PDF file - 1659KB, Apoptosis and autophagy-associated reaction by BGT226. (A) Apoptotic effect of BGT226. HSC3 cells incubated with either BGT226 or cisplatin for 24 h were analyzed for apoptosis-associated protein expression. Arrows indicated the cleavage-form of protein. (B) Effect of AVOs formation in FaDu cells treated with BGT226. Cells were stained with acridine orange. The extent of staining intensity in tested cells was analyzed and plotted by flow cytometry. The left rows represented cells incubated in DMSO or 3MA. The right row showed cells that were treated with BGT226 120 nM with or without the co-treatment of 3MA. The percentage of detected cells in each quadrant is marked on the panel. (C) Evaluation of cathepsin B/D/L (Cat B/D/L) activity in FaDu cells treated with BGT226. The activity of each factor was assessed by its individual activity kit (BioVisiion, CA). For the preparation, we followed manifacturer's protocol after BGT226 treatment of the cells. The samples were then transferred to 96-well plate, with the result read using fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. The data was then compared to the control, with the ratio ploted and shown in the bar chart.
ARTICLE ABSTRACT
Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently accounts for the tumorigenesis in head and neck cancer. To develop a new treatment, we investigated the effect of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in head and neck cancer cells.Experimental Design: The in vitro antitumor effect of BGT226 was determined in various cancer cell lines. Animal models were also applied to examine drug potency. The inhibitory ability of BGT226 on the PI3K/AKT/mTOR signaling pathway was analyzed.Results: The growth inhibition assay revealed that BGT226 was active against all tested cancer cell lines. Cross-resistance was not observed in the cisplatin-resistant cell line. The activation of the AKT/mTOR signal cascade was suppressed by BGT226 in a concentration- and time-dependent manner. Flow cytometric analysis revealed an accumulation of cells in the G0–G1 phase with concomitant loss in the S-phase. Results of the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay and the analysis of caspase 3/7 and PARP indicated that BGT226 induced cancer cell death through an apoptosis-independent pathway. BGT226 induced autophagy as indicated by the aggregation and upregulation of the microtubule-associated protein light chain 3B-II, and p62 degradation. Gene silencing of Beclin1 or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited the BGT226-induced autophagy and led to the retrieval of colony survival. In a xenografted animal model, BGT226 significantly delayed tumor growth in a dose-dependent manner, along with suppressed cytoplasmic expression of p-p70 S6 kinase and the presence of autophagosome formation.Conclusions: These data indicate that BGT226 is a potential drug in the treatment of head and neck cancer. Clin Cancer Res; 17(22); 7116–26. ©2011 AACR.
