Role And Mechanism Of V-ATPases On Gastric Cancer In Anti Proliferation And Invasion And Effects Of Diphyllin

BackgroundGastric cancer is the leading cause of cancer death in China and the third most frequent cause of cancer-related death in North America and Western Europe (Parkin et al.,2005). Biological parameters are being utilized for their prognostic and treatment values in the management of gastric cancer patients.Recent studies have found that the acidic tumor microenvironment is key to managing cancer progression and metastasis. Vacuolar-ATPases (V-ATPases), as the specific proton pumps of the cell, play an important role in maintaining a relatively neutral pHi (internal pH) and are responsible for tumor microenviroment acidification. They are over expressed in many types of metastatic cancers. Furthermore, blocking the V-ATPases can inhibit the growth and metastasis of human cancer cells.In fact, Some molecules and drugs that inhibit V-ATPases have been identified, such as bafilomycin, NiK12192, FR202126and PPI SB242784. Recently, diphyllin, a new natural compound, has been noted to potently inhibit V-ATPases and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. But there are no relevant researches on cancer thus far. Therefore, we aimed to investigate whether diphyllin could inhibit the V-ATPases of gastric cancer, and to explore further its possible mechanisms.Our study was divided into three parts. Part one aimed to explore the expression of V-ATPases in gastric cancer and analysize its relation with the progress and prognosis of gastric cancer. Part two aimed to investigate the effects of silencing V-ATPases on the biological behaviours of human gastric cancer cells via small interference RNA. Part three aimed to study the effects and possible mechanisms of diphyllin on proliferation and invation of human gastric cancer cells SGC7901.Part One Protein expression of V-ATPases in gastric cancer tissues correlates with clinicopathological parameters and a poor prognosis of gastric cancerObjective:To investigate the gene expression of V-ATPases in gastric cancer tissues and explore its role in the progress and prognosis of gastric cancer. Methods:The protein expression of V-ATPases gene in50normal gastric specimens and50gastric cancer tissues were determined by immunohistochemistry.Results:The positive expression rate of V-ATPases was76%in gastric cancer tissue samples which was much higher than that in normal tissue samples (30%, P<0.05) and correlated to lymph node metastasis (P=0.029), tumor size(P=0.017), and differentiation(P=0.038), but not to the patient age, sex, depth of tumor invasion, TNM. V-ATPases may be one of the factors that affect the prognosis of the gastric cancer.Conclusions:Upregulation of V-ATPases maybe involved in esophageal tumor development and progression, and was related with the prognosis of gastric cancer.Part Two Effects of silencing V-ATPases on the proliferation and invasion of human gastric adenocarcinoma cell lines SGC7901Aims:Our Study of part two mainly aimed to investigate the effects and mechanisms of silencing V-ATPases on the proliferation and invation of human gastric adenocarcinoma cell lines SGC7901, thereby providing the evidence for prevention and therapy of gastric cancer.Methods:Human gastric adenocarcinoma cell lines SGC7901were cultured in RPMI-1640medium supplemented with10%fetal bovine serum and antibiotics in a humidified5%CO2atmosphere at37℃. SiRNA-V-ATPases were transfected into SGC7901cell lines. MTT assay was performed to evaluate the proliferation of gastric cancer cells. Annexin V-FITC-PI double staining was used to detect the apoptosis rate of cells. Transwell tests were conducted to campare the ability of invasion of SGC7901cells. Western blotting was employed to determine the protein expressions of V-ATPases ane the relative proteins of Wnt/β-catenin signaling.Results:Siliencing V-ATPases gene inhibited the proliferation of gastric adenocarcinoma cells in a time-dependent manner. The early and late apoptosis rates of SGC7901were significantly enhanced after siRNA-V-ATPases were transfected(P<0.05). The protein expressions of phosphor-LRP6in SGC7901with siRNA-V-ATPases were significantly higher than that in SGC7901without siRNA-V-ATPases (P<.05). β-catenin in Wnt/β-catenin signaling and its target genes, c-Myc and cyclin-D1, were also decreased with the inhibition of V-ATPases (P<0.05).Conclusions:Siliencing V-ATPases gene inhibited the proliferation and invasion, and induced the apoptosis of human gastric adenocarcinoma cells SGC7901, in which the down-regulation of phosphor-LRP6protein and Wnt/β-catenin signaling relative proteins might be involved.Part three Effects of diphyllin as a novel V-ATPases inhibitor on gastric adenocarcinomaAims:This study was conducted to determine whether diphyllin could inhibit V-ATPases in gastric cancer, and explore its mechanism.Methods:We used human gastric cancer cells, SGC7901, as a model in vitro to study the effect of diphyllin.The in vitro antiproliferative, pro-apoptotic and inhibiting of invasion effects by diphyllin were examined. The pHi value was measured in the monolayers using the pH-sensitive fluorescent probe BCECF, and the BCECF fluorescence intensity was recorded by flow cytometry. The pHe (external pH) values of the culture medium were measured by the pH Meter. The effects of diphyllin on the expression of β-catenin signaling pathways were also studied by western-blot and realtime-PCR. Then in vivo the antiproliferative effect of diphyllin were also detected in nude mice injected with SGC7901cells.Results:The natural compound diphyllin, a cytostatic lignan isolated from Cleistanthus collinus, can dramatically inhibit the proliferation and induce the apoptosis of human gastric cancer cells, SGC7901. Our study found that diphyllin can inhibit the expression of V-ATPases in a dose-dependent manner, decrease the internal pH (pHi) and reverse the transmembrane pH gradient in SGC7901cells. Changes of the pH gradient were positively correlated with diphyllin concentration. Further study found that diphyllin treatment caused a decrease in phosphor-LRP6, but not in LRP6. β-catenin in Wnt/β-catenin signaling and its target genes, c-myc and cyclin-D1, were also decreased with the inhibition of V-ATPases.Conclusion:Diphyllin could be characterized as a new V-ATPases inhibitor in treating gastric cancer and inhibiting the phosphorylation of LRP6in Wnt/β-catenin signaling.