The Role And Mechanism Of KLF8Involved In Hypoxia-induced Multidrug Resistance In Gastric Cancer Cells

Gastric cancer is one of the malignant tumor in our country with high death rate, themain treatment include surgery, radiation therapy, chemotherapy, biological treatment andso on. However, the multi-drug resistance (MDR) is a serious obstacle in the clinicalchemotherapy. Many factors could engender the MDR. Among them, hypoxia, a tumormicroenvironment, is a significant factor which induces the MDR in variety of cancers.Hypoxia is a ubiquitous phenomenon in solid tumors, many factors are involved in thehypoxia-induced MDR, but they still fail to reverse the phenomenon of MDR. The mainreason is the molecular mechanism has not yet clear, So, looking for the moleculesinvolved in the hypoxia-induced MDR and elucidating the molecular mechanism willopen a new way for cancer treatment. We early screened the gastric cancer cells functionthrough cellomics and obtained the KLF8which is a gastric cancer related new factor.Literature suggests that KLF8is high expression in renal clear cell carcinoma with highlevel of HIF-1. So whether KLF8is a key molecule between hypoxia and drug resistanceis the focus of our research. So it is important to illuminate the function and mechanism ofKLF8involved in hypoxia-induced MDR. It is helpful to understand the molecularmechanism of hypoxia-induced MDR in gastric cancer cells.Aims: To research the KLF8expression level in hypoxia conditions and the toleranceof gastric cancer cells exposed to the variety of chemotherapy drugs; Constructing the gastric cancer subline with KLF8high level or low level to find the effect of KLF8involving the hypoxia-induced MDR; To further exploit the regulation function andmechanism between KLF8and drug resistance related genes.Methods:1. MTT、Annexin V/PI、Adriamycin accumulation and retention assayswere used to detect the tolerance of gastric cancer cells exposed to chemotherapy drugs innormoxia and hypoxia;2. The KLF8level was also detected by western blot and RT-PCRassays in gastric cancer cell lines MKN28、MKN45and SGC7901under normoxic andhypoxic conditions;3. The KLF8level was also detected in the gastric cancerdrug-resistant cell lines SGC7901/VCR、 SGC7901/ADR and the parental cell lineSGC7901;4. Exogenous KLF8and KLF8-siRNA were transfected into the experimentalcells, and the KLF8level were detected by western blot and RT-PCR assays;5. MTT、Annexin V/PI、Adriamycin accumulation and retention assays were used to detect thefunction of KLF8involving in the hypoxia-induced phenotype of MDR in gastric cancercells;6. The protein levels of P-gp、Bcl-2and Bax were detected by western blot assay inexperimental cells under normoxic and hypoxic conditions;7. Bioinformatics predictedthe transcription regulatory sites of MDR1, a drug resistance related gene, dual luciferasereport gene assay were used to detect the transcriptional regulation mechanism betweenKLF8and MDR1.Results:1. Hypoxia could enhance the tolerance of gastric cancer cell exposed inchemotherapy drugs, reduce the chemotherapy drugs-induced apoptosis and increase thedrug releasing rate;2. Hypoxia could effectively increase the KLF8expression inMKN28、MKN45and SGC7901gastric cancer cells;3. The levels of KLF8in thedrug-resistant cell lines were higher than in the parental cell lines, the KLF8level inSGC7901/VCR was the most obviously increased;4. Exogenous KLF8could increase theKLF8level observably in gastric cancer cells, and the KLF8-siRNA could reduce theKLF8level effectively in gastric cancer cells in normoxia and hypoxia;5. In normoxia,exogenous KLF8could increase the tolerance of gastric cancer cells in the chemotherapydrugs, reduce hypoxia-induced apoptotic index and increase the adriamycin releasing rate.KLF8-siRNA could reverse the phenomenon in gastric cancer cell under hypoxic condition;6. Exogenous KLF8increase the Bcl-2and P-gp protein levels and decrease theBax protein level, knockout of KLF8reverse the above results in gastric cancer cells;7.There were two KLF8binding sites (CACCC box) in the upstream nearby170bp of theresistance-associated gene MDR1transcription start site, KLF8could mediate thehypoxia-induced resistant phenotype via combining with the CACCCbox and promotingthe MDR1transcriptionConclusions:1. Hypoxia enhance the tolerance of gastric cancer cell inchemotherapy drugs;2. Hypoxia increase the KLF8level observably;3. KLF8participated in the hypoxia-induced MDR in gastric cancer cells;4. KLF8inhibitedhypoxia-induced apoptosis via increasing Bcl-2/Bax ratio, and it augment the drugreleasing index by promoting P-gp expression;5. KLF8regulate the transcription ofresistance-associated gene MDR1directly, further clarify the molecular mechanism ofKLF8involved in MDR in gastric cancer cells.