Molecular Mechanisms Of HIF-1 Mediated Hypoxia-induced Multidrug Resistance (MDR) In Gastric Cancer

Hypoxia, frequently found in the center of solid tumor, is a major obstacle in the development of effective cancer chemotherapy. It was reported that hypoxia decreased the efficacy of chemotherapeutic drugs in solid tumors. Because MDR phenotype is complicated and multifactor mechanisms, especially under the hypoxic condition, the hypoxia-induced MDR remains to be elucidated. It was recently reported that HIF-1αmay be a key contributor involving in drug resistance acquired by hypoxia and partly explained an increased level of intracellular HIF-1αassociated with resistance to therapy. So, further study to illuminate the exact mechanism of HIF-1 in hypoxia-induced MDR may be of some interest for developing new sensitizer based on HIF-1 in gastric cancer treatment. Objective: The purposes of present works are to study HIF-1-dependent hypoxia-induced the chemotherapeutic drug sensitivity, to exploit the mechanism of hypoxia-induced drug resistance in gastric cancer cells, to characterize the role and illuminate the molecular mechanisms of HIF-1 contributing to hypoxia-induced MDR.Methods: 1.Gastric cancer cells exposed to hypoxia, MTT assay was used to determine the sensitivity of treated cells toward chemotherapeutic drugs, Annexin V/PI staining was used to detect chemotherapeutic drug-induced apoptosis, flow cytometry Adriamycin accumulation and retention were performed to analyze intracellular drugs accumulation. 2. By Western blot and Dual luciferase reporter assay, the HIF-1 protein expression and transcriptional activity subjected gastric cancer cells to hypoxia were detected. 3.By semiquantitative RT-PCR and VEGF ELISA assay, the VEGF mRNA and protein level subjected gastric cancer cells to hypoxia were detected. 4.By Western blot assay, phosphorylated AKT and p42/44MAPK (ERK1/2) protein were detected in SGC7901 cells subjected to hypoxia at indicated times. By Western blot assay and Dual luciferase reporter assay, the AKT and p42/44MAPK (ERK1/2) and HIF-1 protein, as well as HIF-1 transcriptional activity were detected in SGC7901 cells pretreated with or without LY294002 and U0126 under hypoxic condition. 5. By semiquantitative RT-PCR and VEGF ELISA assay, the VEGF mRNA and protein level were detected in SGC7901 cells pretreated with or without LY294002 and U0126, then exposed cells to hypoxia. 6.Gastric cancer cell stable transfection of HIF-1 expressed plasmid subjected to normoxia or HIF-1 siRNA vector exposed to hypoxia, MTT assay was used to determine the sensitivity of treated cells toward chemotherapeutic drugs, Annexin V/PI staining was used to detect chemotherapeutic drug-induced apoptosis, flow cytometry Adriamycin accumulation and retention were performed to analyze the intracellular drugs accumulation in the cells treated as above. 7.By semiquantitative RT-PCR and Western blot, the expression of P-gp, MRP, Bcl-2, Bax and MGr1-Ag were measured in the cells treated as above. 8.The Dual luciferase reporter assay was applied to study the induction of MGr1-Ag by HIF-1. The EMSA and ChIP assay were used to confirm the hypoxia binding element in MGr1-Ag promoter. 9. Under hypoxic condition, Cell adhesion assay, MTT assay and Annexin V/PI staining were used to determine the situation of cell adhesion, drug sensitivity in gastric cancer cells introduced MGr1-Ag siRNA vector. 10. Nude transplant tumor assay was used to assess the effect of HIF-1 siRNA as a chemotherapeutic sensitizer in vivo. Gastric cancer cells SGC7901/VCR were inoculated subcutaneously in 4 to 6-week-old male athymic nude mice. Mice were randomly selected for HIF-1 siRNA versus scrambled siRNA oligonucleotide, or PBS were used to reverse the drug resistance of xenograft. Measurement for volume of the transplantated tumor was performed to estimate the effect of the reversal agentias.The expression of HIF-1 in xenograft tissue was detected by immunohistochemistry blot. Western Blot was used to detect the expression of HIF-1 in xenograft tissue.Results: 1.Compare to cell lines exposed to normoxia, hypoxic culture cell lines represent a stronger drug resistance, a decrease apoptosis index of VCR and a decrease accumulation and retention of ADR. 2.Western blot result and Dual luciferase reporter assay revealed an increased HIF-1 expression and transcriptional activity in gastric cancer cells exposed to hypoxia at a time-dependent manner. Semiquantitative RT-PCR and VEGF ELISA assay results revealed an increased VEGF mRNA and protein expression in cells treated as above. 3.Western blot analysis of lysates from cells exposed to hypoxia greatly increased AKT and MAPK phosphorylation at time-dependent manner. The results of Western blot analysis and Dual luciferase reporter assay revealed the cells treated with PI3K-specific inhibitor LY294002 and MEK1/2-specific inhibitor U0126 could block hypoxia-induced AKT and P42/44MAPK (Erk1/2) phosphorylation and subsequently inhibited HIF-1 expression and transcriptional activity. 4. Semiquantitative RT-PCR and VEGF ELISA assay results revealed that treatment of the cells with PI3K-specific inhibitor LY294002 and MEK1/2-specific inhibitor U0126 inhibited hypoxia-induced VEGF mRNA and protein expression. Dual luciferase reporter assay revealed that LY294002 and MEK1/2-specific inhibitor U0126 inhibited hypoxia-induced HIF-1 transcriptional activity. 5. Compared to controlled groups, the SGC7901 cell stable transfectants of HIF-1 expressed plasmid represent a stronger drug resistance, a decrease apoptosis index and a decrease accumulation and retention of ADR. 6. The cells blocking HIF-1 expression by stable transfection of HIF-1 siRNA vector inhibited hypoxia-induced drug resistance, abolished hypoxia-prevented VCR-induced apoptosis and increased accumulation and retention of ADR. 7. Semiquantitative RT-PCR and Western blot results revealed that hypoxia increased MDR1, MRP1, Bcl-2 and MGr1-Ag mRNA and protein expression, as well as decreased Bax mRNA and protein expression. Knockdown of HIF-1αabolished hypoxia-induced MDR1, MRP1, Bcl-2, Bax and MGr1-Ag mRNA and protein changes. 8. Luciferase reporter assay analyzed HIF-1-dependent hypoxia induced MGr1-Ag transcriptional activity. ChIP and EMSA assay confirmed a functional HRE (-16 to -11 from translation start site) in the MGr1-Ag gene promoter. 9. Hypoxia could increase the capacity of cell adhesion to LN, whereas blocking MGr1-Ag expression could decrease it. Compared to the controlled cells, blocking MGr1-Ag expression by stable transfection of MGr1-Ag siRNA vector inhibited hypoxia-induced drug resistance, abolished hypoxia-prevented VCR-induced apoptosis.10.HIF-1-siRNA monotherapy significantly reduced SGC7901/VCR tumor volume by 50% from days 12 to 36 compared to those treated with PBS alone, VCR injection, VCR plus scramble sequence and untreated control. Protein level of HIF-1 was decreased significantly in SGC7901/VCR after treatment with siRNA. The results of immunohistochemistry blot showed that the expression of HIF-1 in xenograft tissue was decreased significantly after treatment of siRNA (p<0.05).Conclusion: 1. Hypoxia could decrease chemotherapeutic drugs sensitivity. 2. Hypoxia induced HIF-1 expression and transcriptional activity via activation of PI3K/AKT and MEK/ERK pathway. 2. Overexpression of HIF-1 by hypoxia or stable transfected HIF-1 plasmid decreased chemotherapeutic drugs sensitivity. Knockdown of HIF-1 abolished the hypoxia-increased drug resistance. 3. HIF-1dependent hypoxia-induced MDR phenotype of gastric cancer cells by two means: i. HIF-1 plays a role in development of gastric cancer MDR by upregulating P-gp and MRP, which decreases the accumulation of chemotherapeutic drugs; ii. HIF-1 confers partial resistance of gastric cancer cells to apoptosis by increasing the Bcl-2 expression while decreasing Bax expression, which is a possible mechanism of escaping from apoptosis mediated by chemotherapeutic drugs. 4. HIF-1 induction of MGr1-Ag/37LRP, a novel drug-resistant molecular identified by our lab, prompted MDR phenotype in gastric cancer. 5. We provided evidence that knockdown of HIF-1αin SGC7901/VCR cells suppressed expression of HIF-1 in vivo and contribute to sensitizing for VCR in treatment of gastric cancer by increasing VCR-induced apoptosis.