The Effects And Mechanisms Of FABP4 In Regulating The Proliferation,Metastasis And Radiosensitivity Of Pancreatic Cancer Cells

Objective:To investigate the biological roles and related mechanisms of fatty acid binding protein（FABP）4 in regulating the proliferation,metastasis and radiosensitivity of human pancreatic cancer cells.Methods:Human pancreatic cancer cell lines PANC1 and Patu8988 were respectively co-cultured with human adipose tissues.The real-time fluorescent quantitative PCR method was applied to detect the mRNA expression of FABP1 to 7before and after the adipose/cancer cell co-culture.The recombinant FABP4 adenovirus system was utilized to obtain FABP4-overexpressed human pancreatic cancer cell modles.The specific FABP4 inhibitor BMS309403 was used to block the function of fatty acid binding of FABP4 in human pancreatic cancer cells,and the optimum concentration of BMS309403 was identified by CCK8 viability assay.The fluorescent dye Bodipy staining was used to determine the effect of FABP4 on cellular adipose synthesis in human pancreatic cancer cells.The proliferation of cancer cells was tested by EdU（5-Ethynyl-2’-deoxyuridine）assay,while the migration and invasion of pancreatic cancer cells were determined by wound healing assay and transwell assays respectively.The in vivo pancreatic cancer metastasis mice model was constructed through caudal vein injection in nude mice,and the small animal imaging system was used to observe the in vivo metastasis of pancreatic cancer cells.Besides,the clonogenic assay was performed to identify the influences of FABP4on the radiosensitivity of pancreatic cancer cells.The flow cytometry assay was used to analyze the effects of FABP4 on cell cycle distribution and cell apoptosis of pancreatic cancer cells after exposed to X-ray irradiation.JC-1 assay was used to explore the changes of mitochondrial membrane potential of pancreatic cancer cells.Immunofluorescence staining of phosphorylatedγ-H2AX foci was utilized to detect the effects of FABP4 on DNA double breaks formation,and the expression of DNA double strand damage repair related proteins（ATM、p-ATM、p-NBSI、Rad50、Mre11）was determined by Western blot assay after X-ray irradiation.Results:Out of 7 tested family members,mRNA expression level of FABP4increased significantly in adipose co-cultured pancreatic cancer cells compared with control group.Neither FABP4 overpression nor functional inhibition could effect the proliferation of the pancreatic cancer cells.Both of FABP4 overpression and adipose co-culture could promote the intracellular adipose accumulation,in vitro migration and invasion of human pancreatic cancer cells.In another hand,after treated with FABP4inhibitor BMS309403,the intracellular adipose accumulation and metastasis of pancreatic cancer cells were significantly inhibited.In vivo animal experiment confirmed that FABP4-overexpression facilitated pancreatic cancer cells transferring to the lung,liver,kidney as compared with control group;the adipose co-cultured cancer cells preferred to migrate to lungs,while BMS309403 treatment almost blocked this kind of metastasis.After exposed to 0,2,4,and 6 Gy of X-rays,the survival fraction of FABP4-overexpressed pancreatic cancer cells significantly increased represented as the increased D₀ value（from 3.669Gy to 4.913Gy）,Dq（from 1.788Gy to 2.484Gy）,as compared with control group.And the sensitivity enhancement ratio（SER）was 0.746.After 4 Gy irradiation for 24 hours,the G2/M cells population was significantly reduced in FABP4-overexpressed pancreatic cancer cells,and apoptotic cells and the mitochondrial membrane potential were both decreased significantly,as compared with the control group.The nuclear phosphorylation ofγ-H2AX in pancreatic cancer cells could be inhibited by either FABP4 overexpression or adipose co-cultured;the protein expression of DNA repair protein such as ATM,phosphorylated ATM and MRN protein etc.in pancreatic cancer cells were significantly decreased,as compared to control cells.And vice versa.Conclusions:FABP4 might affect not the proliferation,but the metastasis of human pancreatic cancer cells both in vitro and in vivo;FABP4 could also influence the radiosensitivity of pancreatic cancer cells.The potential mechanisms might be related to the regulatory roles of FABP4 in regulating the cell cycle,apoptosis,cell energy metabolism and DNA damage repair of pancreatic cancer cells.