Abstract 4393: miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo

Abstract

Abstract Malignant neuroblastoma, which mostly occurs in children, remains incurable with conventional chemotherapeutic agents. Innovative therapeutic strategies must be developed to control the growth of this childhood malignancy. Inhibition of cyclin D3 may induce cell cycle arrest and apoptosis in human malignant neuroblastoma. Also, inhibition of the catalytic component of human telomerase reverse transcriptase (hTERT) may induce apoptosis in many human cancers including malignant neuroblastoma. Decrease in expression of the tumor suppressor microRNA-138 (miR-138) is often associated with increases in expression of hTERT and cyclin D3. We hypothesized that miR-138 overexpression could be a better therapeutic strategy than hTERT knockdown to potentiate pro-apoptotic effect of apigenin (APG), a plant-derived flavonoid, in human malignant neuroblastoma. We found that transfection of miR-138 mimics was more powerful than transfection of hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability in human neuroblastoma SK-N-DZ and SK-N-BE2 cells. We then examined comparative effectiveness of different treatment strategies in reducing tumor growth and inducing apoptosis in SK-N-DZ and SK-N-BE2 xenografts in athymic nude mice. Combination of miR-138 overexpression and APG treatment showed better efficacy than combination of hTERT knockdown and APG treatment in reducing tumor growth and inducing cell death in both neuroblastoma xenograft models. According to the microRNA database (miRDB), miR-138 may target and inhibit expression of cyclin D3 so as to impair cell cylce progression. Combination of miR-138 overexpression and APG treatment caused the highest inhibition of expression of cyclin D3, CDK4, and CDK6, which were required for G1/S transition in cell cycle. Down regulation of cyclin D3 could inhibit phosphorylation of the tumor suppressor Rb and impair cell cycle progression. We delineated that apoptosis occurred with induction of molecular components of extrinsic and intrinsic pathways in the xenografts. Combination of miR-138 overexpression and APG treatment most effectively activated caspase-8 and cleaved Bid to tBid to induce extrinsic pathway of apoptosis and also raised Bax:Bcl-2 ratio to trigger intrinsic pathway of apoptosis with mitochondrial release of Smac into the cytosol to neutralize a set of endogenous inhibitor-of-apoptosis proteins such as Survivin so as to facilitate activation of caspase-3. Activation of both calpain and caspase-3 caused proteolysis of α-spectrin to generate calpain-specific 145 kD SBDP and caspase-3-specific 120 kD SBDP, respectively, in course of apoptosis in the xenografts. In conclusion, miR-138 overexpression was more powerful than hTERT knockdown in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastomas in vivo. Citation Format: Mrinmay Chakrabarti, Walden Ai, Swapan K. Ray. miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2014-4393