The Expression Of TRAIL And Its Receptors In Osteosarcoma Cells And The Apoptosis Effect Of A Combination Of TRAIL, Adriamycin And IFN-γ On MG-63 Cells In Vitro

PARTⅠExperimental Study on Apoptosis-inducing Effect ofTRAIL,Adriamycin,Cisplatin and IFN-γrespectively onMG-63 cellsOBJECTIVE To investigate the synergistic induction of cytoxicity and apoptosis bytumor necrosis factor related apoptosis inducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γrespectively on osteosarcoma cell line MG-63.METHODS Methyl thiazolyl tetrazolium(MTT) assays were used to evaluate thecytotoxic effect induced by TRAIL,Adriamycin,Cisplatin and IFN-γrespectively onosteosarcoma cell line MG-63,Flow cytometric analyses were used for the assessment ofapoptosis rates after different TRAIL,Adriamycin,Cisplatin and IFN-γrespectivelytreating osteosarcoma cell line MG-63.DNA gel electrophoresis was used for theassessment of apoptosis rates after TRAIL treating osteosarcoma cell line MG-63.RESULTS a) ALL concentrations of TRAIL had different degree effect onosteosarcoma cell line MG-63.When the concentration was under 1000ng/ml,TRAIL did not show statistically significant effect on osteosarcoma cell line MG-63,with theconcentration increasing,the correlation among groups which have different concentrationunder the same stimulating periods had statistically significant(P＜0.05);b) adriamycin(ADM) could kill osteosarcoma cell line MG-63,which show a dose-dependent cytotoxiceffect relationship between drug and cells.When the concentration was under 4.3μl/ml,adriamycin did not show statistically significant effect on osteosarcoma cell lineMG-63,with the concentration increasing,the correlation among groups which havedifferent concentration under the same stimulating periods had statistically significant(P＜0.05);c) cisplatin(DDP) could kill osteosarcoma cell line MG-63,which show adose-dependent cytotoxic effect relationship between drug and cells.When theconcentration was under 1μl/ml,cisplatin did not show statistically significant effect onosteosarcoma cell line MG-63,with the concentration increasing,the correlation amonggroups which have different concentration under the same stimulating periods hadstatistically significant(P＜0.05);d) IFN-γshow statistically significant effect after treatingosteosarcoma cells MG-63 24 hours.CONCLUSION a) TRAIL show statistically significant effect on osteosarcoma cellsMG-63,when the concentration was higher than 1000ng/ml;b) adriamycin showstatistically significant effect on osteosarcoma cells MG-63,when the concentration washigher than 4.3μl/ml;c) cisplatin show statistically significant effect on osteosarcoma cellsMG-63,when the concentration was higher than 1μmol/L;d) IFN-γshow statisticallysignificant effect after treating osteosarcoma cells MG-63 24 hours.PARTⅡExperimental Study on Apoptosis-inducing Effect ofdifferent combinations of TRAIL,Adriamycin,Cisplatin andIFN-γon MG-63 cellsOBJECTIVE To investigate the synergistic induction of cytoxicity and apoptosis bythe different combinations of tumor necrosis factor related apoptosis inducing ligand (TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γin osteosarcoma cell lineMG-63METHODS Methyl thiazolyl tetrazolium(MTT) assays were used to evaluate thecytotoxic effect by the different combinations of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γonosteosarcoma cell line MG-63,Flow cytometric analyses were used for the assessment ofapoptosis rates after different combinations of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γtreatingosteosarcoma cell line MG-63.RESULTS a)The combination of tumor necrosis factor related apoptosis inducingligand(TRAIL),adriamycin(ADM),and IFN-γinduced significantly higher apoptosisand cytotoxicity than exposure to TRAIL,ADM or IFN-γalone(P＜0.05),but thecombination of two of the three agents did not show significantly synergistic effect onosteosarcoma cell line MG-63.b)The combination of tumor necrosis factor relatedapoptosis inducing ligand(TRAIL),cisplatin(DDP),and IFN-γinduced significantlyhigher apoptosis and cytotoxicity than exposure to TRAIL,DDP or IFN-γalone(P＜0.05),but the combination of two of the three agents did not show significantly synergistic effecton osteosarcoma cell line MG-63.CONCLUSION a)The combination of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),and IFN-γinduced significantly higherapoptosis and cytotoxicity than exposure to TRAIL,ADM or IFN-γalone;b)Thecombination of tumor necrosis factor related apoptosis inducing ligand(TRAIL),cisplatin(DDP),and IFN-γinduced significantly higher apoptosis and cytotoxicity thanexposure to TRAIL,DDP or IFN-γalone.PARTⅢThe expression of TRAIL and its receptors inosteosarcoma cells and the apoptosis effect of a combination of TRAIL,adriamycin and IFN-γon osteosarcoma cell lineMG-63OBJECTIVE Tumor necrosis factor related apoptosis-inducing ligand(TRAIL) is adeath receptor ligand that can induce apoptosis in a variety of cancer cell lines,but not allcancer cells.Past studies suggested that some chemotherapeutic drugs intensified thesensitivity of TRAIL induced apoptosis.We investigated the effect and potentialmechanism of combined TRAIL,adriamycin and IFN-γon apoptosis of humanosteosarcoma cell line MG-63 in vitro.Methods Paraffin embedded surgical specimens from 39 cases of human osteosarcomaare used to detect DR5 and YY1 proteins by immunohistochemical staining method.Expression of DR5 and Yin Yangl(YY1)mRNA after different combinations of TRAIL,Adriamycin and IFN-γwas examined by RT-PCR.Expression of DR5 and YinYangl(YY1)mRNA after different combinations of TRAIL,Adriamycin and IFN-γwasexamined by western blot.RESULT a) DR5 protein was detected in 53.8 %(21/39) cases in human osteosarcoma,YY1 protein was detected in 38.5%(15/39) cases in human osteosarcoma:nosignificant correlate expression of DR5 proteins and YY1 protein was found in humanosteosarcoma.No obvious correlations were found between the expression of DR5 andYY1 and the tumor staging,grading,major vascular involvement and lung metastasis inhuman osteosarcoma.B) The combination of the three treatments induced significantlyhigher apoptosis and cytotoxicity than exposure to TRAIL,ADM or IFN-γalone,but thecombination of two of the three agents did not show synergistic effect.Conclusions a) DR5 and YY1 down regulation seem to be an important event in tumorgenesis and progression in human osteosarcoma.In human osteosarcoma,none of thesetwo tumor inhibiting genes seems to play important role.b) There might be a synergisticalinteraction between paclitaxel in terms of cytotoxicity and its mechanisms might beinvolved in the up-regulation of DR5 gene and the down-regulation of YY1.