Riproximin mediated effects on transcriptomic profile of PI3K-AKT-mTOR pathway genes in breast cancer cell lines

Abstract

Background: Riproximin, a type II ribosome inactivating protein, is a substantial antineoplastic agent and can be used as alternative to chemotherapy specifically to kill tumour cells. Since discovery, this protein has been tested against various cancer cell lines and animal models to explore its anti-neoplastic activities. In this study, riproximin has been tested to unfold its impact on multiple genes related to PI3K/Akt/mTOR signaling pathway. This pathway has its significant importance in regulation of cellular functions which include metabolism, proliferation, survival and growth of cancerous cells. For it valuable importance during carcinogenesis, this pathway is also being targeted for therapeutic purpose.

Materials and Methods: MTT dye reduction assay was used to find out toxicity of riproximin by exposing the selected breast cancer cell lines (MDA-MB-231 and MCF-7) with different concentrations (1-50ng/ml) of the protein. Afterwards, in a separate experiment, the cell lines were exposed to different concentrations (MDA-MB-231: 1-50ng/ml, MCF-7: 1-20ng/ml) of riproximin followed by total RNA extraction, cDNA synthesis and expressional profiling of 10 genes related to PI3K-AKT-mTOR signaling by using real-time PCR. Fold changes were identified via Livak method while comparing the data with untreated cells grown in parallel.

Results: MTT assay results showed that riproximin induced significant toxic effects in the breast cancer cell lines, where MDA-MB-231 cells were less sensitive (IC50: 10.2ng/ml) as compared to MCF-7 (IC50: 1.8ng/ml) cells. Real-time PCR demonstrated a substantial potential of riproximin to alter the expression of cell cycle associated genes. Among these targets, most effective de-regulations in MDA-MB-231 cells were found in FOS (16fold) followed by JUN (6fold) and NFKB1 (4fold). In MCF-7 cells, most substantial modifications were observed in NFKB1 (14fold), CD14 (9fold) and PDK1 (6fold).

Conclusion: Riproximin bears significant cytotoxic potential against primary and metastatic breast cancer cell lines. Substantial expressional modulations in PI3K/Akt/mTOR signaling pathway related genes are imposed by riproximin in the cancer cells. Further detailed in vitro and in vivo studies are required to understand the precise impact of riproximin exposure on PI3K-AKT-mTOR signaling, which ultimately will pave the way for its clinical utilization.