Background: Cytokine-induced killer cells (CIKs) are nonspecific antitumor effectors with superior advantages. CD4+ CIKs can induce Fas-dependent apoptosis in sensitive Raji cells. Here, a Fas-dependent apoptosis was detected in resistant breast cancer MDA-MB-231 cells, and underlying mechanisms were discriminated.
Methods: Amplification of CIKs and purification of CD4+ CIKs were performed in 15 patients with malignant solid tumors. The expression of CD40L and soluble cytokines in CD4+ CIKs were analyzed. The apoptotic rates of tumor cells and the expression of Fas on membranes were detected using flow cytometry assay. The specific blocking antibodies against FasL, CD40L, and interferon-gamma (IFN-gamma) were added to abolish their effects. The changes of 4 apoptosis-related genes (Bcl-2, Bax, Fas-associating protein with death domain [FADD], and FLICE inhibitory protein [c-FLIP]) in MDA-MB-231 cells cocultured with CD4+ CIKs were measured by real-time quantitative reverse-transcriptase polymerase chain reaction after 6 hours and 24 hours with or without blocking antibodies.
Results: Upregulated expression of membrane-attached CD40L and dramatically increased secretion of soluble CD40L and IFN-gamma were identified in CD4+ CIK. The susceptibility to Fas-mediated apoptosis of insensitive MDA-MB-231 cells was elevated after being pretreated with supernatants from CD4+ CIK. After coculture with CD4+ CIK, apoptosis in MDA-MB-231 cells paralleled with enhanced expression of Fas was blocked fully by either anti-FasL or anti-CD40L, but only partly by anti-IFN-gamma antibodies. The anti-CD40L monoclonal antibody (McAb) rather than anti-IFN-gamma McAb induced significant increase of c-FLIP, which negatively correlated with the apoptosis observed in MDA-MB-231 cells.
Conclusions: Apoptosis in MDA-MB-231 cells induced by CD4+ CIK is Fas-dependent. The reversion of Fas resistance is mediated through CD40/CD40L ligation rather than IFN-gamma stimulation by inhibiting synthesis of c-FLIP.