The Effects And Mechanism Of Pseudomonas Aeruginosa Mannose Sensitive Hemagglutination Vaccine On Cell Cycle Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma (HCC) is one of the most commonly occurring cancers in the world. It is the second most frequent cause of death from cancer in our country. Surgical therapy is still the most effective manner in the treatments of HCC. However, the high recurrence and metastasis rate after surgery have become the bottlenecks of further improving the prognosis of HCC patients, and to reduce the high incidence of recurrence and metastasis is the key to cure HCC. The elevation of high mannose glycans is a common feature of malignant cells, and has been suggested to be the basis for alternative cancer therapy for several years. Pseudomonasaeruginosa mannose sensitive hemagglutination vaccine (PA-MSHA) is a genetically engineered PA strain, and it can bind to mannose oligosaccharides on the surface of tumor cells to exert effects on activities of killing tumor cells. Our previous studies have suggested that PA-MSHA could induce apoptosis and inhibit metastasis of HCC. This study is to investigate the effects and mechanism of PA-MSHA on cell cycle of HCC and to provide evidence for clinical use of PA-MSHA.Methods:(1) Two cell lines (MHCC97L and HepG2) were used in this experiment, and six groups of cells were divided: the control group, the100mM mannose treatment group, the2×108/ml heat-killed PA treatment group, the1×108/ml PA-MSHA treatment group, the2×108/ml PA-MSHA treatment group and the2×108/ml PA-MSHA+100mM mannose treatment group.(2) MHCC97L and HepG2cells were cultured and treated with different dose of PA-MSHA and mannose. MTT and flow cytometry were used respectively to measure the cell proliferation and cell cycles. The expressions of the cell cycle related proteins of Cyclin D1, CDK2, PCNA, p21and p27were evaluated by western blot.Results:(1) PA-MSHA suppressed the proliferation of HCC cells in a concentration and time dependent manner.Compared with control group, the effect of PA on proliferation of HCC cell was little (P>0.05). However, the proliferation of HCC cell in PA-MSHA treated group was significantly inhibited, PA-MSHA suppressed HCC cells proliferation in a time and concentration dependant manner (P<0.05).(2) PA-MSHA induced the HCC cell cycle arrest in a dose dependant manner.There was no statistical difference between the control group and PA group in cell cycle of HCC cells (P>0.05). Compared with control group, HCC cells in PA-MSHA treated group were significantly arrested in the G0-G1and G2-M phase of the cell cycle, thereby decreasing the proportion of cells in the S phase (P<0.05). The induction effect on cell cycle arrest of PA-MSHA in high concentration was much stronger than the effect in low concentration(P<0.05).(3) Addition of mannose reversed the effects of PA-MSHA on inhibiting proliferation and arresting cell cycle of HCC cells.Though mannose alone had little effect on proliferation and cell cycle of HCC cells, however, compared with2×108/ml PA-MSHA treatment group, the2×108/ml PA-MSHA+100mM mannose treatment group had a significant decrease in effects on inhibiting proliferation and arresting cell cycle of HCC cells(P<0.05). This phenomenon indicated that the addition of mannose which can bind to the mannose sensitive fimbriae of PA-MSHA reversed the inhibition of proliferation and the arrest of cell cycle of HCC cells.(4)PA-MSHA inhibited the expressions of CyclinDl, CyclinE, CDK2, CDK4and PCNA, and promoted the expression of p21and p27in a concentration dependent manner, the addition of mannose decreased this inhibitory or promotional effects.Compared with control group, PA-MSHA could significantly inhibit the expressions of cell cycle related proteins such as CyclinDl, CyclinE, CDK2, CDK4which are promoting factors of the cell cycle, and decrease the expressing level of PCNA which is closely related to the synthesis of DNA. It could also enhance the expression of p21and p27. And these roles were concentration dependant. Compared with2×108/ml PA-MSHA treatment group, the2×108/ml PA-MSHA+100mM mannose treatment group had a significant decrease in effects on suppressing the expression of CyclinDl, CyclinE, CDK2, CDK4, PCNA and enhancing the expression of p21and p27, which indicated that adding mannose would markedly reduce the effects of PA-MSHA on cell cycle of HCC cells.Conclusion:(1) Through regulating the expression of cell cycle related proteins, PA-MSHA significantly induced HCC cell cycle arrest and inhibited cell proliferation.(2)The effects of PA-MSHA on cell cycle and proliferation of HCC cells were mediated through recognizing and combining to the mannose which was on the surface of HCC cells.(3)Due to the effect of specifically combining to the mannose which was on the surface of tumor cells,PA-MSHA can be utilized for targeted therapy of HCC.