochondrial membrane potential decreased and mitochondrial functions were abnormal. 2.2 DAPI staining was revealed that blue signal significantly enhanced in SW480 and HT-29 after treatment with increasing LCL161 concentrations, and there were more than two staining site in some cell nucleuses, showing pyknosis and nuclear cracking appeared in SW480 and HT-29, which were induced by LCL161. 2.3 PI staining confirmed these findings. Cell death was minimal in untreated(Control) cultures. More apoptosis induced by LCL161 in experience group illustrated LCL161 lead to apoptosis in SW480 and HT-29. The changes were interrelated to the concentration of LCL161. 3 LCL161 induced c IAP1 degradation, promoted the synthesis of TNF-α, thus induced apoptosis of colon cancer cells 3.1 Western blot was used to analyze the expressions of inhibitor of apoptosis proteins(IAPs) in the colon cancer cells with LCL161. From the experimental results, we can be observed that with the increase of drug concentration, the expression of c IAP1 protein has a gradually decreasing trend in the two cell lines. And at the same method and time points, the changes in the expressionof c IAP2 and XIAP protein was not obvious. 3.2 Western blot was used to analyze the expressions of TNF-α in the colon cancer cells with LCL161.With the increasing of LCL161 concentration, the bands of TNF- αfrom two cell lines on PVDF membrane showed the gradually deep trend, which proved that LCL161 could promote the expression of TNF-α.Conclusion: 1 Smac mimetics LCL161 can significantly inhibited the growth and proliferation ability of human colon cancer SW480 and HT-29 cell line,and has potential research value, as a new targeted drug for clinical treatment of colon cancer. 2 The possible mechanism of LCL161-induced proliferation inhibition in human colon cancer cells were interrelated to cell apoptosis induced by LCL161 in colon cancer cells, whether there are other pathways still need further study. 3 Smac analogues LCL161 may regulate the apoptosis of colon cancer cells by binding and degradation of c IAP1 and stimulating the synthesis and secretion of TNF-α. Detailed mechanisms and pathways need to be confirmed further.