Bilal Rah

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.

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.

Anticancer activity, toxicity and pharmacokinetic profile of an indanone derivative

The present study describes anticancer effect of gallic acid based indanone derivative (1). Indanone 1 exhibited in vivo anticancer activity against Erhlich ascites carcinoma in Swiss albino mice by inhibiting tumor growth by 54.3% at 50 mg/kg b.wt. It showed antitubulin effect by inhibiting tubulin polymerase enzyme. In cell cycle analysis, it inhibited G2/M phase and induced apoptosis. It significantly suppressed VEGF-R1, VEGF-R2 and HIF-α in human breast cancer MCF-7 cells, thus exhibiting antiangiogenic activity. In acute oral toxicity, indanone 1 was well tolerated and was found to be non-toxic up to 1000 mg/kg b.wt. in Swiss albino mice. Pharmacokinetic studies in rabbits revealed rate of absorption, half life, volume of distribution with high plasma and blood clearance after i.v. administration. Indanone 1, is a safe and moderately active anticancer agent.

Design and synthesis of novel N,N′-glycoside derivatives of 3,3′-diindolylmethanes as potential antiproliferative agents

A library of 34 compounds containing the DIM core have been synthesized and tested for their anticancer efficacy by measuring their cytotoxicity to cancer cell lines A549, HeLa and MCF-7. Some of the selected derivatives were N-glycosylated to increase their efficacy. Compound 7d, an N-glycosylated DIM derivative, was found to be effective at 1.3, 0.3 and 0.9 μmol concentrations against A549, HeLa and MCF-7, respectively. Immunochemistry studies revealed that it could induce apoptosis by upregulating a pro-apoptotic protein Par-4 and concomitantly diminishing the expression of pro-survival proteins Bcl-2 and GRP78. Flow cytometry studies showed that the compound arrested cells in the G1 phase of the cell cycle and significantly abrogated the motility of HeLa cells. Computer docking simulations of 7d with GRP78 suggested its involvement in two H-bonds with Asp78, two H-bonds with Arg290, one with Arg367, and one water mediated H-bond interaction. The interaction patterns also demonstrated that the presence of bromide in the vicinity (within 3.5 A) of Lys294 generates the possibility of a halogen bond, which may also contribute in providing some extra stability to the complex. Hence, compounds of this class will be useful for the design of new anticancer agents.

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.

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 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.

Isolation and characterization of Streptomyces tauricus from Thajiwas glacier—a new source of actinomycin-D

The aim of the present study was to isolate potent anticancer compound from actinomycetes strain SRP18 isolated from soil of high altitude region of Thajiwas glacier, Sonamarg Kashmir—India. The potential isolate SRP18 was identified as Streptomyces tauricus on the basis of 16S rDNA sequence technique. The large scale cultivation of potent strain, Streptomyces tauricus SRP18 and subsequent isolation and purification by a series of chromatographic techniques resulted in isolation of potent anticancer compound. Structure elucidation of potent compound by using various spectroscopic techniques including NMR was confirmed to be actinomycin–D. As such, this is the first report of a strain of S. tauricus capable of producing this bioactive compound. The isolated compound was evaluated for its cytotoxicity potential against four human cancer cell lines like HeLa, PC-3, THP-1, and Caco-2 and showed a potent activity with IC50 range of 4.91–7.25 µM.