Abstract

Background: Apoptosis induced by chemotherapeutical agents can use different signaling pathways and can be suppressed by various mechanisms of resistance depending on individual tumor biology. Gemcitabine is used in palliative chemotherapeutical protocols in NSCLC therapy. Here we studied this drug in the human NSCLC cell line KNS62 with special regard to the CD95-induced apoptotic pathway and the role of Bcl-xL, an anti-apoptotic protein. Material and methods: Apoptosis in cell line KNS62 induced by gemcitabine or CD95-activating antibody CH11 was evaluated in JAM assay and Dapi-staining analysis. The impact of caspase inhibitor zVAD towards gemcitabine-induced apoptosis was quantified. In Bcl-xL over-expressing cells activation of caspase 3, and -8 by gemcitabine and CH11 was tested. For evaluation of the mitochondrial pathway of apoptosis, cleavage of BID, Caspase 9 as well as cytocrome-c delivery and mitochondrial transmembrane potential (MTP) were measured. In an orthotopic murine xenotransplant model tumor growth of KNS62-wt, -Bcl-xL, and -EGFP vector controls were evaluated after three weeks of i. p. gemcitabine therapy. Results: In JAM assays and Dapi staining analysis apoptosis induced by gemcitabine therapy as well as after CD95 activation by CH11 was confirmed. The cell death was found to be caspase dependent, since zVAD inhibited gemcitabine-triggered apoptosis. In KNS62-Bcl-xL clones, apoptosis after gemcitabine therapy as well as CD95-induced apoptosis was significantly inhibited. Interestingly, in vitro Bcl-xL mediated protection against apoptosis was significantly more effective in CD95 mediated than in gemcitabine induced apoptosis. Cleavage of caspase 8 was shown after CH11-, but almost not after gemcitabine application. Gemcitabine treatment, as well as stimulation of CD95, resulted in cleavage of effector-caspase 3 as well as its substrate PARP and caspase 9. Release of cytochrome C and loss of mitochondrial transmembrane potential were suppressed in Bcl-xL clones, in contrast to KNS62-wt variant and vector controls. In vivo, an orthotopic murine model of lung cancer confirmed a complete resistance of KNS62-Bcl-xL tumors against gemcitabine therapy. Conclusion: The current study reveals an activation of type II apoptosis by gemcitabine in NSCLC cell line KNS62. In contrast to CD95 induced apoptosis, activation of initiator caspases was not detectable upon gemcitabine treatment. Bcl-xL over-expression induced significant resistance against gemcitabine. The anti-apoptotic effect of Bcl-xL under gemcitabine treatment was higher in vivo than in vitro. The study may implicate further investigations on expression of pro- and anti-apoptotic factors in NSCLC tumors in correlation with their response on chemotherapeutical substances.