The Construction Of The Expression Vector Of Transmembrane TNF α Containing The PPRE Promotor And Its Synergistic Effect With ω-3PUFA On Tumor Cells

ω-3 polyunsaturated fatty acid (ω -3PUFA) is a group of very important lipids mainly from the cold-water fish. Clinical trials have reported they can decrease the concentration of TAG and VLDL in plasma, can induce HDL production and inhibit TAX2 production, and are beneficial for the prevention and treatment of cardiovascular diseases. Epidemiological and experimental evidences have shown that ω -3PUFA exerts protective effects against diverse types of tumor cells. Multiple mechanisms are involved in these chemopreventive and chemotherapeutic activities, including cell growth inhibition, differentiation and apoptosis induction. The nuclear transcription factor peroxisome proliferator-activated receptors (PPARs) are special ω -3PUFA nuclear receptors, and the transcriptional activation of them correlates with lipid homeostasis and cell proliferation through regulation of special gene expression with upstream peroxisome proliferator responsive element (PPRE).Transmembrane tumor necrosis factor a (tmTNF a ),an important cytokine with various biological functions, is produced by various types of immune cells. Many studies have reported tmTNF a acts chiefly through cell-to-cell contact; it induces tumor cell apoptosis directly and indirectly via different pathways and has a broader oncolytic spectrum than secreted TNF a (sTNF a ). But the exact mechanisms by which tmTNF a influences tumor cells are not well known.Many researches and clinical trials have already suggested that a conditional expression system of exogenous gene induced by hormones or drugs maybe more effective and special in gene therapy. In order to provide the theoretical support for further research and clinical gene therapy of tumor, in this paper, we constructed a tmTNF a expression vector induced by ω -3 PUFA and studied the synergistic effects of ω-3PUFA and tmTNF a on the proliferation and apoptosis of tumor cells and their possible molecular mechanisms.Firstly, we constructed an eGFP report vector containing a PPRE-tk promotor and found that the expression of eGFP in transfecant cells was up-regulated induced byω-3PUFA in a dose and time-dependent way.Further, tmTNFa eukaryotic expression vector containing the same promoter was constructed and transfected into the human leukemia HL-60 cells and human breast cancer MCF-7 cells. The expression of exogenous tmTNF a gene induced by 3PUFA was investigated by RT-PCR and immunofluorescence. The effects of 3PUFA and/or exogenous tmTNF a on cell proliferation, cell cycle distribution and the apoptotic rate were measured by growth curve, MTT test and flow cytometry, at the same time, the DNA ladder was detected. As it is well known that caspase network is the central role of the apoptotic machinery, the activity of caspase-8 was examined, as well as RT-PCR and western blotting were used to analyze the expression of caspases (1,3,8 and 9). In addition, to confirm the function of caspase-8 activation in 3PUFA and exogenous tmTNF a -induced apoptosis, Ac-IETD-CHO, an inhibitor of caspase-8 and caspase-3, was used when cells were treated with 3PUFA.The results and conclusions are summarized as follows:l.The pPPRE-tmTNFa expression vector containing a special PPRE-tk promoter was successfully constructed by recombination DNA technology and then transfected into the MCF-7 and HL-60 cells. The production of exogenous tmTNF a gene was located in the cell membrane. After treatment with 3PUFA, the expression of exogenous tmTNF a in transfecant was significantly increased in a dose and time-dependent way,too. These results suggested that conditional expression system of tmTNF a induced by 3PUFA was successfully constructed.2.In HL-60 cells treated with 6.0 10mol/Lw-3PUFA, the proliferation capacity was decreased, the apoptotic rate was increased and cell cycle progression was arrested at the G0/G1 phase. These changes were more striking in transfected HL-60 cells whereas in -3PUFA-treated MCF-7 cells only the growth suppression was found. In transfected MCF-7 cells after treatment with 3PUFA, not o...