[Impact of RNA interference targeting hypoxia-inducible factor-1alpha on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia]

Abstract

Objective: To investigate the impact of RNA interference (RNAi) targeting hypoxia-inducible factor 1alpha (HIF-1 alpha) on chemosensitivity of esophageal squamous cell carcinoma cells under hypoxia.

Methods: Human esophageal squamous cell carcinoma cells of the line EC9706 were cultured and divided into 3 groups: untransfected group, added with cobalt chloride (CoCl(2)), a chemical hypoxia inducer, for 8 h so as to establish a hypoxia model; control siRNA transfected group, transfected with control siRNA, and 30 h after the transfection exposed to CoCl(2) for 8 h; and HIF-1 alpha siRNA-transfected group, transfected with HIF-1 alpha siRNA, and 30 h later exposed to CoCl(2) for 8 h. Western blotting was used to detect the protein expression of HIF-1 alpha. Another EC9706 were cultured and divided into 3 groups to be treated as mentioned above, and then exposed to cisplantin or platixal under normoxic or hypoxic condition. 24 hours later 3-(4, 5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay was used to detect the inhibition rates of the cells. Further another EC9706 cells were cultured and then divided into 5 groups: cultured under normoxic condition, cultured under hypoxic condition for 8 h, transfected with control siRNA for 30 h and then under hypoxic condition for 8 h, transfected with HIF-1 alpha siRNA for 30 h and then under hypoxic condition for 8 h. The cell cycle was measured by flow cytometry.

Results: The HIF-1alpha protein expression of the HIF-1alpha siRNA group was significantly lower than those of the untransfected and control siRNA transfected groups. The inhibition rates of the EC9706 cells of the groups treated by cisplatin of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.01). The inhibition rates of the EC9706 cells of the groups treated by platixal of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.05) Under hypoxic condition, the inhibition rates of the HIF-1alpha siRNA transfected EC9706 cells treated by cisplatin and platixal of different concentrations were all significantly higher than those of the control siRNA transfected and untransfected EC9706 cells (all P < 0.05). Flow cytometry showed that under hypoxic condition the proportion of cells in G(1)-phase of the EC9706 cells was significantly higher, and the proportion of S-phase cells was significantly lower than those of the normoxic group (both P < 0.05), and under the same hypoxic condition the proportion of the EC9706 cells in G(1)-phase was significantly lower, and the proportion the EC9706 cells in S-phase was significantly higher than those of the normoxic group (all P < 0.05).

Conclusion: The cell cycle arrest induced by HIF-1alpha may be the mechanism of the resistance to anticancer drugs of the esophageal squamous cell carcinoma cells under hypoxic condition. Blocking HIF-1alpha in esophageal squamous cell carcinoma cells may reverse the multidrug resistance of the tumor cells, so it may offer an avenue for gene therapy.