Inhibition Effects Of Hypoxia And Metformin On The H₂₂ Solid Tumor In Mice

Objectives:To investigate the effects of intermittent hypoxia and metformin on the growth of solid tumor in H₂₂ tumor-bearing mice and its mechanisms,providing for theoretical and experimental basis for the prevention and treatment of liver cancer.Methods:1.To establish the solid tumor model by inoculating subcutaneously H₂₂ cells into the Km mice,and observe the effects of 10% hypoxic environment?1,2 and 4 h/d?and metformin?100 and 200 mg/kg/d?on the growth of tumor,respectively;The tumor volume was measured every 2 days and the weight of tumor were weighed at the 15 th day after inoculation,and then the inhibitory rate of tumor growth were calculated.2.The mice were divided into four group: normoxia group,metformin-treated group,hypoxia group,hypoxia and metformin-treated group.The effects of 100mg/kg/d of metformin and intermittent hypoxia for 2 h on the growth of solid tumors were observed.The tumor tissues and blood samples were collected and detected at the 15 th day after inoculation.3.The serum levels of VEGF was determined by ELISA assays;the expression of HIF-1?,MMP-2 protein and CD34 in tumor tissues were detected by western blot and immunohistochemistry.Routine pathology and immunohistochemistry were used to observe the tumor infiltration,microvascularogenesis and lung metastasis.Results:1.The tumor growth were significantly inhibited when 1,2,4 h/d of hypoxia intervention?P<0.05,vs normoxia group?;And the antitumor intensity in 2 and 4 h/d of hypoxia group were significantly higher than that of 1 h/d of hypoxia group;But there was no significant difference between 4 h/d and 2 h/d hypoxia group?P>0.05?.The groups treated with metformin had significant anti-tumor effects?P<0.01,vs NS group?;but there was no significant difference between 100 and 200 mg of metformin groups?P>0.05?.2.The inhibitory rate of tumor growth in hypoxia group,metformin-treated group and hypoxia and metformin-treated group were significantly higher than that of the normoxia group?P<0.05?;And the antitumor effect was the highest in metformin-treated group.The serum levels of VEGF and the expression of HIF-1?,MMP-2 and CD34 in the experimental groups were significantly lower than those in the normoxia group?P<0.05?;and the levels of VEGF,HIF-1? and CD34 in hypoxia and metformin-treated group were significantly lower than those in normoxia group and metformin group?P<0.05?.3.The results of histopathology and immunohistochemistry showed that the treatement with hypoxia,metformin and metformin combined with hypoxia could significantly inhibit tumor invasion and spontaneous lung metastasis,and reduce the number of microvessel.Conclusion:1.Intermittent hypoxia and metformin could coordinated suppress the growth,metastasis and angiogenesis of the solid tumor in H₂₂ tumor-bearing mice2.Mechanically,down-regulated expression of HIF-1?,VEGF,MMP-2 and CD34 could be involved in the inhibitory effects of intermittent hypoxia and/or metformin on H₂₂ tumor-bearing mice.