Effects Of Hypoxia On C-met Protein Expression And Cell Proliferation In Human Laryngeal Cancer Hep-2 Cells

Objective: Hepatocyte growth factor receptor (c-met) can regulate cell proliferation, differentiation, morphogenesis and invasiveness and so on, and is closely related to the occurrence and development of variety of human tumor. This experiment simulates the hypoxia environment of human solid tumor in ritro to detect human laryngeal squamous carcinoma Hep-2 cell's proliferation, apoptosis and the expression of c-met and HIF-1 a (hypoxia-inducible factor1) protein. The purpose of the present study is to explore the effects of hypoxia on c-met protein expression and cell proliferation in human laryngeal cancer Hep-2 cells. Thus, it provides the theoretical basis and experimental foundation for the mechanism of tumor and anti-tumor targeting therapy.Methods:1. Cell Culture Environment: the culture conditions of hypoxic group is 37℃, 5% CO2, 2% O2, 93% N2 with saturated humidity. The culture conditions of normoxia control group is 37℃, 5% CO2, 2% O2, 93% N2 with saturated humidity.2. We identified the expression of c-met protein in human laryngeal cancer Hep-2 cell with immunocytochemical methods. 3. Expression of c-met, HIF-1a protein and apoptosis rate in laryngeal squamous cell lines Hep-2 were detected by flow cytometry Detected Ways under both the hypoxia and normoxia conditions at 6h, 12h, 24h and 36h.4. The proliferation rate was detected in the hypoxic environment and normoxic environment at 6h, 12h, 24h, 36h; the cell proliferation rate of adding 40ng/ml HGF group and without HGF group was detected at 6h, 12h, 24h and 36h using MTT method.Results:1.Through the immunocytochemical method, compared with negative control group, brownish-yellow particles, which is c-met protein, can be seen on the cell membrane in the experimental group.2. By flow cytometry detection methods, levels of c-met protein by FI at 6h, 12h, 24h, 36h in normoxia are 1.062±0.087,1.057±0.034,1.082±0.018 and 1.111±0.056; the FIs of c-met protein at 6h, 12h, 24h, 36h in hypoxia are 1.079±0.042,1.088±0.036,1.167±0.058 and 1.109±0.029;the FIs of HIF-1a protein at 6h, 12h, 24h, 36h in normoxia are 1.002±0.027,1.119±0.021,1.140±0.030 and 1.161±0.065; the FIs of HIF-1a protein at 6h, 12h, 24h, 36h in hypoxia are 1.163±0.072,1.214±0.011,1.268±0.011 and 1.149±0.085, respectively, expression of HIF-1ɑand c-met protein was gradually increased at 6h, 12h, 24h in hypoxia. Their peaks of expression were at 24h, but they gradually decreased at 24h to 36h. Expressions of two proteins were stable under normoxia, without time-dependent manner. Expression of HIF-1a at 6h, 12h, 24h and 36h group were markedly enhanced in hypoxia than in normoxia. Expression of c-met at 12h, 24h in hypoxia was higher than in normoxia. It shows that hypoxia can promote the expression of HIF-1a and c-met. The expression of HIF-1 a and c-met are positively correlated (r = 0.710, p <0.05) at 6h, 12h, 24h in hypoxia, and the expression of HIF-1a is in preference to the expression of c-met, which reveals that HIF-1a can regulate c-met.3. The proliferation rate showed a trend of gradual increase in normoxia and hypoxia. The normoxic without HGF group compared with hypoxic without HGF group has significant difference (p <0.01) at 24h, but not at 6h, 12h, 36h; In normoxic conditions ,comparisons of HGF expressions between each two time points were significantly different (p <0.01),In hypoxic conditions , comparisons of HGF expressions between each two time points were significantly different (p <0.01). Cell proliferation rate and the HIF-1a protein in hypoxia-6h, 12h, 24h was positively correlated (r = 0.811, p <0.01). This indicates that hypoxia can promote cell proliferation, and cell proliferation rate can be increased gradually with time.4. Cell proliferation rate of both normoxic HGF (40ug / L) group and hypoxia HGF (40ug / L) groups were gradually increased with time. In normoxic condition, cell proliferation rates in the non-HGF group and HGF40ug / L group are different (p <0.05) at 12h, and at 24h (p <0.01). In hypoxic condition, the non-HGF group and HGF40ug/L group have significant difference (p <0.05) in the proliferation rate at 24h. Cell proliferation rate and the HIF-1a protein was positively correlated (r = 0.682, p <0.05) in hypoxia at 6h, 12h and 24h. This shows that HGF/ c-met can promote cell proliferation, and cell proliferation rate can be increased gradually with time.5. In normoxic environment, cell apoptosis rate was no significant difference (p> 0.05) at 6h, 12h, 24h and 36h. In hypoxic environment, the apoptosis rate was no significant difference at 6h, 12h, 24h, but 36h group had difference with 6h, 12h and 24h groups. It had increased apoptosis rate trends at 36h.Conclusion:1. The Hep-2 cells indeed have substantial expression of c-met protein.2. In the hypoxic environment similar to the solid tumor in vivo, hypoxic can promote the expression of HIF-1a and c-met protein of tumor cells. In normoxic environment, the expressions of these two proteins were relatively stable, and it was probable that hypoxia regulate the expression of c-met gene.The increasing expression of c-met gene is regulated by the increasing HIF-1a mRNA expression. The expression level of c-met protein increased with a high level of HIF-1a. They are positively correlated.But the expression of HIF-1a was in preference to c-met, whereas, the late long-term sustainability of hypoxic resulted in the decreased expression of two proteins.3. In hypoxic conditions, the cell proliferation rate was significantly higher than that in normoxic conditions.The expression of HIF-1a was active in hypoxia. And cell proliferation rate increased with the increased expression of HIF-1a protein in hypoxia. It illustrates that hypoxia can promote proliferation of Hep-2 cell, and HIF-1a plays an important role in this promotion process.4. We applicated external intervention (HGF) to stimulate the expression of c-met protein of Hep-2 cells. It was that the cell proliferation rates after adding HGF are elevated both in normoxic or hypoxic environment environment.This demonstrates that HGF/c-met can promote cell proliferation. In the hypoxic environment, the cell proliferation rate increased with the increased expression of c-met protein, which further illustrates that the c-met protein can promote the Hep-2 cell's proliferation.5. The apoptosis rate of Hep2 cells in hypoxia was significantly lower than that in normoxia, which demonstrates that hypoxia can inhibit apoptosis.But the long-term sustainabile hypoxia led to increased apoptosis rates, demonstrating that hypoxia can also promote cell apoptosis under prolonged time of hypoxia.