Embelin Induces G2/M Phase Arrest Of Gastric Cancer Cells By Activation Of AKT Pathway

Gastric cancer is a malignant tumor in the stomach epithelial tissue.Approximate80million people were diagnosed for gastric malignancyworldwide per year.It has taken the second place in the mortality of allmalignant tumors, accounting for9%. The advanced treatment of gastric canceris surgical excision and an active comprehensive therapy. With thedevelopmental science and tumor biology, it has been recognized that themajority of malignant tumors are systemic disease, and surgery is the onlyeffective treatment. The comprehensive treatment of immune therapy, such asdrug therapy and gene therapy, has been used frequently in clinics. Thedevelopment of gastric cancer often goes through multiple stages, including avariety of oncogenes, tumor suppressor genes, changes in accumulation of cellcycle regulatory genes. Therefore, the mechanism of gastric cancer related genein activition of gastric cancer can provide the ideas and methods of treatmentand improve the clinical outcome of gastric cancer.Inhibitor of apoptosis protein (IAP) family plays a very important role inthe regulation of apoptosis and proliferation of tumor cells. The x-linkedinhibitor of apoptosis protein (XIAP), as an inhibitor of apoptosis protein familymember, is the strongest inhibitor of apoptosis in IAP family. It can regulate thecell cycle and inhibit apoptosis through a various ways. The XIAP oftenoverexpresses in most tumor cell lines, which is closely related with tumorresistance to chemotherapy, progression and recurrence. Embelin is a small molecule inhibitor of XIAP specifically. Thereby, it canaffect cell proliferation and apoptosis in many tumors. The relationship betweenXIAP and ovarian cancer, lung cancer, liver cancer, prostate cancer has beenrevealed by many reports. However, the impact of XIAP in gastric cancer cells israrely elucidated. Our previous research showed that embelin could make thegastric caner cells arrest in the G2/M stage of cell cycle, in which XIAP washighly expressed.The close relationship of XIAP and AKT has been proved by many studiespreviously. AKT, as protein kinase B, ususlly engages in the synthesis of protein,and carbohydrate or lipid metabolism. It can mediate the cell survival as a factorof cell growth. Studies have pointed out that Embelin can inhibit cell growing bydown-regulation XIAP and AKT protein in B lymphocytes. The p-AKT1couldbe inhibited by XIAP expression in granulocyte layer prickle cell of rats, whichwould restrain the cells growing. In addition, less expression of p-AKT1couldbe achieved by down regulation of XIAP in uterine cancer cells. Therefore, wehypothesize that the AKT pathway may play a key role in gastric cancer cellG2/M arrest by down regulating XIAP. This present study focused on themechanism of Embelin in gastric cancer cells G2/M arrest. We used Embelin ofdifferent concentrations (0,10,20,40,80μM) to affect gastric cancer cell AGSand N87. Westernblot was perfomed to detect AKT1, p-AKT1, and related cycleprotein expression level of CDK1, CyclinB1. RT-PCR was carried to testexpression levels of AKT mRNA. We found that AKT1protein and mRNAexpression did not change obviously, whereas p-AKT1and cyclin expressiondecreased dramatically. Furthermore, the expression of these proteins decreasedgradually followed by the increasd concentration of Embelin. Then AKT specific inhibitors were added into the two gastric cancer cells. The MTTdetermined the half cell growth concentration of AKT specific inhibitor API-2which was6μm. Forty-eight hours later, XIAP protein and mRNA expressionlevels did not change significantly. Gastric cancer cells no longer arrested at theG2/M phase.It can be concluded that XIAP located in the upstream of AKT inEmbelin induced G2/M phase arrest process of gastric cancer cells. In order todetect the further potential of AKT in gastric cancer cell cycle, the Embelin wasadded into gastric cancer cells once more after transfection by designed AKT1plasmid. The results showed that gastric cancer cells did not arrest at the G2/Mphase any longer, although the gastric cancer cells transfected by the emptyplasmid still arrested at the G2/M phase. Finally, we concluded thatdown-regulated XIAP, induced by Embelin, could make the gastric cancer cellsarrest at the G2/M phase by AKT pathway.