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F19. Inhibition of PI3K/Akt and histone deacetylase activity induces apoptosis in non-small cell lung cancer in vitro and in vivo
Brian K Rundall, Chadrick E. Denlinger, David R. Jones; Charlottesville, VA
OBJECTIVE: We have previously shown that resistance to histone deacetylase inhibitor (HDI) therapy in non-small cell lung cancer (NSCLC) is through upregulation of the pro-apoptotic transcription factor NF-κB. It has been suggested that HDIs activates NF-κB transcription through PI3K/Akt-dependent pathways. We hypothesize that small molecule inhibition of Akt followed by treatment with the HDI butyrate will sensitize NSCLC cells to apoptosis in vitro, as well as in an in vivo NSCLC xenograft model.
METHODS: The tumorigenic NSCLC cell lines A549, H358, and H157 were treated with nothing, butyrate, the PI3K/Akt inhibitor LY294002, or both compounds. Following treatment, NF-κB transcriptional activity was assessed by Gal4/p65 reporter gene assay as well as with RT-PCR of the NF-κB dependent genes cIAP-2, Bfl/A1 and MnSOD. GAPDH was used as a control. Cell survival was determined by crystal violet staining, and apoptosis by caspase-3 and DNA fragmentation assays. NHBE cells were used as a control for the above experiments. A549 NSCLC xenografts in athymic nude mice were created and allowed to reach a size of 0.5 cm3. Tumor-bearing mice (N = 5/group) were treated with nothing, butyrate (po), LY294002 (i.p.) or both compounds, and tumor volume was calculated every other day for 21 days. Tumors were harvested and analyzed by western blot for phospho-Akt, MnSOD, cIAP-2, and histone H3 and for apoptosis by TUNEL. ANOVA was used for statistical analysis when indicated.
RESULTS: Butyrate activated NF-κB transcription and the addition of LY294002 abrogated this effect (p < 0.0004). mRNA levels of the NF-κB regulated genes cIAP-2, Bfl/A1, and MnSOD were elevated following treatment with butyrate, and these levels normalized to basal with the addition of LY294002. Combined treatment induced significant cell death compared to either drug alone by crystal violet assay (p < 0.01) and by caspase-3 activation (p < 0.01). Xenografts treated with butyrate and LY294002 had significant regression of tumor size compared to either drug alone (p = 0.001). Additionally, there was more apoptosis on TUNEL analysis with the combined treatment than with either drug alone. There was evidence of target validation in that phospho-Akt levels were dramatically decreased and histone H3 was hyperacetylated in the xenografted tumors treated with butyrate and LY294002. Finally, MnSOD and cIAP-2 protein levels were dramatically reduced with the combined therapy.
CONCLUSIONS: Combined histone deacetylase and NF-κB inhibition via the PI3K/Akt pathway decreased levels of NF-κB-regulated anti-apoptotic genes and increased cell death in vitro, and perhaps more importantly in vivo. These results suggest that this combined biologic therapy warrants further investigation as a viable treatment strategy in NSCLC.
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