Experimental Research Of Induction Of Apoptosis By Curcumin And Its Mechanism In Hep2 Cell Line In Vitro

Part ? Experimental research of the mechanism of TRAIL signal pathway in apoptosis of hep2 cells induced by CurcuminBackground and Objective: Curcumin could lead cancer cells to apoptosis has been tested already.TRAIL signal pathway can induce apoptosis of tumour cells as well as do no harmful to normal organizes and cells as a exogenous way of apoptosis.The two have synergistic effect or not is unclear though their effects are similar to each other.Method: We detected the expression level of TRAIL through RT-PCR;The expression of death receptor DR4,decoy receptor Dc R1 and Dc R2 on hep2 cells after the effect of Curcumin were detected by western blot assays;the apoptosis of TRAIL effect on hep2 cells were analyzed by flow cytometry.Result: TRAIL have a steady expression level in hep2 cells;the apoptosis of hep2 cells increased gradually,but the expression level of death receptor DR4,decoy receptor Dc R1 and Dc R2 have no abvious change;there is no abvious apoptosis occur after 12h?24h?48h ' stimulate of 50nmol/L TRAIL both observed by microscope and analyzed by flow cytometry.Conclusion: Our results suggest that hep2 cells are TRAIL-resistantce cells,.The expression of TRAIL receptors have no abvious change while hep2 cells could be induced to apoptosis by Curcumin,indicated that Curcumin induces apoptosis through some other pathways.Part ? Relationship between mt DNA Defect and Apoptosis in Human Epithelial Type 2 CellsBackground and purpose: To establish the ?0 cells of laryngeal squamous cell carcinoma(LSCC)human epithelial type 2 cells and investigate the relationship between Mitochondrial DNA(mt DNA)defect and apoptosis.Method: Cells deficient mt DNA(?0 cells)were aquired from human epithelial type 2 cells(hep2 cells)through continuous passage culture in DMEM supplemented with 50ng/ml EB,100ug/ml pyruvate and 50ug/ml uridine;mt DNA copies of the two cell lines were detected by real-time PCR after treated by EB for a month;apoptosis induced by Curcumin were analyzed using flow cytometry at different time(4th,8th and 12 th day)after treated by EB.Result: The ?0 cells of hep2 were successfully established.The results detected by real-time PCR indicated there is no mt DNA expression in hep2 cells after treated by EB for a month.The results analyzed by flow cytometry indicated that apoptosis induced by Curcumin was decreased gradually with the lesion of mt DNA as cell division.Conclusion: Our results suggest that mt DNA may participate in the inducement of apoptosis in laryngeal squamous cell carcinoma lines of human epithelial type 2 cells.Part ? The Research on Curcumin Induces Apoptosis through the injure of ROS in hep2 cellsBackground and purpose: Many species of medicine studied for carcinoma as the deeper explore in carcinoma,the majority of carcinomas cannot be cured effectively yet.Our research aimed at exploring the mechanisms of Curcumin effects on carcinoma,we cultured laryngeal squamous cell carcinoma(LSCC)human epithelial type2 cells for research,hope to provide effectively bases for clinical treatment.Method: Established the ?0 Cells of hep2;the expression of mt DNA in ?0-hep2 cells were detected by real-time PCR;the outputs of ROS in ?0-hep2 cells and hep2 cells after effected by Curcumin were analyzed by fluorescence microplate;we choosed JC-1 as the probe to detect the mitochondrial membrane potential(??m)on ?0-hep2 Cells and hep2 cells after effected by Curcumin;we used ultraviolet spectrophotometer to detect the mitochondrial permeability transition pore(MPTP)on ?0-hep2 Cells and hep2 Cells after effected by Curcumin;the location of Cytochrome C were detected by Western blot assays;the activity of caspase 9 in ?0-hep2 Cells and hep2 cells after effected by Curcumin were detected by microplate reader;the rates of apoptosis were analyzed by flow cytometry.Result:cells deficient mt DNA(?0 cells)can resistant to apoptosis induced by Curcumin to some extent;the ?0-hep2 cells product less ROS after effect of Curcumin;the tests of mitochondrial permeability transition pore(MPTP),mitochondrial membrane potential(??m),location of Cyto C,activity of caspase9 were proved that apoptosis occurred by the effect of Curcumin.Conclusion: Our results suggest that Curcumin induces apoptosis through the injure of ROS.