Abstract 4129: Green tea polyphenols modulate expression of specific microRNAs for induction of molecular mechanisms of apoptosis in malignant neuroblastoma SH-SY5Y and SK-N-DZ cells

Abstract

Abstract Neuroblastomas are extracranial solid tumors that mostly occur in children. Specific microRNAs (miRs) act as oncogenes or tumor suppressors in different malignancies. Modulation of expression of specific oncogenic miRs (onco-miRs) and tumor suppressor miRs (TS-miRs) may control growth of malignant neuroblastomas. The green tea polyphenolic compounds (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG) possess anticancer effects. We studied changes in expression of onco-miRs (miR-92, miR-93, and miR-106b) and TS-miRs (miR-7-1, miR-34a, and miR-99a) in human malignant neuroblastoma SH-SY5Y and SK-N-DZ cells after treatment with EGC or EGCG. We also examined the effects of overexpression of miR-93 or miR-7-1 and treatment with EGC or EGCG on apoptosis. First, cells were treated with EGC or EGCG and subjected to MTT assay to determine residual viability, Annexin V-FITC/PI staining and flow cytometry to detect apoptotic populations, and Wright staining to determine morphological features of apoptosis. Then, differential expression of specific miRs was examined by RT-PCR. Further, Western blotting was performed to detect molecules involved in receptor and mitochondria mediated apoptotic pathways. Both EGC and EGCG dose-dependently reduced cell viability in both cell lines. EGC was less efficacious than EGCG in inducing apoptosis, as determined by Annexin V-FITC/PI staining and further confirmed by Wright staining. Significant decreases in expression of three onco-miRs (miR-92, miR-93, and miR-106b) and increases in expression of three TS-miRs (miR-7-1, miR-34a, and miR-99a) occurred after treatment with 50 μM EGC or 50 μM EGCG. Western blotting showed that EGCG was much more potent than EGC for upregulating pro-apoptotic Bax and down regulating anti-apoptotic Bcl-2 resulting in an increase in Bax:Bcl-2 ratio, which could trigger mitochondrial release of pro-apoptotic factors such as Smac and AIF into the cytosol. Both EGC and EGCG decreased expression of survivin, the endogenous caspase inhibitor, and activated calpain and caspase-3 to generate calpain-specific 145 kD spectrin break down product (SBDP) and caspase-3-specific 120 kD SBDP. Increased proteolytic activity of caspase-3 also generated ICAD fragment leading to activation of CAD for completion of apoptosis. To confirm the onco-miR role of miR-93 and TS-miR role of miR-7-1, we transfected the cells with the mimic of miR-93 or miR-7-1 and then treated with EGC or EGCG and determined apoptotic features by flow cytometry and Western blotting. In conclusion, our results showed that green tea polyphenols modulated expression of specific miRs for induction of apoptosis and established that overexpression of miR-7-1 and treatment with EGCG could be used as an effective combination therapy 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 4129. doi:1538-7445.AM2012-4129