Arsenic trioxide reduces chemo-resistance to 5-fluorouracil and cisplatin in HBx-HepG2 cells via complex mechanisms

Background: Multidrug resistance is one of the major reasons chemotherapy-based treatments failed in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). Hypoxia is generally associated with tumor chemo-resistance. The aim of the study was to investigate the effect of Arsenic trioxide (As2O3) on the hypoxia-induced chemo-resistance to 5-FU or cisplatin and explored its underlying mechanism in the HBx-HepG2 cells. Methods: MTT assay was used to examine the cell viability. Mitochondrial membrane potential (MMP) and cell cycle was examined by flow cytometry. qRT-PCR was employed to observe the mRNA expression level; and western blot assay was used to determine the protein expression level. Results: Our results showed that transfection of HBx plasmid established the HBx-HepG2 cells expressing HBx, and the expression of HBx was confirmed by qRT-PCR and western blot. Exposure of HBx-HepG2 cells to hypoxia (5 % O-2, 3 % O-2, 1 % O-2) for 48 h increased the chemo-resistance to 5-fluorouracil (5-FU) (50-1600 mu M) and cisplatin (25-800 mu M), reduced MMP, and caused the cell cycle arrest at G(0)/G(1) phase in a concentration-dependent manner. Hypoxia also concentration-dependently (5 % O-2, 3 % O-2, 1 % O-2) reduced mRNA expression level of P-glycoprotein (P-gp), multidrug resistance protein (MRP1), lung resistance protein (LRP), and decreased the protein expression level of hypoxia-inducible factor-1 alpha (HIF-1 alpha), P-gp MRP1, and LRP. Following pretreatment with As2O3 at a non-cytotoxic concentration re-sensitized the hypoxia (1 % O-2)-induced chemo-resistance to 5-FU and cisplatin in HBx-HepG2 cells. As2O3 pretreatment also prevented MMP reduction and G(0)/G(1) arrest induced by hypoxia. Meanwhile, As2O3 antagonized increase of HIF-1 alpha protein induced by hypoxia, and it also suppresses the increase in expression levels of P-gp, MRP1, and LRP mRNA and proteins. In addition, As2O3 in combination with 5-FU treatment caused up-regulation of DR5, caspase 3, caspase 8, and caspase 9, and down-regulation of BCL-2, but had no effect of DR4. Conclusions: Our results may suggest that As2O3 re-sensitizes hypoxia-induced chemo-resistance in HBx-HepG2 via complex pathways, and As2O3 may be a potential agent that given in combination with other anti-drugs for the treatment of HBV related HCC, which is resistant to chemotherapy.