Souneek Chakraborty

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.

Design and Synthesis of Antitumor Heck-Coupled Sclareol Analogues: Modulation of BH3 Family Members by SS-12 in Autophagy and Apoptotic Cell Death.

Sclareol, a promising anticancer labdane diterpene, was isolated from Salvia sclarea. Keeping the basic stereochemistry-rich framework of the molecule intact, a method for the synthesis of novel sclareol analogues was designed using palladium(II)-catalyzed oxidative Heck coupling reaction in order to study their structure-activity relationship. Both sclareol and its derivatives showed an interesting cytotoxicity profile, with 15-(4-fluorophenyl)sclareol (SS-12) as the most potent analogue, having IC50 = 0.082 μM against PC-3 cells. It was found that SS-12 commonly interacts with Bcl-2 and Beclin 1 BH3 domain proteins and enhances autophagic flux by modulating autophagy-related proteins. Moreover, inhibition of autophagy by autophagy inhibitors protected against SS-12-induced apoptosis. Finally, SS-12 effectively suppressed tumor growth in vivo in Ehrlichs ascitic and solid Sarcoma-180 mouse models.

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.

A journey beyond apoptosis: new enigma of controlling metastasis by pro-apoptotic Par-4

Prostate apoptotic response 4 (Par-4) is coined as a therapeutic protein since owing to its diverse physiologically relevant properties, especially in the cancer perspective. Albeit, Par-4 expression is not restricted to any specific tissue/organ, apart from cell death promotion (due to challenging threats), the other biological role of Par-4 is convincingly emerging. In the recent years, several laboratories have intended to dissect the signaling or mechanisms involved in Par-4 activation to augment apoptosis cascades but new developments in Par-4 research have widened its therapeutic potential. One of these important avenues is the prevention of metastasis by pro-apoptotic Par-4. In this review, we will focus on the therapeutic perspective of Par-4 with a special reference to its (Par-4) virgin prospect of devastating metastasis control.

Dual role of Par-4 in abrogation of EMT and switching on Mesenchymal to Epithelial Transition (MET) in metastatic pancreatic cancer cells

Epithelial‐mesenchymal transition (EMT) is a critical event that occurs during the invasion and metastatic spread of cancer cells. Here, we conceive a dual mechanism of Par‐4‐mediated inhibition of EMT and induction of MET in metastatic pancreatic cancer cells. First, we demonstrate that 1,1′‐β‐D‐glucopyranosyl‐3,3′‐bis(5‐bromoindolyl)‐octyl methane (NGD16), an N‐glycosylated derivative of medicinally important phytochemical 3,3′‐diindolylmethane (DIM) abrogates EMT by inducing pro‐apoptotic protein Par‐4. Induction of Par‐4 (by NGD16 or ectopic overexpression) strongly impedes invasion with inhibition of major mesenchymal markers viz. Vimentin and Twist‐1 epithelial marker‐ E‐cadherin. Further, NGD16 triggers MET phenotypes in pancreatic cancer cells by augmenting ALK2/Smad4 signaling in a Par‐4‐dependent manner. Conversely, siRNA‐mediated silencing of endogenous Par‐4 unveil reversal of MET with diminished E‐cadherin expression and invasive phenotypes. Additionally, we demonstrate that intact Smad4 is essential for Par‐4‐mediated maintenance of E‐cadherin level in MET induced cells. Notably, we imply that Par‐4 induction regulates E‐cadherin levels in the pancreatic cancer cells via modulating Twist‐1 promoter activity. Finally, in vivo studies with syngenic mouse metastatic pancreatic cancer model reveal that NGD16 strongly suppresses metastatic burden, ascites formation, and prolongs the overall survival of animals effectively.