Suppression of mTOR Expression by siRNA Leads to Cell Cycle Arrest and Apoptosis Induction in MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Mammary carcinogenesis, being ranked second in cancer-related mortality and the inadequacy of existing chemotherapy advocates the development of a novel treatment approach targeting its molecular signalling. Hyperactivation of mammalian target of rapamycin (mTOR) has a critical role in developing invasive mammary cancer and it can be a potential target. OBJECTIVE: This experiment was to explore the efficacy of mTOR-specific siRNA on therapeutic targeting of the mTOR gene, assess its proficiency in suppressing in vitro breast cancer and determine underlying molecular mechanisms. METHODS: Specific siRNA targeting mTOR was transfected into MDA-MB-231 cells and mTOR downregulation was validated through qRT-PCR and western blot analysis. Cell proliferation was analysed by MTT assay and confocal microscopy. Apoptosis was studied through flow cytometry and S6K, GSK-3ß and caspase 3 expression were estimated. Further, the effect of mTOR blockade on cell cycle progression was determined. RESULTS: Following transfection of mTOR-siRNA into the MDA-MB-231 cells, cell viability and apoptosis were examined which indicates that clinically relevant concentration of mTOR-siRNA inhibited cell growth and proliferation and promote apoptosis, resulting from the suppression of mTOR. This leads to the downregulation of mTOR downstream S6K and upregulation of GSK-3ß. An increased level of caspase 3 symbolises that the apoptotic activity is mediated through caspasedependent pathway. Further, mTOR downregulation causes cell cycle arrest in G0/G1 phase as observed in the flow cytometry study. CONCLUSION: With these results, we can conclude that mTOR-siRNA exerts direct 'anti-breast cancer' activity propagated by the S6K-GSK-3ß- caspase 3 mediated apoptosis and by inducing cell cycle arrest.

Therapeutic silencing of mTOR by systemically administered siRNA-loaded neutral liposomal nanoparticles inhibits DMBA-induced mammary carcinogenesis.

BACKGROUND: Mammary carcinogenesis possesses great challenges due to the lack of effectiveness of the multiple therapeutic options available. Gene therapy-based cancer treatment strategy provides more targeting accuracy, fewer side effects, and higher therapeutic efficiency. Downregulation of the oncogene mTOR by mTOR-siRNA is an encouraging approach to reduce cancer progression. However, its employment as means of therapeutic strategy has been restricted due to the unavailability of a suitable delivery system. METHODS: A suitable nanocarrier system made up of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) has been developed to prevent degradation and for proficient delivery of siRNA. This was followed by in vitro and in vivo anti-breast cancer efficiency analysis of the mTOR siRNA-loaded neutral liposomal formulation (NL-mTOR-siRNA). RESULTS: In our experiment, a profound reduction in MCF-7 cell growth, proliferation and invasion was ascertained following extensive downregulation of mTOR expression. NL-mTOR-siRNA suppressed tumour growth and restored morphological alterations of DMBA-induced breast cancer. In addition, neutral liposome enhanced accumulation of siRNA in mammary cancer tissues facilitating its deep cytosolic distribution within the tumour, which allows apoptosis thereby facilitating its anti-tumour potential. CONCLUSION: Hence, the current study highlighted the augmented ground for therapies aiming toward cancerous cells to diminish mTOR expression by RNAi in managing mammary carcinoma.

LC-MS characterized methanolic extract of zanthoxylum armatum possess anti-breast cancer activity through Nrf2-Keap1 pathway: An in-silico, in-vitro and in-vivo evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC (Rutaceae) containing flavonoids, alkaloids, coumarins, lignans, amides and terpenoid is well-known for its curative properties against various ailments including cancer. In the current research, phytochemicals present in the methanolic extract of Zanthoxylum armatum bark (MeZb) were characterized by LC-MS/MS analysis and chemotherapeutic potential of this extract was determined on DMBA-induced female Sprague Dawley rats. MATERIALS AND METHODS: A simple and fast high-performance liquid chromatography-mass spectroscopy (LC-MS/MS) of MeZb was established followed by in-vitro antioxidant assays. This was followed by in-silico docking analysis as well as cytotoxicity assessment. Successively in-vivo study of MeZb was performed in DMBA-induced Sprague Dawley rats possessing breast cancer along with detailed molecular biology studies involving immunofluorescence, RT-qPCR and Western blot analysis. RESULTS: LC-MS/MS investigation revealed the presence of compounds belonging to flavonoid, alkaloid and glycoside groups. MeZb revealed potential antioxidant activity in in-vitro antioxidant assays and strong binding energy of identified compounds was seen from the in-silico study with both HO1 and Keap1 receptor. Furthermore, the antioxidant action of MeZb was proven from the in-vivo analysis of antioxidant marker enzymes (lipid peroxidation, enzymic and non-enzymic antioxidants). This study also revealed upregulation of protective Nrf-2 following downregulation of Keap1 after MeZb treatment with respect to untreated cancerous rats. CONCLUSION: These results exhibited anti-breast-cancer potential of MeZb through Nrf2-Keap1 pathway which may be due to the flavonoids, alkaloids and glycosides present in it.

Strategic Developments & Future Perspective on Gene Therapy for Breast Cancer: Role of mTOR and Brk/ PTK6 as Molecular Targets.

Breast cancer is a serious health issue and a major concern in biomedical research. Alteration in major signaling (viz. PI3K-AKT-mTOR, Ras-Raf-MEK-Erk, NF-kB, cyclin D1, JAK-STAT, Wnt, Notch, Hedgehog signaling and apoptotic pathway) contributes to the development of major subtypes of mammary carcinoma such as HER2 positive, TNBC, luminal A and B and normal-like breast cancer. Further, mutation and expression parameters of different genes involved in the growth and development of cells play an important role in the progress of different types of carcinoma, making gene therapy an emerging new therapeutic approach for the management of life-threatening diseases like cancer. The genetic targets (oncogenes and tumor suppressor genes) play a major role in the formation of a tumor. Brk/PTK6 and mTOR are two central molecules that are involved in the regulation of numerous signaling related to cell growth, proliferation, angiogenesis, survival, invasion, metastasis, apoptosis, and autophagy. Since these two proteins are highly upregulated in mammary carcinogenesis, this can be used as targeted genes for the treatment of breast cancer. However, not much work has been done on them. This review highlights the therapeutic significance of Brk and mTOR and their associated signaling in mammary carcinogenesis, which may provide a strategy to develop gene therapy for breast cancer management.