Suberoylanilide hydroxamic acid combined with gemcitabine enhances apoptosis in non-small cell lung cancer.

BACKGROUND

We have shown that non-small cell lung cancer (NSCLC) is resistant to the histone deacetylase inhibitor (HDI) suberoylanilide hydroxamic acid (SAHA) through upregulation of the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB). HDIs also promote chromatin remodeling, potentially making the DNA more accessible to chemotherapy. We hypothesize that combined SAHA and gemcitabine sensitizes NSCLC to apoptosis.

METHODS

Three NSCLC cell lines (A549, H358, H460) were untreated, or treated with SAHA, gemcitabine, or both agents. NF-kappaB-dependent transcription was determined by reporter gene assays, reverse transcriptase-polymerase chain reaction RT-PCR, and Western blot analysis for the NF-kappaB-regulated antiapoptotic gene MnSOD. Survival of NSCLC cells overexpressing Bfl/A1, Bcl-X(L), or MnSOD and treated with SAHA and gemcitabine was determined in the presence or absence of NF-kappaB. Survival of treated cells overexpressing HDAC-1, 2, 3 or p/CAF was determined. Apoptosis was determined by fluorescence-activated cell sorter analysis, DNA fragmentation, and caspase-3 activity. Colony formation assays were performed on cells treated concurrently and sequentially with SAHA and gemcitabine. Assays were performed in triplicate, and the Student t test was applied as appropriate.

RESULTS

SAHA-activated NF-kappaB (P <or= .05) and gemcitabine inhibited these effects (P <or= .01). Increased cell survival was observed after overexpression of antiapoptotic genes, as well as in cells overexpressing HDAC-1, -2, and -3. Fluorescence-activated cell sorter analysis, DNA fragmentation, and caspase-3 assays all showed enhanced apoptosis with combined therapy, compared with single-agent therapy (P <or= .01). Sequential treatment offered no improvement over concurrent treatment.

CONCLUSIONS

Combined SAHA and gemcitabine sensitized NSCLC cells to apoptosis. Potential "proapoptotic" mechanisms for this finding include gemcitabine inhibition of SAHA-induced NF-kappaB activation and chromatin remodeling mediated by the inhibition of histone deacetylases.