Inhibition of Breast Cancer Progression by Multi Targeting Approach Using RNAI as a Tool: An in Vitro Approach

Abstract

Bcl-2 is an anti apoptotic protein that plays an important role in rendering cancer cells resistant to death signals. This increases longevity of cancer cells, which then proliferate and metastasise to far flung areas to find an appropriate niche. RhoGTPases are molecular switches regulating a plethora of cellular functions such as migration, transformation and invasion which are important during metastasis. Therapeutic targeting of Bcl-2 and RhoGTPAses has attracted many cancer researchers. Many small molecule inhibitors are currently under various levels of clinical trials. This study was designed to identify and select an effective shRNA sequence that can shut down the availability of functional Bcl-2/ RhoGTPases transcripts by using plasmid based expression system. To achieve the set objectives, bioinformatics tools based identification of siRNAs against Bcl-2, RhoA, RhoC, transfection into breast cancer cell lines, quantitative reverse transcription PCR (SYBR green & TaqMan), morphological assessment (by DAPI), caspase assay, cell viability assay (MTT), caspase 3 activation assay, membrane leakage (LDH) assay, gap closure and anchorage independence assays were performed. Silencing of Bcl-2 resulted into activation of caspase 3 which reduced cell viability. Cell death was seen in culture after silencing. Similarly, RhoC silencing decreased cell migration and reversed anchorage independence phenotype. Moreover, these were more prominent in multiple vector transfected cells. This multiple vector had capacity to produce shRNAs against all the three genes. This study revealed that sequence specific downregulation can decrease effective concentration of otherwise overexpressed oncogenes. This study also suggests using RNAi as a modern therapeutic strategy rather than just loss of function screens.