Study on the proapoptotic effect of CD40L in apoptosis of Fas-resistant breast cancer cells induced by CD~4+CIKs

Objective:To study the proapoptotic effect of CD40/CD40L cross-linking induced by CD4+CIKs on Fas-resistant breast cancer cells and the underlying mechanisms.Methods:After large scale of amplification of CIKs in vitro,the subset of CD4+CIK cells was isolated by magnetic bead separation columns.The typical Fas-mediated apoptosis of MDA-MB-231 cells induced by anti-Fas agonist CH11 was measured at pretreatmunt with or without supernatants of CD4+CIKs.Apoptotic rates of MDA-MB-231 cells cocultured with CD4+CIK for 24 hours at different ratio of E/T were evaluated by AnnexinV staining and expression of Fas was compared by flowcytometry as well.Then the antibodies against FasL,CD40L,IFNγ were added in the coculture,respectively.Apoptotic rates and the expression of Fas on MDA-MB-231 cells were detected.The mRNA expression of Bax、Bcl-2、FADD、c-FLIP in MDA-MB-231 cells after cocultured with CD4+CIK for ether 6 h or 24 h was analyzed by real-time quantitative RT-PCR method,and the antibody blocking experiment was studied.Results:The susceptibility to Fas-mediated apoptosis of insensitive MDA-MB-231 cells increased after being pretreated with supernatants of CD4+CIK.After co-culturing with CD4+CIK,the apoptotic rates of MDA-MB-231 cells increased,which significantly correlated with the enhanced expression of membrane Fas(r=0.618,P0.01).The elevated apoptotic rates were blocked fully by either anti-FasL or anti-CD40L,and partly by anti-IFN-γ antibodies,which decreased from(33.70±2.77)% to(7.57±1.15)%,(7.80±0.26)% and(14.21±1.70)%.However the Fas expression on MDA-MB-231 cells inhibited equally by either anti-CD40L or anti-IFN-γ,which dropped from(25.90±2.45)% to(6.93±1.56)% and(8.73±4.70)%.The antibody blocking experiment showed that anti-CD40L rather than anti-IFN-γ antibody induced significant increase of c-FLIP during 6 h after coculturing with CD4+CIKs,which was significantly negatively correlated with the apoptosis induced in MDA-MB-231 cells(r=0.898,P0.05).Conclusion:Both CD40L and IFN-γ participate the Fas-dependent apoptosis of MDA-MB-231 cells induced by CD4+CIK.However,reversion of Fas-resistance is mediated mainly through CD40/CD40L ligation rather than IFN-γ stimulation by inhibiting synthesis of c-FLIP.