Therapeutic Efficacy By Targeting Correction Of Notch1-induced Aberrants In Uveal Tumors

Abstract

Purpose: : Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. Deregulation of the Notch pathway is implicated in carcinogenesis. We extended this potential role to UM, observing high expression of Notch1 in the UM cell lines OCM1 and VUP. The recombinant oncolytic adenovirus H101 has been approved by the Chinese State Food and Drug Administration. We seek to explore a potential synergy of inhibiting Notch signaling combined with H101 oncolytic adenovirus therapy on UM.

Methods: : The chemosynthetic Notch1-siRNA (siNotch1) and control siRNA (siNC) oligonucleotide were synthesized and purified by Shanghai Genepharma (Genepharma, Shanghai, China). Recombinant oncolytic adenovirus H101 was kindly provided by Shanghai Sunway Biotech (Sunwaybio, Shanghai, China). The expression of Notch1 after transfection with siNotch1 and/or H101 was determined by RT-PCR and Western blot. Appropriate multiplicity of H101 infection and cell proliferation were measured by MTT assay. Cell cycle and apoptotic activity of UM cells were analyzed by flow cytometry.

Results: : Semiquantitative RT-PCR and Western blot for Notch1 confirmed suppression by siNotch1 but not siNC or by H101 alone. Notch1 expression was markedly inhibited by siNotch1 or siNotch1 plus H101. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) produced substantial growth inhibition on UM cells. The combined treatment H101-Notch1-siRNA greatly enhanced apoptosis and cell cycle arrest as compared to treatment with H101 or siNotch1 alone.

Conclusions: : Notch pathway deregulation could be a feature of UM, and could also be a promising therapeutic target. Current observations are consistent with the previous analyses we demonstrated that blocking Notch1 signaling via RNA interference inhibited HeLa cell growth (2007 Int J Gynecol 17: 511-516). We previously used a "double target" approach to antitumor therapy by combining H101 with siRNA that targeted Bcl2 (2009 Mol Ther 17: 57-64). In this study, we explored the potential synergy of inhibiting Notch signaling combined with H101 oncolytic adenovirus therapy on UM cell lines OCM1 and VUP. This is the first report of this combination treatment of UM cell lines.

Keywords: melanoma • adenovirus • gene transfer/gene therapy