Abstract 1101: Modulation of expression of specific oncogenic and tumor suppressor microRNAs enhanced therapeutic efficacy of 4-HPR and EGCG in human malignant neuroblastoma cells
Authors: Chakrabarti Mrinmay , Norris James S. , Banik Naren L. , Ray Swapan K. 
Published: August 2012

Abstract
Malignant neuroblastomas are extra cranial solid tumors that mostly occur in children. Studies show modulation of expression of specific microRNAs (miRs) in the pathogenesis of malignant neuroblastomas. So, suppression of specific oncogenic miRNAs (onco-miRs) or overexpression of specific tumor suppressor miRs (TS-miRs) may provide opportunity for controlling growth of malignant neuroblastomas. We explored whether N-(4-hydroxyphenyl) retinamide (4-HPR, a synthetic retinoid) and (-)-epigallocatechin-3-gallate (EGCG, a flavonoid from green tea) could be effective in modulating expression of specific onco-miRs and TS-miRs for increasing differentiation and apoptosis in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells. We treated the cells with different doses of 4-HPR, EGCG, and combination of both, subjected to MTT assay, and found that combination of 0.5 μM 4-HPR and 50 μM EGCG acted most synergistically to reduce cell viability in both cell lines. Combination therapy increased morphological and biochemical features of neuronal differentiation and apoptotic death. Increases in apoptosis in both cell lines were associated with activation of extrinsic and intrinsic caspase cascades. Increased cytosolic level of AIF also indicated activation of caspase-independent pathway of apoptosis. Activation of calpain and caspase-3 produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Increased activity of caspase-3 also caused ICAD fragmentation leading to completion of apoptotic process. Our RT-PCR experiments indicated that significant changes in expression of three onco-miRs (miR-92, miR-93, and miR-106b) and three TS-miRs (miR-7-1, miR-34a, and miR-99a) could be associated with increases in differentiation and apoptosis in both cell lines after treatment with combination of 4-HPR and EGCG. Then, we investigated the effects of overexpression of an onco-miR (miR-93) and a TS-miR (miR-7-1) on the therapeutic efficacy of the drugs. We transfected the cells with miR-93 and miR-7-1 mimics and subjected to treatment with 4-HPR and EGCG alone and in combination for evaluation of the effects on induction of apoptotic death by flow cytometry and Western blotting. Overexpression of the onco-miR (miR-93) reduced the efficacy of the combination therapy while overexpression of the TS-miR (miR-7-1) increased the efficacy of the combination therapy for induction of apoptosis in both cell lines. Collectively, results showed that combination of 4-HPR and EGCG acted synergistically to increase differentiation and apoptosis, modulated the expression of specific onco-miRs and TS-miRs for induction of apoptosis, and overexpression of the TS-miR (miR-7-1) in combination with 4-HPR and EGCG could be used as the most potent therapeutic regimen for controlling growth of 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 1101. doi:1538-7445.AM2012-1101