One of the most lethal cancers is non-small cell lung carcinoma (NSCLC), which is resistant to chemotherapy- and irradiation-induced programmed cell death or apoptosis. Our objective is to define processes in NSCLC opposing apoptosis. Previously, we showed IFNγ potently inhibits proliferation of human NSCLC A549 cells under optimal growth conditions by a process involving transcription factor STAT1. Here we investigated whether IFNγ made A549 cells susceptible to activation of the death receptor FAS.
A549 cells grown with 10% serum were treated as follows: vehicle, 10 ng/ml IFNγ, 70 ng/ml activating FAS antibody, or 10 ng/ml IFNγ and 70 ng/ml FAS antibody. After 48 h, apoptosis was measured by DNA laddering, annexin V binding, and Western immunoblotting for PARP and caspase 3 cleavage. FAS was measured by Westerns, protein by BioRad DC assay. Cells were transfected with siRNA STAT1 using lipofectamine. Statistical significance (P < 0.05) was determined by ANOVA.
IFNγ did not induce apoptosis of A549 cells as indexed by PARP or caspase 3 cleavage, DNA laddering, or annexin V binding. Neither did the agonistic anti-FAS antibody. However, together, they induced marked apoptosis as indexed by all 4 assays and loss of protein from culture dishes. The permissive effect of IFNγ on FAS-induced apoptosis was not due to upregulation of FAS, as A549 cells expressed FAS and its expression was not affected by IFNγ. To assess the role of STAT1, cells were transfected with STAT1 siRNA or lipofectamine alone (control) prior to treatment. Although STAT1 expression was downregulated with siRNA, PARP and caspase 3 cleavage were still observed with the combination of IFNγ and FAS antibody.
IFNγ plays a permissive role in FAS-mediated apoptosis of NSCLC A549 cells by a process downstream of FAS and independent of STAT1. Further studies are underway to determine which STAT1-independent mechanism sensitizes A549 cells to apoptosis.
The combination of IFNγ and activating FAS antibody could be a novel therapeutic strategy in the treatment of non-small cell lung cancer.
Christopher Spradley, Product/procedure echnique that is considered research and is NOT yet approved for any purpose. The combination of IFNγ and activating FAS antibody in the treatment of non-small cell lung cancer.