RNAI MEDIATED GENE SILENCING OF EIF3A: A POSSIBLE SOLUTION TO CONTROL BREAST CANCER

Objective: The eukaryotic translational initiation factor 3A (eIF3A) is reported to be over expressed in most breast cancer cells. In the present study, our aim is to suppress the over expression of eIF3A in human breast cancer MCF-7 cell line using gene silencing technique (RNA interference (RNAi)).Methods: The artificial microRNA (amiRNA) targeting eIF3A gene was constructed by incorporating short interference RNA (siRNA) sequences against eIF3A gene into endogenous microRNA30 (miR-30) and cloned into pcDNA3.1 vector. The amiRNA containing plasmid was then transfected into MCF-7 cell line and the expression of eIF3A was examined by RT-PCR. The cytotoxicity of plasmid with amiRNA targeting eIF3A on MCF–7 cells was evaluated by MTT assay.Results: The amiRNA construct significantly inhibited eIF3A gene expression and reduce the cell viability of MCF-7 cell line.Conclusion: The usage of modified endogenous amiRNA in vector based expression system with significant gene silencing efficiency suggests that RNAi based gene silencing method can be considered as one of the effective means to control cancer.Â

Reporter Protein Complementation Imaging Assay to Screen and Study Nrf2 Activators in Cells and Living Animals

NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferase reporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents’ for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators preclinically in small animal models by noninvasive molecular imaging approach

Proteomic Identification of Pterostilbene-Mediated Anticancer Activities in HepG2 Cells

In the present study, we attempt to shed light on the underlying molecular mechanism of the anticancer activity of pterostilbene (PTS) in HepG2 cells through the proteomic approach. PTS was found to induce apoptosis by altering the expression of apoptotic genes and the G2/M phase of cell cycle arrest. Further, the 2-DE map showed the expression of 72 differentially regulated proteins in PTS-treated HepG2 cells, of which 8 spots with >2 fold up- or down-regulated level were identified by MALDI-TOF analysis, which has a regulatory role in apoptosis. These findings for the first time offer valuable insights into the mechanism of apoptotis by PTS in HepG2 cells

Carvacrol induces cytotoxicity in human cervical cancer cells but causes cisplatin resistance: Involvement of MEK-ERK activation

Carvacrol has been shown to possess anticancer activity but the mechanism is unknown, as well as the possibility of interaction with anticancer drugs. The aim of this study was to investigate the role of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling in carvacrol-induced human cervical cancer HeLa cell cytotoxicity. In addition, we studied sensitization of HeLa cells to cisplatin (CP) by carvacrol. Both carvacrol and CP showed dose-dependent cytotoxicity against HeLa cells and activated ERK1/2. The MEK inhibitor PD325901 suppressed ERK expression and further increased cytotoxicity of carvacrol but increased viability of CP-treated cells by modulating apoptosis. The MEK inhibitor also increased microtubule-associated protein 1A/1B- light chain 3 (LC3B) expression in CP treatment. Co-treatment with CP and carvacrol resulted in increased viability of the cancer cells compared to CP treatment, which was associated with the suppression of apoptosis. MEK inhibition decreased the cell viability, without changes in apoptosis. Concomitantly, carvacrol increased CP-induced expression of LC3B, which was enhanced by MEK inhibition. The results of the current study suggest the opposite role of ERK1/2 in carvacrol and CP- induced HeLa cell cytotoxicity. Interestingly, carvacrol induced CP resistance in HeLa cells through ERK1/2-independent suppression of apoptosis and ERK1/2-dependent modulation of autophagy