Preparation, characterization and toxicological investigation of copper loaded chitosan nanoparticles in human embryonic kidney HEK-293 cells.

Metallic nanoparticles often attribute severe adverse effects to the various organs or tissues at the molecular level despite of their applications in medical, laboratory and industrial sectors. The present study highlights the preparation of copper adsorbed chitosan nanoparticles (CuCSNPs), its characterization and validation of cytotoxicity in human embryonic kidney HEK-293 cells. Particle size of the CuCSNPs was determined by using Zetasizer and the copper loading was quantified with the help of ICP/MS. Further characterization of CuCSNPs was carried out by FT-IR analysis to determine the formation of nanoparticles and SEM was conducted for the morphological analysis of the CuCSNPs. The CuCSNPs exhibited pronounced cytotoxic effects towards HEK-293 cells as analyzed by MTT assay. Moreover, the CuCSNPs inhibited the colony formation and induced nuclear damage at the dose of 100 µg/mL, much more effectively than the in built control copper sulfate (CuSO4). At the molecular level, the CuCSNPs were found to be triggering reactive oxygen species (ROS), activating effector caspases and subsequent PARP cleavage to induce cell death in HEK-293 cells.

Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E.

The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.

Par-4 dependent modulation of cellular ß-catenin by medicinal plant natural product derivative 3-azido Withaferin A.

Here, we provide evidences that natural product derivative 3-azido Withaferin A (3-AWA) abrogated EMT and invasion by modulating ß-catenin localization and its transcriptional activity in the prostate as well as in breast cancer cells. This study, for the first time, reveals 3-AWA treatment consistently sequestered nuclear ß-catenin and augmented its cytoplasmic pool as evidenced by reducing ß-catenin transcriptional activity in these cells. Moreover, 3-AWA treatment triggered robust induction of pro-apoptotic intracellular Par-4, attenuated Akt activity and rescued Phospho-GSK3ß (by Akt) to promote ß-catenin destabilization. Further, our in vitro studies demonstrate that 3-AWA treatment amplified E-cadherin expression along with sharp downregulation of c-Myc and cyclin D1 proteins. Strikingly, endogenous Par-4 knock down by siRNA underscored 3-AWA mediated inhibition of nuclear ß-catenin was Par-4 dependent and suppression of Par-4 activity, either by Bcl-2 or by Ras transfection, restored the nuclear ß-catenin level suggesting Par-4 mediated ß-catenin regulation was not promiscuous. In vivo results further demonstrated that 3-AWA was effective inhibitor of tumor growth and immunohistochemical studies indicated that increased expression of total ß-catenin and decreased expression of phospho-ß-catenin and Par-4 in breast cancer tissues as compared to normal breast tissue suggesting Par-4 and ß-catenin proteins are mutually regulated and inversely co-related in normal as well as cancer condition. Thus, strategic regulation of intracellular Par-4 by 3-AWA in diverse cancers could be an effective tool to control cancer cell metastasis. Conclusively, this report puts forward a novel approach of controlling deregulated ß-catenin signaling by 3-AWA induced Par-4 protein.

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells.

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.

A therapeutically relevant, 3,3'-diindolylmethane derivative NGD16 attenuates angiogenesis by targeting glucose regulated protein, 78kDa (GRP78).

Angiogenesis remain a critical procedure for tumor progression and malignancy. Anticancer agents targeting angiogenic cascades have been proved to be an effective strategy in the field of cancer therapeutics. The current study aims to explore the mechanistic prevention of angiogenesis and cancer cell proliferation by 1,1'-ß-d-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), a novel N-glycosylated derivative of 3,3'-diindolylmethane (DIM). NGD16 suppressed the viability of prostate cancer (PC-3), pancreatic adenocarcinoma (MiaPaca-2), colorectal cancer (COLO-205) and human umbilical vein endothelial cells (HUVECs) effectively with IC50 values 0.8 µM, 2.8 µM, 5.3 µM and 2.5 µM respectively. Abrogation of angiogenesis by NGD16 was promising in in vivo mouse Matrigel plug assay as well as in ex vivo sprouting of rat thoracic aorta. At the molecular level, NGD16 inhibited the expression of glucose regulated protein, 78 kDa (GRP78), vascular endothelial growth factor receptor-2 (VEGFR2) and matrix metalloproteinase-9 (MMP-9) expression, the main mediators of angiogenesis and neovessel formation. Overexpression of GRP78 upregulated the expression of MMP-9 and VEGFR2 in PC-3 and HUVECs. Antibody blocking of GRP78 further potentiated NGD16 in attenuating angiogenesis through inhibition of MMP-9. NGD16 depicted its promising biodistribution profile in a pharmacokinetic study with 46.9% intraperitoneal bioavailability. Our findings suggest NGD16 is a potent inhibitor of neo-angiogenesis with a desirable pharmacokinetic profile, which can be taken forward in its development as an anticancer drug.