Abstract 4113: Combination of LC3 II shRNA and genistein completely inhibited rapamycin-induced autophagy and promoted apoptosis in human malignant neuroblastoma cell lines
Authors: Nishant Mohan, Besim Ogretmen, Naren L. Banik, Swapan K. Ray
Journal: Cancer Research (2012)

Abstract Neuroblastoma is an extracranial solid tumor that mainly occurs in children. It is derived from embryonic neural crest cells of the peripheral sympathetic nervous system. The overall survival rate of malignant neuroblastoma patients is very poor despite multi-modal therapy including surgery, radiotherapy, and chemotherapy. Efficacy of chemotherapy is often compromised due to presence of autophagy, which is a survival mechanism in solid tumors. Autophagy is a catablolic process for lysosomal degradation of cytoplasmic contents for recycling and it is activated during stress such as nutrient starvation and growth factor deprivation. The hallmark of autophagy is generation of double membrane structure called autophagosome that contains the microtubule-associated protein light chain 3 form II (LC3 II). In this study, we used rapamycin to mimic starvation-induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR32 cells and then investigated capability of the combination of LC3 II knockdown and genistein (GST) treatment for inhibition of autophagy and promotion of apoptosis. First, we treated both cell lines with 200 nM rapamycin for 6, 12, and 24 h and performed acridine orange (AO) staining, flow cytometric analysis, RT-PCR, and Western blotting to establish time-dependent increases in autophagic features such as formation of acidic vesicular organelles (AVO) and LC3 II. Then, we carried out dose-response studies by transfecting the rapamycin-exposed cell lines with various concentrations of LC3 II short hairpin RNA (shRNA) plasmid and treating them with several doses of GST alone and in combination. MTT assays showed the highest synergistic efficacy in inhibiting cell viability using the combination of 50 nM LC3 II shRNA and 25 µM GST in SK-N-BE2 cells while the combination of 100 nM LC3 II shRNA and 25 µM GST in IMR32 cells. Our AO staining and flow cytometric quantitation of AVO confirmed that combination of LC3 II shRNA and GST completely inhibited rapamycin-induced autophagy to promote apoptotic death. In situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy most effectively induced apoptosis in both cell lines when compared with control shRNA, LC3 II shRNA, or GST alone. We unraveled the molecular mechanisms for inhibition of autopphagy and induction of apoptosis by Western blotting of the proteins involved in these processes. Down regulation of LC3 II and upregulation of mTOR in both cell lines showed inhibition of autophagy. Combination therapy promoted mitochondrial pathway leading to activation of caspase-3 and degradation of PARP for inducing apoptosis. Collectively, our results clearly show that combination of LC3 II knockdown and GST treatment serves as promising therapeutic strategy for inhibition of autophagy and induction of apoptosis in different human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4113. doi:1538-7445.AM2012-4113