Effects of hypoxia on malignant phenotype of HCC cells / 中华普通外科杂志

ABSTRACT

Objective To investigate the effects of hypoxia on malignant phenotype of a hepatocellular carcinoma(HCC)cell line and its molecular basis.Methods Human HCC cell line with highly metastatic potential(MHCC97H)was grown under hypoxia(induced by 100 μmol/L CoCl_2)and normoxic conditions respectively.To observe the effects of hypoxia on MHCC97H cells was observed,the tumorigenicity was carried out by soft agar cloning method in vitro and inoculation in nude mice in vivo;Invasive and metastatic potential were measured.Experiments with and/or without Matrigel in vitro and a metastatic human HCC orthotopic nude mice model by HAL in vivo.To clarify the molecular background,the proliferation of cells were analyzed by MTT assay and the cell cycle was detected by flow cytometry;The expression of embryonic stem cell(ES)-like genes(Oct4,Nanog and Sox2)were detected by real-time RT-PCR;CD90~+ and CD133~+ subpopulation from MHCC97H cells were isolated by flow cytometry and the expressions of CD90 and CD133 of MHCC97H cells were analyzed by immunofluorescence.Results Hypoxia increased tumor migration(t=2.792,P=0.023),invasiveness(t=7.624,P＜0.0001)and clone forming ability(t=3.292,P=0.011)of MHCC97H cells in vitro,and promoted tumorigenesis(x~2=8.571,P=0.015)and pulmonary metastasis(x~2=5.507,P=0.031)in vivo.The proliferation of MHCC97H cells arrest under hypoxic conditions accompanied with increased proportion of cells in G1 phase,the expressions of Oct4,Nanog,Sox2 significantly increased in response to hypoxia,but the fluorescence intensity and number of CD90~+ and CD133~+ MHCC97H cells decreased or unchanged.Conclusion Hypoxia increases aggressiveness of MHCC97H cells,which was correlated with the acquisition of embryonic stem cell-like characteristics.