title: Combination of N-(4-hydroxyphenyl) retinamide and apigenin suppressed starvation-induced autophagy and promoted apoptosis in malignant neuroblastoma cells.
abstract: Autophagy is a catabolic process for recycling of cellular contents in response to metabolic stress in malignant tumors. We explored efficacy of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the isoflavonoid apigenin (APG) in the serum-starved human malignant neuroblastoma cells. Combination of 0.5 μM 4-HPR and 50 μM APG synergistically decreased cell viability in the serum-starved neuroblastoma SH-SY5Y, SK-N-BE2, and IMR-32 cells. Acridine orange (AO) staining and LC3 II upregulation showed that serum-starvation for 12 and 24h progressively increased the formation of acidic vesicular organelles (AVO) and autophagy in SH-SY5Y cells. Further, AO staining and flow cytometry showed blockage of formation of AVO and accumulation of auophagic population, respectively, following the treatment of the serum-starved SH-SY5Y cells with combination of 0.5 μM 4-HPR and 50 μM APG. Combination therapy downregulated autophagy inducing proteins such as Beclin 1, LC3 II, TLR-4, and Myd88 while upregulated autophagy inhibitory p-Akt/mTOR singaling pathway. Consistent with the hypothesis that inhibition of autophagy could induce apoptosis, we noticed inhibition of autophagy and induction of apoptosis in the serum-starved SH-SY5Y cells with the suppression of the survival factor NF-κB, upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2, activation of caspase-3, and degradation of poly(ADP-ribose) polymerase (PARP) after combination therapy. Collectively, combination of 4-HPR and APG worked synergistically to suppress autophagy and promote apoptosis in human malignant neuroblastoma cells.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, USA.
authors.forename: Nishant
authors.initials: N
authors.lastname: Mohan
authors.forename: Naren L
authors.initials: NL
authors.lastname: Banik
authors.forename: Swapan K
authors.initials: SK
authors.lastname: Ray
mesh_terms: D047310:Apigenin / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D016895:Culture Media, Serum-Free; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D017313:Fenretinide / Q000494:pharmacology*; D006801:Humans; D057054:Molecular Imaging / Q000379:methods; D015388:Organelles / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; D016895:Culture Media, Serum-Free; D017313:Fenretinide; D047310:Apigenin