4-Hpr And Apigenin Together Suppressed Autophagy And Cell Survival Pathways To Increase Apoptosis In Human Malignant Neuroblastoma Sh-Sy5y Cells

Malignant neuroblastomas are extracranial tumors that affect mainly children. These tumors are not yet cured or successfully treated and thus associated with poor prognosis. In this study, we employed a unique combination of N-(4-hydroxyphenyl) retinamide (4-HPR) and apigenin (APG) to enhance cell death in human malignant neurobalstoma SH-SY5Y cells. Autophagy or ‘self eating’ is a genetically programmed cell death process and its manipulation may prevent cancer growth and enhance efficacy of chemotherapy. We found that combination of 4-HPR and APG inhibited key autophagy pathway (TLR-4/MyD88) and cell survival pathway (mTOR/Akt/NF-κB) leading to induction of apoptosis in SH-SY5Y cells. We assessed residual cell viability following treatment of the cells with various concentrations of 4-HPR and APG alone and in combination. MTT assay revealed that combination of 0.5 µM 4-HPR and 50 µM APG effectively decreased cell viability when compared with control and monotherapy groups. By using flow cytometry and Western blotting, we confirmed the expression of TLR-4 (Toll Like Receptor-4), a key inducer of autophagy, in SH-SY5Y cells. After 24 h treatment of SH-SY5Y cells with 4-HPR and APG alone and in combination, we observed a decrease in expression of TLR-4 in cells treated with the combination. Expression of MyD88, a signaling adaptor of TLR-4, was also decreased, suggesting inhibition of autophagic signaling pathways. Previous studies reported that suppression of autophagy could lead to increase in susceptibility of cancer cells towards apoptosis. We further examined the effect of combination therapy on cell survival pathway (mTOR/Akt/NF-κB) and found down regulation of the survival signaling molecules. Phase contrast microscopy following treatments showed the morphological features of apoptosis such as cell shrinkage, membrane blebbing, and chromatin condensation. To further understand the mode of cell death after treatments, we performed Annexin V/PI staining and analyzed the cells by flow cytometry. Results from our flow cytometric analysis demonstrated a significant increase in Annexin V positive cells after treatment with combination of 4HPR and APG, suggesting an increase in apoptosis due to combination therapy. Molecular mechanism of apoptosis was confirmed by analyzing the ratio of expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. An increase in Bax:Bcl-2 ratio in the cells treated with combination therapy indicated that cell death occurred by apoptosis. Taken together, our results showed that 4-HPR and APG together could effectively control the growth of human malignant neuroblastoma cells by suppressing autophagy and cell survival pathways so as to increase their apoptotic death. This investigation was supported in part by the R01 grant (NS-57811) from the National Institutes of Health (Bethesda, MD). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2011-2139