Inhibitory Mechanism Of BEZ235on HepG2Cells And Clinicopathological Significance Of Six1Overexpression In HCC

Aims:To investigate the inhibitory molecular mechanism of BEZ235on angiogenesis and apoptosis of HepG2cells in vitro and the clinicopathological significance of Sixl protein overexpression in hepatocellular carcinoma (HCC).Methods:HepG2cells were treated with BEZ235in vitro, and the cell viability was detected by using MTT assay. Then, the cell morphology changes were observed by phase-contrast microscope. Western blot and immunofluorescence staining were used to detect the location and expression of migration maker Ezrin and angiogenesis maker Sixl, VEGF and HIF. Meanwhile HepG2cells were treated with BEZ235, and the apoptotic rate was detected by using flow cytometry and hoechst staining. Series of molecular techniques, such as colony formation, wound-healing and transwell assay were used to determine the proliferation, migration and invasion in HepG2cells with and without treatment. Additionally, total162cases of HCCs,87of peripheral nontumor tissues and35of normal liver tissues were selected for immunohistochemical staining of Sixl protein, and the clinicopathological significance was also analyzed according to the detailed clinical information of HCCs.Results:1. Phosphor-Akt expression levels were significantly downregulated in HepG2cells with BEZ235treatment, indicating that BEZ235could effectively inhibit the PI3K/Akt signaling pathway in HepG2cells.2. BEZ235could effectively inhibit the cell proliferation of HepG2cells in vitro. The cultured HepG2cells showed tight cell-cell adhesion and significant cell polarity by phase-contrast microscopy observation. However, the cells treated by BEZ235at500nM for48h showed spindle shapes, and scattered and loose cell-cell interaction. Meanwhile, MTT assay found that the cell viability was significantly decreased in HepG2cells treated100nM BEZ235, and showed the dose dependent manner. Moreover, colony-forming assay also indicated that HepG2cells were significantly inhibited by BEZ235. The expression level of Skp2dramatically downregulated in HepG2cells treated with BEZ235compared with the control untreated cells, indicating that Skp2played an important role on the inhibitory effect of BEZ235on HepG2cells.3. BEZ235could effectively inhibit the HepG2cells via suppressing the motility and invasiveness. And the mechanism is mainly to be through the downregulation of Ezrin and Six1.4. BEZ235induces the apoptosis of HepG2cells by caspases-3/9and PARP activation in vitro. Flow cytometry assay revealed that apoptotic rate was significantly increased in the treated cells (7.42%and12.84%in250nM and500nM, respectively) compare with the control untreated cells (2.98%). Additionally, Hoechst33342staining showed an even distribution of the stain and round homogeneous nuclei, however, the cells treated by BEZ235showed many apoptotic cells, the nucleus displayed typical changes including reduction of cellular volume, bright staining and condensed or fragmented nuclei. The cleaved caspase-3/9and PARP were significantly increased in the HepG2cells treated by BEZ235compared with the control untreated cells.5. BEZ235could effectively inhibit the angiogenesis of HepG2cells by downregulating Sixl, VEGF and HIF. Western blot and immunofluorescence staining showed that the protein levels of Sixl, VEGF and HIF were significantly downregulated in the cells treated with BEZ235compared with the control group, and showed the dose dependent manner.6. Sixl expression prompted the poor prognosis of hepatocellular carcinoma. The strongly positive rate of Sixl protein in HCC was significantly higher than peripheral nontumor tissues and normal liver tissues. Six1protein overexpression was significantly correlated with the tumor size, pTNM Stage and venous infiltration of HCC. Importantly, it was found that Sixl overexpression also emerged as an independent prognostic factor in HCC.Conclusions:BEZ235could effectively suppress the HepG2cells in vitro via inhibiting the cell proliferation, migration, angiogenesis and apoptosis. Also, Sixl played an important role in the progression of HCC, the overexpression of Sixl protein might predict the poor prognosis of patients with HCC, and Six1might be an independent marker for risk of HCC.