Studies On The Specific Antagonists Of Tumor Anti-Apoptotic Factor C-FLIP And Anti-Tumor Effect Of Panax Notoginseng Saponins(PNS)

Part one Studies on the Specific Antagonists of Tumor Anti-apoptotic Factor c-FLIPTumor necrosis factor-related apoptosis-inducing ligand TRAIL can selectively induce apoptosis of transformed cells,tumor cells and virus-infected cells without apparent toxic effects on normal cells.Since it was discovered,TRAIL has been a focus of intensive research.However,from recent studies,we have found that many tumor cells are resistant to TRAIL-induced apoptosis due to the high levels expression of anti-apoptotic proteins such as IAPs,Bcl-2 and c-FLIP.Currently,many inhibitors of IAP and Bcl-2 have been discovered,and many of them have entered into phase Ⅰ,Ⅱ clinical trials.However,there is little coverage about c-FLIPs as a target of cancer therapy except the RNA interference technique.The c-FLIP specific inhibitors or antagonists suitable for clinical study have not been reported yet.The main purpose of my works is to find c-FLIP specific antagonists as a therapy for cancer treatment,or in combination with other anticancer drugs to enhance the sensitivity of tumor cells to apoptosis to finally achieve the purpose of killing tumor cells.In summary,we believe that there is such a small molecule with a conformation mimic to a part of DED of FADD,thus can combine with the c-FLIP but not procaspase-8/10.The DED of FADD is composed of six a-helixs.These six spirals peptides were synthesized and separately coupled to activated sepharose to generate six affinity coliumns with six different helix peptides.Affinity chromatography technique was used to screen out the helix that has the highest affinity to c-FLIP.Conclusion:1.c-FLIPS-pET28a(+)prokaryotic expression vector was contracted successfully;2.The optimum conditions of inducible expression are 4h,0.6mM IPTG,32℃;3.The peptide "SMLLEQ" in the Helix4 of FADD’DED has a strong affinity with c-FLIP which was detected by the affinity screening system in vitro.Meanwhile,we detected the similar results for fluorescence polarization(FP);4.In the detection of small peptides on cell viability effect of experiments,We found three peptides,including AC-SMLLEQ-NH2、AC-SMLLE-NH2、AC-MLLEQ-NH2,potentiated apoptotic effects of TRAIL plus AT406 in cells pretreated with pH6.5 DMEM briefly.In the future study,we will continue to modify these peptides and to improve the cell penetration method such as electroporation to obtain better apoptotic effects.Part two Studies on Anti-tumor Effects of Panax Notoginseng Saponins(PNS)Panax Notoginseng is a traditional Chinese herb medicine,its main components are Panax notoginseng saponins(PNS),dencichine,flavonoids,volatile oil,amino acids,carbohydrates and so on.Because of its positive clinical efficacy,it has received an extensive attention.Recently,it is claimed to plays a major role in many systems,such as cardiovascular system,nervous system,immune system,blood system and anti-inflammatory,anti-tumor,anti-aging and so on.One of important components is PNS,it promotes blood flow and resolves blood stasis.In recent years,there are a few reports on the anti-tumor effects of PNS,but just few researchers in domestic or abroad focus on its specific anti-tumor activity.Therefore,it is an important for clinical treatment to find out the anti-tumor effect of PNS.This assay mainly study the PNS effects on survival rates of normal cells(pulp cells,Pulp),tumor cells(including glioma cells U87,U373,U251,osteosarcoma cell U2OS,cervical cancer cell Hela,hepatoma cell HEPG2,non-small cell lung cancer cell A549,colon cancer cell HT29,ovarian cancer SKOV-3)by MTT assay.And Western Blot analysises the content of apoptosis-related proteins and finally understands the underlined molecular mechanism.Conclusion:1.From the results of MTT assay,we found PNS had strong effectson cell death for most of tumor cells(especially for glioma cells),but less for normal cells;2.Results of Western Blot showed PNS had large reduction for glioma cell viability,this process maybe relate to autophagy,rather than apoptosis.However,the specific mechanism is still not clearly elucidated.In the future study,we will examined the relationship among the downregulation with necrosis,proliferation by flow cytometry or other methods.Finally,we can clarify the mechanism for the reduction of cell viability produced by PNS.