The Role Of Mst1-JNK Signaling Pathway In Apoptosis Of Colorectal Cancer Cells Induced By Taurine

Colorectal cancer (CRC) is one of the most commonly malignant tumor in the digestive system, and threatens human health seriously. Mammalian sterile 20-like kinase1(Mst1) is highly conserved in yeast, fruit flies, mice and humans. Mst1 has functions of regulating cell cycle, promoting cell apoptosis and inhibiting tumor growth. In the Mst1-JNK signaling pathway, Mst1 activate JNK by mitogen-activated protein kinase 4 (MKK4)/MKK7, then the activated JNK is involved in some cell signaling pathways, such as autophagy and apoptosis. JNK is a main member of the mitogen-activated protein kinase (mitogen activated-protein kinase MAPK) superfamily, and is involved in the regulation of a variety of cell functions, e.g proliferation, differentiation, survival and cell apoptosis. Some researches have shown that Taurine(Tau) have the function of inhibiting proliferation and promoting apoptosis in colon cancer cells, thereby achieving the effect of the prevention and treatment of tumor in a certain degree. Therefore in colorectal cancer cells, we might explore the role of Mst1-JNK signaling pathway in apoptosis of colorectal cancer cells induced by Tau through changing the expression of Mst1 gene and blocking Mst1-JNK pathway at the condition of Tau, then observing the activation of JNK, as well as the effect of apoptosis in colorectal cancer cells.Objective:1. Observing the effect of Tau on apoptosis and Mst1, JNK protein expression and JNK phosphorylation in human colorectal cancer cells;2. Observing the effect of Mst1 gene on apoptosis of colorectal cancer cells induced by Tau, and investigating its possible mechanism;3. Observing the effect of using JNK inhibitor SP600125 on related mRNA and proteins expression of Mst1-JNK signaling pathway of colorectal cancer cells.Methods:1. Western blot detection of Mst1 and JNK protein expression in different colorectal cancer cell lines, including Caco-2, HT-29, HCT-116, SW480, SW620 and LoVo cells, explore whether the level of Mst1 protein expression was related to JNK expression in different cells, and choosing two kind of cells that the highest and lowest expression of Mst1for experiments.2. Observing the effect of Tau at different concentrations on proliferation inhibition rate by MTS assay and apoptosis inhibition rate by flow cytometry in colorectal cancer cells. Using western blot to explore the effect of Tau at high concentrations on the proteins expression of Mst1, JNK and Phospho-JNK(T183 and Y185).3. Transfecting p-EGFP-Mst1 or siRNA into human colorectal cancer cells Caco-2 and SW620 in vitro by Polyfectect reagent. Observing the effect of upregulating or silencing Mst1 gene on proliferation inhibition rate by MTS assay and apoptosis inhibition rate by flow cytometry and Hoechst 33342 staining; Detecting the mRNA level of Mst1, JNK, p53, Bax, Bcl-2 and caspase-3 by RT-qPCR, Detecting the protein expression of Mst1, JNK, Phospho-JNK(T183 and Y185), Bax and Bcl-2 by western blot, exploring the molecular mechanisms of proliferation and apoptosis after upregulating or silencing Mst1 gene in Caco-2 and SW480 cells in vitro, and the relationship between the expression of Mst1 protein and Tau-induced cell apoptosis.4. Using JNK inhibitor SP600125 to block Mst1-JNK signaling pathway, observing the effect of upregulating or silencing Mst1gene on apoptosis by flow cytometry and Hoechst 33342 staining in colorectal cancer cells, Detecting the mRNA level of Mst1, JNK, p53, Bax, Bcl-2 and caspase-3 by RT-qPCR, Detecting the protein expression of Mst1, JNK, Phospho-JNK(T183 and Y185), Bax and Bcl-2 by western blot, exploring the effect of using JNK inhibitor SP600125 on relative mRNA and proteins expression of Mst1-JNK signaling pathway of colorectal cancer cells.Results:1. Mst1 expression has substantially positive correlation (R= 0.8692) to JNK protein expression in six kinds of cells, and the JNK and Mst1 expression was the lowest in Caco-2 cell and the highest in SW620 cell.2. Treating Caco-2 and SW620 cells in vitro with Tau, we found that low concentrations of the drug (10mM,20mM,40mM) promoted cell proliferation; high concentration of the drug (80mM,160mM,320mM) inhibited proliferation and induced apoptosis in a time-dose-dependent effect, calculating the IC50 values of two kinds of cells after 48h treatment with high concentration Tau, we found that The capacity of inhibiting proliferation in SW620 cell is greater than Caco-2 cell; With the concentration increasing in high concentration Tau, protein expression of Mst1 increased unconspicuously, There is no change of JNK protein expression and its phosphorylation.3. p-EGFP-Mst1 plasmid was mediated into Caco-2 cell and SW620 cell in vitro, increased, when Mst1 gene is used in combination with Tau, the apoptosis rate of cells was higher(P<0.01); That Mst1 gene expression is higher can promote the protein expression of JNK, Bax and the mRNA levels of JNK, p53, Bax and caspase-3 increased significantly(P<0.01), inhibit the expression of Bcl-2, Mst1 gene combine with Tau can enhance the effect of promoting apoptisis, they had a synergistic effect.4. Silencing Mst1 gene can reduce apoptosis rate in Caco-2 cell and SW620 cell; the protein expression of JNK, Bax and the mRNA levels of JNK, p53, Bax and caspase-3 was down-regulated, and the expression of Bcl-2 was upregulated after transfected with Mst1-siRNA fragment, Mst1 gene combine with Tau can reverse gene silencing effect to some extent.5. Treatment with JNK inhibitor SP600125 in Caco-2 cell and SW620 cells, we found that There is no obvious effect on expression of Mst1, JNK and p53 (P> 0.05); and the expression of Bax and caspase-3 are significantly increased (P<0.01), while the expression of Bcl-2 is significantly reduced (P<0.01).Conclusion:1.High concentration of Tau can inhibit proliferation and promote apoptosis in colorectal cancer cells SW620 and Caco-2, There is no obvious effect on Mst1, JNK protein expression and JNK phosphorylation in SW620,while they can promote Mst1 protein expression and JNK phosphorylation in Caco-2 cells;2. The mechanisms of promoting colorecal cancer cells apoptosis induced by Tau of Mst1-JNK signaling pay way are transcription-dependent manner and non-transcribed dependent manner at the same time.3. The results of block JNK signaling pathway indicate that there is another pro-apoptotic signaling pathway which Mst1 also acts as one of core elements excepting Mst1-JNK signaling pathway.