Objective: Resistance to histone deacetylase inhibitors in non-small cell lung cancer is mediated in part through activation of nuclear factor-kappaB through a phosphatidylinositol 3-kinase/Akt-dependent pathway. We hypothesize that inhibition of phosphatidylinositol 3-kinase/Akt will sensitize non-small cell lung cancer cells to histone deacetylase inhibitor-induced apoptosis.
Methods: Tumorigenic non-small cell lung cancer cell lines H157, H358, H460, and A549 were treated with nothing, the histone deacetylase inhibitor butyrate, the phosphatidylinositol 3-kinase/Akt inhibitor LY294002, or both compounds. Nuclear factor-kappaB activity was assessed by reporter gene assays and reverse transcriptase-polymerase chain reaction of the nuclear factor-kappaB dependent genes cIAP-2, Bfl/A1, and MnSOD. Whole cell extracts were immunoblotted for phospho-Akt, Akt, and phospho-ser/thr-Akt substrate. Cell death and apoptosis were measured by crystal violet staining, caspase-3 activity, and DNA fragmentation. A549 non-small cell lung cancer xenografts were created in athymic nude mice, and tumor growth was assessed after treatments as noted above. Explanted tumors underwent terminal deoxynucleotide transferase-mediated dUTP nick-end labeling and Western blot analyses for apoptosis assessment and drug target validation, respectively.
Results: Butyrate activated nuclear factor-kappaB-dependent transcription, and LY294002 abrogated this effect. Combined treatment induced more apoptosis and cell death in vitro compared with either drug alone as measured by caspase-3, DNA fragmentation, and clonogenic survival. Combined butyrate and LY294002 was tumoristatic in vivo, but all other xenografts grew. This decreased tumor growth correlated with more apoptosis in the xenografts treated with combined therapy. Tumor levels of phospho-Akt and acetylated histone H3 were decreased and increased, respectively, in xenografts treated with combined therapy.
Conclusions: Combined histone deacetylase inhibitor and phosphatidylinositol 3-kinase/Akt pathway inhibition sensitized non-small cell lung cancer xenografts to apoptosis. Further investigations of this combined therapy are warranted as new pharmacologic phosphatidylinositol 3-kinase/Akt pathway inhibitors are developed.