Hina Amin

A novel MMP-2 inhibitor 3-azidowithaferin A (3-azidoWA) abrogates cancer cell invasion and angiogenesis by modulating extracellular Par-4.

Background Withaferin A, which is a naturally derived steroidal lactone, has been found to prevent angiogenesis and metastasis in diverse tumor models. It has also been recognized by different groups for prominent anti-carcinogenic roles. However, in spite of these studies on withanolides, their detailed anti-metastatic mechanism of action remained unknown. The current study has poised to address the machinery involved in invasion regulation by stable derivative of Withaferin A, 3-azido Withaferin A (3-azidoWA) in human cervical HeLa and prostate PC-3 cells. Methods and Principal Findings Sub-toxic concentration of 3-azidowithaferin A (3-azido WA) inhibited cancer cell motility and invasion in wound healing and Boyden chamber invasion by suppressing MMP-2 activity in gelatin zymography and its expression has proved to be a major obstacle in chemo-sensitivity. We have uncovered a novel mechanism of 3-azidoWA induced extracellular pro-apoptotic candidate tumor suppressor Par-4 protein stimulation in conditioned media and also noticed a concomitant marked reduction in pAkt and pERK signaling by immunoblot analysis. Furthermore, our zymography results suggest 3-azidoWA induced MMP-2 inhibition was mediated through secretory Par-4. The inhibition of apoptosis by 3-azidoWA could not restore MMP-2 gelatinase activity. In addition to this, our in vivo animal experiments data showed 3-azidoWA abrogated neovascularisation in dose dependent manner in mouse Matrigel plug assay. Conclusion/Significance For this report, we found that 3-azidoWA suppressed motility and invasion of HeLa and PC-3 cells in MMP-2 dependent manner. Our in vitro result strongly suggests that sub-toxic doses of 3-azidoWA enhanced the secretion of extracellular Par-4 that abolished secretory MMP-2 expression and activity. Depletion of secretory Par-4 restored MMP-2 expression and invasion capability of HeLa and PC-3 cells. Further, our findings implied that 3-azidoWA attenuated internal phospho-ERK and phospho-Akt expression in a dose dependent manner might play a key role in inhibition of mouse angiogenesis by 3-azidoWA.

Par-4 dependent modulation of cellular β-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 β‐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 β‐catenin and augmented its cytoplasmic pool as evidenced by reducing β‐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‐GSK3β (by Akt) to promote β‐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 β‐catenin was Par‐4 dependent and suppression of Par‐4 activity, either by Bcl‐2 or by Ras transfection, restored the nuclear β‐catenin level suggesting Par‐4 mediated β‐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 β‐catenin and decreased expression of phospho‐β‐catenin and Par‐4 in breast cancer tissues as compared to normal breast tissue suggesting Par‐4 and β‐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 β‐catenin signaling by 3‐AWA induced Par‐4 protein.

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.

Targeting EGFR and IGF 1R: a promising combination therapy for metastatic cancer.

Acute drug resistance, intolerable side effects and non-specific target activation are the crucial barriers for efficient translational outcome of target directed cancer drug discovery. In the last five years, many of the bulls eye drugs failed to obtain FDA approval because of highly complicated mechanisms of the targeting receptors. These receptors include epidermal growth factor receptor (EGFR) and Insulin-like growth factor receptor 1 (IGF 1R), and are considered as pivotal signaling routes in highly transformed metastatic cancers. IGF 1R and EGFR families show homology in their structure and both the receptors share considerable crosstalk in their functions. An aberrant activation of these two pathways is often diagnosed among many cancer patients. Therefore, target based monoclonal antibodies and small molecule tyrosine kinase inhibitors, either in combination or co-targeting these two receptors may provide a new era of promising therapy and can help in remarkable progress among cancer patients.

A new cytotoxic quinolone alkaloid and a pentacyclic steroidal glycoside from the stem bark of Crataeva nurvala: study of anti-proliferative and apoptosis inducing property.

Chemical investigation of stem bark of Crataeva nurvala afforded 5,7-dimethoxy-3-phenyl-1-ethyl-1,4-dihydro-4-quinolone and a steroidal glycoside with unprecedented pentacyclic ring system named crataemine (1a) and crataenoside (2) respectively. The structures of the compounds were determined by spectroscopic analysis. A series of compounds with modification at position 1 of 1a (1a-1c) were prepared. All compounds were screened for cytotoxic activity against HeLa, PC-3 and MCF-7 cells. Only 1a and 2 showed potency against all three cells. Mechanism based study for activity of the compounds demonstrated that it could block the migration of more aggressive HeLa and PC-3 cells and prevent their colony formation ability as well. The compounds potentiated apoptosis in HeLa and PC-3 cells in a significant manner.

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.

Inhibition of Invasion in Pancreatic Cancer Cells by Conjugate of EPA with β(3,3)-Pip-OH via PI3K/Akt/NF-kB Pathway.

The present work describes the anti-invasive effect of conjugate BC06, a novel conjugate of EPA, (2E,4E)-4-(benzo[d][1,3]dioxol-5-ylmethylene) hex-2-enoic acid with β,β-disubstituted-β-amino acid, β(3,3)-Pip-OH (2-(4-aminopiperidin-4-yl)acetic acid), in human pancreatic carcinoma. The conjugate BC06 inhibited invasion and migration of PANC-1 cells in wound healing, matrigel invasion, and gelatin degradation assays. Apart from suppressing PI3K/Akt/NF-kB signaling, which is involved in the up-regulation of matrix metalloproteinases, our study also demonstrated that dose-dependent treatment of BC06 results in the upregulation of TIMP-1 and E-cadherin expression. Further, BC06 was found to be inhibiting the metastatic ability of PANC-1 cells by reducing MMP-2 and MMP-9 expression. These findings suggest that EPA conjugate with β(3,3)-Pip-OH, BC06, may be used as an anti-invasive agent against human pancreatic carcinoma.

Differential regulation of NM23-H1 under hypoxic and serum starvation conditions in metastatic cancer cells and its implication in EMT

Multiple stresses are prevalent inside the tumor microenvironment rendering tumor growth, neighboring invasion and metastasis of the cancer cells to distant organs. NM23-H1 is the first metastasis suppressor gene identified and known to be implicated as an important regulator of stress-induced metastasis. Herein, we demonstrated that prototypical NM23-H1 expression diminished during hypoxia and serum starvation in Panc-1/MDA-MB-231 cells, but converse invasion patterns were obtained in these two diverse stresses. Supportingly, a compelling discrete difference in mRNA and protein levels of NM23-H1 was achieved in hypoxia as well as serum starvation. Knockdown of NM23-H1 activates EMT whereas the similar effects are subdued in serum starvation where NM23-H1 down-modulation prompted E-cadherin upregulation. Stable NM23-H1 expression augmented E-cadherin levels along with retardation in invadopodea formation and invasion. In hypoxia/serum starvation excess NM23-H1 effectively modulated the Twist1 promoter activity. Thus, differential regulation of NM23-H1 may corroborate/abrogate EMT depending on the nature of stress, tumor microenvironment and cellular context.

Inhibition of Twist1-mediated invasion by Chk2 promotes premature senescence in p53-defective cancer cells

Twist1, a basic helix–loop–helix transcription factor is implicated as a key mediator of epithelial–mesenchymal transition (EMT) and metastatic dissemination in p53-deficient cancer cells. On the other hand, checkpoint kinase 2 (Chk2), a major cell cycle regulatory protein provides a barrier to tumorigenesis due to DNA damage response by preserving genomic stability of the cells. Here we demonstrate that Chk2 induction proficiently abrogates invasion, cell scattering and invadopodia formation ability of p53-mutated invasive cells by suppressing Twist1, indicating Chk2 confers vital role in metastasis prevention. In addition, ectopic Chk2, as well as its (Chk2) induction by natural podophyllotoxin analog, 4′-demethyl-deoxypodophyllotoxin glucoside (4DPG), strongly restrain Twist1 activity along with other mesenchymal markers, for example, ZEB-1, vimentin and Snail1, whereas the epithelial markers such as E-cadherin and TIMP-1 expression augmented robustly. However, downregulation of endogenous Chk2 by siRNA as well as Chk2 selective inhibitor PV1019 implies that 4DPG-mediated inhibition of Twist1 is Chk2-dependent. Further, mechanistic studies unveil that Chk2 negatively regulates Twist1 promoter activity and it (Chk2) interacts steadily with Snail1 protein to curb EMT. Strikingly, Chk2 overexpression triggers premature senescence in these cells with distinctive increase in senescence-associated β-galactosidase (SA-β-gal) activity, G2/M cell cycle arrest and induction of senescence-specific marker p21waf1/Cip1. Importantly, stable knockdown of Twist1 by shRNA markedly augments p21 expression, its nuclear accumulation, senescence-associated heterochromatin foci (SAHF) and amplifies the number of SA-β-gal-positive cells. Moreover, our in vivo studies also validate that 4DPG treatment significantly abrogates tumor growth as well as metastatic lung nodules formation by elevating the level of phospho-Chk2, Chk2 and suppressing Twist1 activity in mouse mammary carcinoma model. In a nutshell, this report conceives a novel strategy of Twist1 suppression through Chk2 induction, which prevents metastatic dissemination and promotes premature senescence in p53-defective invasive cancer cells.

Cristacarpin promotes ER stress-mediated ROS generation leading to premature senescence by activation of p21(waf-1).

Stress-induced premature senescence (SIPS) is quite similar to replicative senescence that is committed by cells exposed to various stress conditions viz. ultraviolet radiation (DNA damage), hydrogen peroxide (oxidative stress), chemotherapeutic agents (cytotoxic threat), etc. Here, we report that cristacarpin, a natural product obtained from the stem bark of Erythrina suberosa, promotes endoplasmic reticulum (ER) stress, leading to sub-lethal reactive oxygen species (ROS) generation and which eventually terminates by triggering senescence in pancreatic and breast cancer cells through blocking the cell cycle in the G1 phase. The majority of cristacarpin-treated cells responded to conventional SA-β-gal stains; showed characteristic p21waf1 upregulation along with enlarged and flattened morphology; and increased volume, granularity, and formation of heterochromatin foci—all of these features are the hallmarks of senescence. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly reduced the expression of p21waf1, confirming that the modulation in p21waf1 by anti-proliferative cristacarpin was ROS dependent. Further, the elevation in p21waf1 expression in PANC-1 and MCF-7 cells was consistent with the decrease in the expression of Cdk-2 and cyclinD1. Here, we provide evidence that cristacarpin promotes senescence in a p53-independent manner. Moreover, cristacarpin treatment induced p38MAPK, indicating the ROS-dependent activation of the MAP kinase pathway, and thus abrogates the tumor growth in mouse allograft tumor model.