The Molecular Mechanism Of MGI2Enhanced Human Hepatocarcinoma Cell Lines Sensitivity To Staurosporine-Induced Apoptosis

Hepatocellular carcinoma (HCC) is a highly malignant tumor, lethal primary tumor. The predominant cause of poor prognosis is the invasion and metastasis to surrounding normal tissue, which are correlated with multiple abnormal biological behaviors, including cell proliferation, apoptosis. The generation and development of HCC have been associated with the reduction of cell apoptosis and alteration of related signaling molecules.Membrane-associated guanylate kinase inverted2(MAGI2), also known as synaptic scaffolding molecule (S-SCAM), is a multi-domain scaffold protein functioned in recruiting and anchoring cellular signaling proteins. MAGI2is highly expressed in brain tissue, which associated with the formation and maintenance of the vertebrate central nervous system glutamatergic synapse, resulting in the generation and development of some neurodegenerative diseases. Meanwhile, MAGI2also exists in hepatocarcinama cells as a tumor suppressor. Staurosporine (STS) is an inhibitor of potent protein kinase C (PKC) and other protein kinases which promotes the growth of nervous synapses, inhibits the upregulating of vascular endothelial growth factor (VEGF) of tumor cell and induces cell apoptosis. However, STS has not been widespread utilized as a clinical chemotherapy drugs. Still, the causal mechanism that how STS-induced cell apoptosis has not been elucidated. It is known that MAGI2inhibits cell migration and proliferation, but how influence tumor cell apoptosis has not been reported. Our research is mainly to explore how MAGI2enhances STS-induced apoptosis in hepatocarcinoma cells. It have been demonstrated that phosphatase and tensin homologue deleted on chromosome ten (PTEN) binds to MAGI2through an interaction between the C-terminal PDZ-binding motif of PTEN and the second PDZ domain of MAGI2, the complex of PTEN and MAGI2may promote a series of biological functions. PTEN is a tumor suppressor molecule with protein phosphatase, which inhibits cell growth, migration and proliferation, induces cell apoptosis and regulates cell cycle. We have previously shown that MAGI2inhibits cell migration and proliferation in human hepatocelluar carcinoma cells, which was closely correlated with the upregulation of PTEN. PI3K/AKT signaling pathway plays a fundamental role in cell proliferation and survival. Multifactor regulates PI3K/AKT signaling pathway, the negative feedback is modulated by PTEN. PTEN prevents elevated level of PIP3via dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), then downregulates the phosphorylation of PI3K/AKT.The study mainly explored that MAGI2enhanced human hepatocarcinoma cell lines sensitivity to staurosporine-induced apoptosis via PTEN. The research included two steps, in first step we studied the effect of MAGI2in human hepatocarcinama cell apoptosis induced by STS. We transfected the plasmid of vector and myc-MAGI2into BEL-7404human hepatocarcinoma cells respectively, named as vector-7404and myc-MAGI2-7404cells. Compared with vector-7404cell, myc-MAGI2-7404cell sensitized BEL-7404cells to apoptosis in response to STS stimulus in a time-and dose-dependent manner. In the second step, we investigated the molecular mechanism of MAGI2enhanced STS-induced cell apoptosis. We confirmed that PTEN protein level was increased and the degradation rate was inhibited in myc-MAGI2-7404cells, but no change was found in PTEN mRNA level. Then we demonstrated that MAGI2co-localization with PTEN using Immunofluorescence microscopy, and adding to STS enhanced the complex formation of MAGI2and PTEN. The results showed that MAGI2binding to PTEN may enhance MAGI2-PTEN complex formation and restrain the degradation of PTEN. In order to investigate the mechanism of MAGI2promoted cell apoptosis furtherly, we tested the hypothesis by treating with PTEN RNAi resulting to downregulation of PTEN. We found that knowndown of PTEN expression decreased MAGI2enhanced STS-induced cell apoptosis. Furthermore, we observed the function of AKT on MAGI2promoted STS-induced cell apoptosis. These results suggested that MAGI2enhance cell apoptosis through binding of PTEN and decreasing the phosphorylation of AKT.In conclusion, we proof that MAGI2enhances PTEN protein expression through interacting with PTEN and inhibiting PTEN protein degradation. PTEN involves MAGI2enhanced STS-induced human hepatocarcinoma cell apoptosis by downregulates the phosphorylation level of AKT. MAGI2promotes cell apoptosis via PTEN signaling pathway, which realizes its tumor-suppressive function. Our research might provide a novel target for molecular interference therapy. It is valuable to understand the essence of tumor formation and development and supply promising therapeutic strategies for hepatocarcinoma in the future.