Investigation Of Cardamonin Based On The Target Of MTOR

ObjectiveTo investigate the proliferation and expression of 70 kDa ribosomal protein s6 kinase (p70S6K1) and interleukin-2 (IL-2) in human adenocarcinoma cell cancer A549 after tansfection with mammalian target of rapamycin (mTOR) gene and FK506 binding protein 12 (FKBP12) gene. To explore possible mechanism of antiproliferation effect of cardamonin (CAR) on A549 cells and A549 transfection cells.Methods1 Construction of eukaryotic expression vectorsHuman FKBP12 gene was amplified by RT-PCR method from human smooth muscle cells. Eukaryotic expression vector pcDNA3.1/Hygro(+) with products of PCR was constructed. Eukaryotic expression plasmid of the pcDNA3.0-mTOR was provided by Professor Shou Weinian (University of Indiana, America).2 Identification of recombinant plasmidsThe plasmids of recombinant pcDNA3.1/Hygro(+)-FKBP12 and pcDNA3.0-mTOR were transformed into competent cells (DH-a Escherichia coli). DNA was extracted with the alkaline lysis. The recombinant plasmids were determined by PCR, digestion of restriction enzymes and sequencing analysis.3 Transfected stably cell linesA549 cells were transfected with pcDNA3.1/Hygro (+)-FKBP12 and pcDNA3.0-mTOR by Lipofectamine 2000 reagent, respectively. The clones of positive cells were selected with antibiotics (hygromycin B and G418). The protein expressions of FKBP12, mTOR, IL-2, P-p70S6K1 were determined by Western blotting method. The survival rates of different cells were assayed by MTT (methyl thiazolyl tetrazolium) method.4 Certification of CAR mechanismTransfected cells and non-transfected cells were divided into the following groups:blank control group (CG), solvent control group (SG), RAP group (RAPG,10-7 mol·L-1), CAR1 group (CARG1,3×10-5 mol·L-1), CAR2 group (CARG2,10-6 mol·L-1). MTT assay was performed to examine the proliferation of A549 cells. The protein expressions of P-mTOR, P-p70S6K1, FKBP12 and IL-2 were determined by Western blotting analysis, respectively.Results1 Construction and identification of recombinant plasmidsThe concentration of pcDNA3.1/Hygro(+)-FKBP12 and pcDNA3.0-mTOR plasmids extracted from DH5αwere 379 ng·μL-1 (OD260/OD280=1.93) and 306 ng·μL-1 (OD260/OD280= 1.89). The recombinant plasmids were identified by PCR, double digestion with enzymes and nucleic acid sequencing. It is comformed that the transfected plasmids were correct.2 Establishment of stably transfected cell lines(1) The clones of the positive cells were selected with hygromycin B (250μg·mL-1) and G418 (300μg·mL-1). Clones of A549-FKBP12 cell line and A549-mTOR cell line were transfected stably and successfully.(2) Protein expressions of FKBP12 and IL-2 were increased significantly in the transfected cells with FKBP12 gene (P<0.01, n=3). Similaritly, protein expressions of P-mTOR and P-p70S6K1 increased significantly in transfected cells with mTOR gene (P<0.01, n=3).(3) The velocity of cells growth and proliferation was obviously increased in the transfected cell lines.3 Certification of mechanism of cardamomin(1) Effects of CAR on proliferation of three cell lines(1) The inhibitory effect of cell proliferation of RAP in transfected cell lines (FKBP12 gene and mTOR gene) was stronger than that of A549 cell lines (0.503±0.023 vs 0.426±0.056,0.465±0.032 vs 0.426±0.056, P<0.01, respectively).(2) Antiproliferation of CAR on transfected (mTOR gene) cells was higher than that of A549 cells (0.456±0.023 vs 0.363±0.054,0.316±0.028 vs 0.266±0.512, P<0.01, respectively), however, antiproliferation of CAR on transfected (FKBP12 gene) cells was not affected comparison with A549 cells.(2) Probable mechanism of CAR on antiproliferation①Protein expressions of P-mTOR, P-p70S6K1, FKBP12 and IL-2 were decreased by RAP in transfected cells and non-transfected cells, respectively.②Protein expressions of P-mTOR and P-p70S6K1 were decreased and that of FKBP12 and IL-2 were not affected by CAR in the transfected cells.Conclusion1 Cell proliferation of A549 was obviously increased in vitro by transfection of mTOR gene and FKBP12 gene, respectively.2 Cell antiproliferation of A549 by CAR and RAP was involved in mTOR-p70S6Kl signal pathway.3 The mechanism of CAR on antiproliferation is directly associated with target of mTOR, it may not depend on the activation of FKBP12.