| Aims:Pancreatic ductal adenocarcinoma (PDAC) is known for its high malignancy, rapid progression and poor prognosis. It is one of the major causes of cancer death worldwide, and the fourth leading cause of cancer-related deaths in European and American countries. The morbidity and mortality rates in our country have increased significantly, ranking the6th among deaths related to malignant tumors. The poor prognosis of PDAC patients is due to its late clinical presentation with symptoms, early and aggressive local invasion, and high metastatic potential. The pathogenesis is not yet clear and there have been no breakthroughs in treatments till now. Thus, it is important to make further research on the molecular biology of pancreatic cancer. We investigated the role of NVP-BEZ235(BEZ235) in inhibiting pancreatic cancer cells in vitro via PI3K/Akt signal pathway and the molecular mechanism to provide possible molecular targets and inhibitors for biological treatments of pancreactic cancer, and also studied the clinicopathological significance of SIX1protein expression in the biological behaviour of pancreatic cancer.Materials and methods:Pancreatic cancer cells, i.e., Panc-1cells, were cultured in vitro. The inhibitory role of BEZ235, the selective inhibitor of PI3K/AKT, for Panc-1was detected by using MTT assay. The cell morphology changes before and after adding the drug (0h,48h) were observed by inverted microscope; series of molecular techniques, such as wound-healing and transwell assay were used to determine the migration and invasion of Panc-1cells before and after treatment with BEZ235. After that Affymetrix GeneChip miRNA Arrays were used to detect the expression of metastatic related genes, including Ezrin and SIXl, in Panc-1cells before and after treatment with BEZ235. Western blot and immunofluorescence staining were used to detect the location and expression of migration marker Ezrin and angiogenesis marker SIX1before and after treatment with BEZ235. Moreover, the expression of SIXl protein was tested in103cases of pancreatic cancer tissues and45cases of normal pancreas tissues using immunohistochemical staining, and the clinical significance was analyzed.Results:1. BEZ235can inhibit PI3K/Akt signal pathway in Panc-1cells. When Panc-1cells were treated with different doses of BEZ235for48hours, phospho-Akt expression levels showed a dose-dependent decrease in50nM group, indicating that BEZ235could effectively inhibit the PI3K/Akt signaling pathway in Panc-1cells.2. BEZ235can inhibit the proliferation of Panc-1cells. According to MTT assay, BEZ235affected Panc-1cell viability. The inhibitory effect was apparent when the Panc-1cells were treated with250nM,500nM and1000nM BEZ235for48hours (P<0.01). Panc-1cells that were treated with100nM,250nM and500nM BEZ235for48hours were observed by inverted microscope. The observation revealed that Panc-1cells that were tight and plump before BEZ235treatment became scattered, deformed, deranged or dead after BEZ235treatment and these changes were BEZ235dose-dependent.3. BEZ235can inhibit the migration and invasion of Panc-1cells. Wound-healing test results showed that Panc-1cells, after being treated with BEZ235for24h,48h and72h, had obviously inhibited migration compared with the control group cells untreated with BEZ235. Transwell test showed that Panc-1cells, after being treated with250nM BEZ235for48h, had obviously inhibited invasion compared with the control group cells untreated with BEZ235. Western blot showed that migration related factor Ezrin and phosphorylated Ezrin (p-Ezrin) expression decreased dose-dependently, and the expression level of upstream SIX1also showed a dose-dependent decrease. In addition, immunofluorescence assay also demonstrated that BEZ235could inhibit the expression of Ezrin and its upstream SIX1protein in Panc-1cells.4. BEZ235markedly inhibits Ezrin and SIX1gene expression. Affymetrix(?) GeneChip(?) miRN A Arrays confirmed that the expression of Ezrin and SIX1in Panc-1cells had significant differences before and after BEZ235treatment, and their expression levels decreased dose-dependently.5. SIX1protein overexpression indicates poor prognosis of pancreatic cancer patients. The expression of SIX1protein in pancreatic cancer tissues was markedly enhanced than "that in normal pancreas tissues. SIX1protein overexpression was significantly correlated with poor prognosis of pancreatic cancer, i.e., SIX1protein expression was associated with the tumor size, clinical stage and histological grading of pancreatic cancer. Univariate and multivariate analysis showed that SIX1overexpression could be used as an independent prognostic factor for determining the prognosis of PDAC.Conclusions:BEZ235can effectively inhibit the migration and invasion of Panc-1cells; SIX1plays an important role in the progression of PDAC and its overexpression of SIX1protein may predict the poor prognosis of patients with pancreatic cancer, and SIX1may become an independent marker for evaluating the risk of PDAC. |
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