Jab1-siRNA Induces Cell Growth Inhibition and Cell Cycle Arrest in Gall Bladder Cancer Cells via Targeting Jab1 Signalosome.

BACKGROUND: The aberrant alteration in Jab1 signalosome (COP9 Signalosome Complex Subunit 5) has been proven to be associated with the progression of several carcinomas. However the specific role and mechanism of action of Jab1 signalosome in carcinogenesis of gall bladder cancer (GBC) are poorly understood. OBJECTIVE: The main objective of our study was to elucidate the role and mechanism of Jab1 signalosome in gall bladder cancer by employing siRNA. METHODS: Jab1 overexpression was identified in gall bladder cancer tissue sample. The role of Jab1-siRNA approach in cell growth inhibition and apoptotic induction was then examined by RT-PCR, Western Blotting, MTT, ROS, Hoechst and FITC/Annexin-V staining. RESULTS: In the current study, we have shown that overexpression of Jab1 stimulated the proliferation of GBC cells; whereas downregulation of Jab1 by using Jab1-siRNA approach resulted incell growth inhibition and apoptotic induction. Furthermore, we found that downregulation of Jab1 induces cell cycle arrest at G1 phase and upregulated the expression of p27, p53 and Bax gene. Moreover, Jab1-siRNA induces apoptosis by enhancing ROS generation and caspase-3 activation. In addition, combined treatment with Jab1-siRNA and gemicitabine demonstrated an enhanced decline in cell proliferation which further suggested increased efficacy of gemcitabine at a very lower dose (5µM) in combination with Jab1-siRNA. CONCLUSION: In conclusion, our study strongly suggests that targeting Jab1 signalosome could be a promising therapeutic target for the treatment of gall bladder cancer.

Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma.

BACKGROUND: Plant sterols have proven a potent anti-proliferative and apoptosis inducing agent against several carcinomas including breast and prostate cancers. Jab1 has been reported to be involved in the progression of numerous carcinomas. However, antiproliferative effects of sterols against Jab1 in gall bladder cancer have not been explored yet. OBJECTIVE: In the current study, we elucidated the mechanism of action of stigmasterol regarding apoptosis induction mediated via downregulation of Jab1 protein in human gall bladder cancer cells. METHODS: In our study, we performed MTT and Trypan blue assay to assess the effect of stigmasterol on cell proliferation. In addition, RT-PCR and western blotting were performed to identify the effect of stigmasterol on Jab1 and p27 expression in human gall bladder cancer cells. We further performed cell cycle, Caspase-3, Hoechst and FITC-Annexin V analysis, to confirm the apoptosis induction in stigmasterol treated human gall bladder cancer cells. RESULTS: Our results clearly indicated that stigmasterol has up-regulated the p27 expression and down-regulated Jab1 gene. These modulations of genes might occur via mitochondrial apoptosis signaling pathway. Caspase-3 gets activated with the apoptotic induction. Increase in apoptotic cells and DNA were confirmed through annexin V staining, Hoechst staining, and cell cycle analysis. CONCLUSION: Thus, these results strongly suggest that stigmasterol has the potential to be considered as an anticancerous therapeutic agent against Jab1 in gall bladder cancer.

Molecular Interaction and Computational Analytical Studies of Pinocembrin for its Antiangiogenic Potential Targeting VEGFR-2: A Persuader of Metastasis.

BACKGROUND: Designing a novel antagonist against VEGFR-2 is being applied currently to inhibit cancer growth and metastasis. Because of the unexpected side effects incurred by the contemporary anticancer medications, the focus has been laid towards identifying natural compounds that might carry the potential to inhibit tumor progression. VEGR-2 remains an important target for anticancer drug development as it is the master regulator of vascular growth. OBJECTIVE: The study focuses on virtual screening of compounds from plants of Asteraceae family that bears antiangiogenic potential and thus, inhibiting VEGFR-2 using a computational approach. MATERIALS AND METHODS: Structures of phytochemicals were prepared using ChemDraw Ultra 10 software and converted into its 3D PDB structure and minimized using Discovery Studio client 2.5. The target protein, VEGFR-2 was retrieved from RCSB PDB. Lipinski's rule and ADMET toxicity profiling were carried out on the phytochemicals of the Asteraceae family and the filtered compounds were further promoted for molecular docking and MD simulation analysis. The study extends towards the SOM analysis of Pinocembrin to predict the possible toxic and non-toxic in vivo metabolites via in silico tools (Xenosite Web and PASS online server). RESULTS: The docking results revealed promising inhibitory potential of Pinocembrin against VEGFR-2 with binding energy of -8.50 kcal/mole as compared to its known inhibitors Sorafenib and YLT192 having binding energy of -6.49 kcal/mole and -8.02 kcal/mol respectively. Further, molecular dynamics (MD) simulations for 10ns were conducted for optimization, flexibility prediction, and determination of folded VEGFR-2 stability. The Hsp90-Pinocembrin complex was found to be quite stable with RMSD value of 0.2nm. Pinocembrin was found to be metabolically stable undergoing phase I metabolism with non-toxic metabolites compared to the standard drug Sorafenib and YLT192. CONCLUSION: Obtained results propose Pinocembrin as a multi-targeted novel lead compound that bears outstanding antiangiogenic potential against VEGFR-2.

Elucidation of Antiangiogenic Potential of Vitexin Obtained from Cucumis sativus Targeting Hsp90 Protein: A Novel Multipathway Targeted Approach to Restrain Angiogenic Phenomena.

BACKGROUND: Angiogenesis involves the process of sprouting of microvessels from preexisting microvasculature and is held responsible for the growth, malignancy and metastasis of cancer. Heat shock protein Hsp90 has been proven responsible for indirectly inducing multiple pathways leading to angiogenesis and metastasis in cancer. Recent researches shift towards proposing novel phytochemicals as possible antiangiogenic agents. OBJECTIVE: The study aims towards Virtual screening of compounds from Cucurbitaceae family and their Druglikeliness and PreADMET filtering in search of potent lead as Vitexin, targeting Hsp90 and hence restraining angiogenesis. MATERIALS AND METHODS: Structures of phytochemicals from Cucurbitaceae family were retrieved from PubChem database and were converted into suitable 3-D structures. The target protein, Hsp90 was retrieved from RCSB Protein Data Bank. Phytochemicals of Cucurbitaceae family were filtered through enumerated Lipinski's rule of five and ADMET toxicity profiling and the filtered compounds were further taken forward for molecular docking analysis and interaction studies using AutoDock Tools 4.0. RESULTS: The docking results revealed Vitexin, a prominent glycosylated natural flavonoid, showing promising inhibitory potential against Hsp90 with binding energy of -8.80 kcal/mole and Ki 353.24 nM as compared to its known inhibitor Ganetespib having binding energy of -7.33 kcal/mole and Ki 4260 nM. Vitexin also exhibits better drug having properties with satisfactory ADMET profiling in relation to Ganetespib. CONCLUSION: The result proposes Vitexin to hold prominent antiangiogenic potential surpassing different in silico parameters and thus expected to be a multi-targeted novel antiangiogenic lead.