Akt Signaling Pathway Mediated VEGF-C/-D Promote Lymphangiogenesis In Gastric Cancer

Background and objective:With the progress of basic anticancer research, lymphangiogenesis has beenshown to play more and more important role in the cancer prognosis. Gastric cancercan have a metastasis to lymphnode even in a very early stage which leading to a verypoor outcome of these patients. So nowadays, more and more insights have beenfocused to the mechanism of the lymphnode metastasis and lymphangiogenesis ofgastric cancer. Previous studies revealed that protein kinase B (Akt) played a key rolein tumor progress and prognosis and it has a close correlation with tumor lymphnodemetastasis. But the role of Akt played in the lymphangiogenesis of stomach cancer isstill unknown. In the current study, we analysed the relationship of the proteinexpression of Akt/mTOR signaling pathway with lymphangiogenic factorVEGF-C/-D and also with lymph vessel density (LVD) in situ and in vitro andeventually to clarify whether a Akt/mTOR/VEGF-C/-D signaling pathway exist inthe gastric cancer.Methods:1. In situ gastric cancer tissue experiments:55fresh gastric cancer tissues withmatched normal gastric mucosas from patients with disparate pathological stageswere collected and fresh-frozen in liquid nitrogen after surgical resections performedat the first affiliated hospital of Nanchang University. Of these,42were male and13were female. None of the patients had received chemo-, radio-or immuno-therapybefore resection. Part of each specimen were routinely processed, fixed in10%buffered formalin, and embedded in paraffin for histopathological analysis(hematoxylin and eosin stain) and for immunohistochemical staining. The expressionstatus of p-Akt, p-mTOR, D2-40, VEGF-C and–D was detected byimmunohistochemistry.2. In vitro experiments: SGC7901stomach cancer cells were divided intoLY294002intervention group and Rapamycin intervention group. MTT method wasused to determine the effects of these two drugs on SGC7901stomach cancer cells surviving. Western blot was used to detected the expression level of Akt, p-Akt,mTOR, p-mTOR, VEGF-C and-D after the above two drugs intervention.Results:1.Immunohistochemical expression and relationship of p-Akt, p-mTOR,VEGF-C,VEGF-D in gastric cancer tissue.The positive expression rates of p-Akt, p-mTOR, VEGF-C, VEGF-D in gastriccaner were74.5%,85.45%,72.73%and58.18%, respectively. The expression level ofp-Akt was correlated with p-mTOR, VEGF-C and-D expression levels, respectively(P <0.05or0.01). At the meanwhile, the expression level of p-mTORwas alsocorrelated with VEGF-C and–D (P <0.05or0.01).2. Immunohistochemical expression of p-Akt,p-mTOR,VEGF-C,VEGF-D ingastric cancer tissue and their relationships with LVD.The LVD was deteceted by immunostain of D2-40. The results revealed that themean LVD in gastric cancer was94.18±72.965, ranged from0-263.The mean LVDwas23.31±21.569in normal gastric tissue, ranged from0-95, which has a significantdifference when compared with that of cancer tissue (P <0.001). A closelyrelationship was found between LVD and p-Akt, p-mTOR, VEGF-C, VEGF-D,respetively(P <0.05or0.01). We also found that the expression level of p-mTOR,VEGF-C and VEGF-D were different among the p-Akt's negative group, positivegroup,and strongly positive group (P <0.05).The expression level of VEGF-C andVEGF-D were also significantly different among the p-mTOR's negative group,positive group and strongly positive group (P <0.05).3. The effects of Akt activation inhibitor LY294002and the mTOR activationinhibitor Rapamycin on the growth of gastric cancer SGC7901cells.LY294002and Rapamycin can effectively inhibit the growth of stomach cancerSGC7901cells. MTT assay indicated that the viability of SGC7901cells wassignificantly decreased in a concentration-and time-dependent manner afterLY294002and Rapamycin treatment, P <0.001.4.The inhibitory effects of LY294002and Rapamycin on the expression ofVEGF-C and-D in gastric cancer cell line SGC7901. LY294002and Rapamycin can effectively downregulate the phosphorylation ofAkt and mTOR, respectively. The expression level of VEGF-C and VEGF-D wasalso downregulated either in LY294002intervention group or Rapamycinintervention group.The inhibitory expression level of p-Akt, p-mTOR are postivelyrelated with the downregulatory expression levels of VEGF-C and–D (P <0.05).Conclusion：The expression levels of p-Akt,p-mTOR,VEGF-C and–D were all positivelycorrelated with lymph vessel density in gastric cancer. The blockage of Akt and/ormTOR activation could downregulate the expression of VEGF-C and–D. TheAkt/mTOR/VEGF-C/-D signaling pathway might exist in gastric cancer to regulatethe lymphangiogenesis and eventually to regulate the lymphatic metastasis of thecancer.