Enhancement of Drug Sensitivity by Knockdown of HIF-1α in Gastric Carcinoma Cells

Abstract

In this study, the effects of hypoxia-inducible factor-1α (HIF-1α) on gastric carcinoma (GC) drug resistance through apoptosis-related genes are investigated. First, HIF-1α-specific siRNA was synthetized and transfected into drug-resistant GC cell line OCUM-2MD3/L-OHP. Then MTT assay was applied to test the inhibition rate of GC cells by 5-fluorouracil (5-FU) and oxaliplatin (L-OHP). After that, flow cytometry (FCM) was applied to measure apoptosis rate. qPCR and Western blot assay were employed to detect HIF-1α and apoptosis-related genes. Results showed that HIF-1α in OCUM-2MD3/L-OHP cells was higher than that in OCUM-2MD3 and gastric epithelial cells. After HIF-1α-siRNA transfection, inhibition rates of 5-FU and L-OHP to tumor cells increased significantly. FCM results showed that apoptosis rate of OCUM-2MD3/L-OHP cells increased significantly. After HIF-1α-siRNA transfection, survivin and Bcl-2 decreased, whereas Bax, caspase 3, and caspase 8 increased significantly. Results from this study seem to confirm that HIF-1α getting involved in GC drug resistance is possibly due to its regulation of some apoptosis-related genes. HIF-1α may be a potential target to reverse drug resistance of GC.

Figure 1

HIF-1α expression levels in gastric cell lines. Gastric cell lines GES1, OCUM-2MD3, and OCUM-2MD3/L-OHP were subjected to qPCR (A) and Western blot (B) assays to determine the expression of HIF-1α. Values are shown as means ± SD for cell samples n = 4 in each group. *p < 0.01 versus GES-1 group; #p < 0.01 versus OCUM-2MD3 group.

Figure 2

HIF-1α-siRNA downregulated HIF-1α expression in CUM-2MD3/L-OHP cells. Cells were transfected with different amounts of sequence of HIF-1α-siRNA for 48 h (A, B); the expression of HIF-1α was identified by qPCR (A) as well as Western blot (B). *p < 0.01 versus NS-siRNA control group. Con, blank control group; Non, NS-siRNA control group.

Figure 3

The effects of HIF-1α-siRNA on the apoptosis of gastric carcinoma cell line OCUM-2MD3/L-OHP with FCM. Cells were transfected with HIF-1α-siRNA or control NS-siRNA, and then tested by FCM. Apoptosis rates of three groups are shown. *p < 0.01 versus NS-siRNA control group. Con, blank control group; Non, NS-siRNA control group.

Figure 4

The effects of HIF-1α-siRNA on the expression of apoptosis-related genes in gastric carcinoma cell OCUM-2MD3/L-OHP. Cells were transfected with HIF-1α-siRNA or control NS-siRNA and then subjected to qPCR (A) or Western blot assays (B) to detect mRNA or protein expression levels of survivin, Bcl-2, Bax caspase 3, and caspase 8. The mRNA or protein expression levels are represented as columns in (A) and (B). *p < 0.01 versus NS-siRNA control group. Con, blank control group; Non, NS-siRNA control group.