Abstract 2194: KLF4 overexpression and apigenin treatment inhibited cell migration and induced apoptosis in human malignant neuroblastoma SK-N-DZ and IMR32 cells
Authors: Mohan Nishant , Ai Walden , Yu Feng , Chen Hexin , Banik Naren L. , Ray Swapan K. 
Published: August 2012

Abstract
Neuroblastoma is a childhood tumor that arises from neuroblasts of the sympathetic nervous system and mostly occurs in adrenal gland. Despite major advances in our understanding of its biology and treatment strategies, malignant neuroblastoma still remains mostly incurable. So, identification of novel molecular targets and exploration of new therapeutic agents are urgently needed. Krüpple-like factor 4 (KLF4) is a transcription factor that shows divergent functions in different malignancies; however, the precise function of KLF4 in malignant neuroblastoma is unclear. In this study, we examined the effects of KLF4 overexpression and apigenin (APG) treatment in human malignant neuroblastoma SK-N-DZ and IMR32 cell lines. We transfected malignant neuroblastoma cells with the plasmid vector overexpressing KLF4 or the control vector and monitored the KLF4 overexpression by staining the cells with FITC conjugated KLF4 antibody and DAPI followed by laser scanning confocal microscopy. Further, KLF4 overexpression in both SK-N-DZ and IMR32 cell lines was confirmed by Western blotting. Then, we determined the reduction in cell viability after transfecting the cells with various concentrations of KLF4 expression vector and treating the cells with APG alone and in combinations. We found that 100 nM KLF4 expression vector and 25 µM APG acted synergistically for most effectively inhibiting the growth of SK-N-DZ and IMR32 cells. We also performed in vitro cell migration assay to monitor inhibition of cell migration and found that combination of KLF4 and APG was highly effective in inhibiting the migration of both neuroblastoma cell lines. Further, we examined the efficacy of combination of KLF4 and APG in induction of apoptosis in SK-N-DZ and IMR32 cells by in situ Wright staining and Annexin V-FITC/PI staining. Combination of KLF4 and APG significantly increased the amounts of apoptosis in both cell lines when compared with control vector or single treatment. We performed more Western blotting to determine the molecular mechanisms for inhibition of cell migration and induction of apoptosis. Matrix metalloproteinases (MMPs), especially MMP2 and MMP9, are responsible for proteolytic processing of extracellular matrix structural proteins and highly implicated in cancer cell migration. We found down regulation of both MMP2 and MMP9 after treatment with combination KLF4 and APG. We also noticed that combination therapy decreased expression of the anti-apoptotic protein Mcl-1, increased expression of the pro-apoptotic proteins Noxa and Puma, and caused activation of caspase-3 for cleavage of ICAD (inhibitor of caspase-activated DNase) leading to completion of apoptosis machinery. Collectively, our results strongly suggest that KLF4 functions as a tumor suppressor and potentiates anti-cancer activities of APG 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 2194. doi:1538-7445.AM2012-2194