Alteration of HIF1a target genes led to 5FU resistance in gastric cancer MKN45 cells under hypoxic condition

Abstract

Purpose Gastric cancer (GC) is reported as the fifth most commonly diagnosed cancer and is the fourth most common cause of cancer-associated death in the world. Tumor cell hypoxia is one of the main factors causing 5FU resistance in gastric cancer cells. Herein, we intend to evaluate the efficacy of the 5FU on the MKN45 cell line by establishing an in vitro hypoxic environment and comparing them with results from the normoxic condition. This study aims to evaluate the efficacy of the 5FU on the MKN45 cell line in normoxic and hypoxic conditions. Methods The MKN45 gastric cancer cell line was cultured in normoxic and hypoxic conditions. The cells were treated with various concentrations of 5-FU for 72 h. Then, cell viability was analyzed by the MTT method. Also, the induction of apoptosis was analyzed by flow cytometry. The expression levels of HIF-1 a, P53, BAX, Bcl2, MRP1, and Casp3 genes were quantified by real-time PCR. A statistical analysis of the results was done using SPSS software. Results Our study showed that a hypoxic condition leads to a higher resistance against 5-FU toxicity in MKN45 cells compared to normoxia. As a result of this drug resistance, we also found significantly low apoptotic cells in hypoxic conditions. Data of gene expression in 5-FU treated MKN45 cells, indicated significant up-regulation of HIF1a in hypoxic conditions. We also showed an elevated level of pro-apoptotic genes (Bax and casp3) in the normoxic and hypoxic groups, but this elevation was significantly lower in hypoxia. In contrast, significant down-regulation of the anti-apoptotic gene (Bcl2) was detected just in the normoxic group, while the Bcl2 gene was significantly up-regulated in the hypoxia versus normoxia group. In the case of p53 and MRP1 genes, we found a higher level of gene expression in MKN45 cells treated under normoxic and hypoxic conditions compared to control, while this increase was more significant in hypoxic conditions. There was no significant difference in the level of expression of the MRP1 gene in hypoxic conditions compared to normoxia. Conclusion Altogether, our results demonstrated that the resistance to 5-FU in MKN45 gastric cancer cells might be due to the upregulation of the HIF-1α gene and its regulated downstream target gene under hypoxic conditions.