Aashiq Hussain

Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

Background: Pharmacological investigations position withanolides as important bioactive molecules demanding their copious production. Results: Differential transcriptional and translational expression of three oxidosqualene cyclases leads to redirection of metabolic fluxes. Conclusion: Negative regulator channelizes substrate pool toward cycloartenol synthase at subdividing junction leading to enhanced withanolide production. Significance: Understanding the regulatory role of oxidosqualene cyclases on withanolide accumulation could serve as a prognostic tool for metabolic engineering. Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

Interaction of natural products with cell survival and signaling pathways in the biochemical elucidation of drug targets in cancer

The use of natural products with therapeutic properties is as ancient as human civilization and for a long time mineral, plant and animal products were the main sources of drugs. Worldwide sales of medicinal plants, crude extracts and finished products amounted to US

Recent Development in Targeting PI3K-Akt-mTOR Signaling for Anticancer Therapeutic Strategies

15 billion in 1999 and it increased to

Understanding Histone Deacetylases in the Cancer Development and Treatment: An Epigenetic Perspective of Cancer Chemotherapy

23 billion in 2002. More interestingly, the influence of natural products upon anticancer drug discovery and design cannot be underestimated. Approximately 60% of all drugs in clinical trials are either a natural product, compounds derived from natural products or contain pharmacophores derived from active natural products. Thus, even today, in the presence of massive numbers of agents from combinatorial libraries, compounds from natural sources are still in the forefront of cancer chemotherapeutics as sources of active drug types, as well as being involved in drug discovery in diseases such as microbial and parasitic infections and the control of cholesterol/lipids, among other functions.

Click chemistry inspired synthesis and bioevaluation of novel triazolyl derivatives of osthol as potent cytotoxic agents.

Cancer is a diverse class of diseases which differ widely in their cause and biology. The aberrant behavior of cancer reflects up regulation of certain oncogenic signaling pathways that promote proliferation, inhibit apoptosis, and enable the cancer to spread and evoke angiogenesis. Phosphoinositide-3-kinase(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls various biological processes that are important for normal functioning of the cell via cell cycle progression, survival, migration, transcription, translation and metabolism. However, PI3K signaling pathway is dysregulated almost in all cancers which is due to the amplification and genetic mutation of PI3K gene, encoding catalytic and regulatory subunit of PI3K isoforms. The current review focuses on the structural features of various PI3K isoforms including Akt and mTOR and their inhibition using specific small molecule inhibitors in an attempt to achieve an attractive target for cancer prevention and chemotherapy.

Therapeutic Targeting of Cancer Cell Metabolism: Role of Metabolic Enzymes, Oncogenes and Tumor Suppressor Genes

Cancer is a pathologic condition that involves genetic and epigenetic events culminating in neoplastic transformation. Alteration in epigenetic events that regulate the transcriptional activity of genes associated with various signaling pathways can influence multiple stages of tumorigenesis. In cancer cells, an imbalance often exists between histone acetyl transferase and histone deacetylase (HDAC) activities, and current research focuses actively on seeking competitive HDAC inhibitors (HDACi) for chemotherapeutic intervention. HDACi are proving useful for cancer prevention and therapy by virtue of their ability to reactivate the expression of epigenetically silenced genes, including those involved in differentiation, cell cycle regulation, apoptosis, angiogenesis, invasion, and metastasis. Furthermore, epidemiological studies suggest that different diets such as intake of cruciferous vegetables may lower the risk of different cancers, and there is growing interest in identifying the specific chemoprotective constituents and mechanistic insights of their action. Interestingly, it has been observed that cancer cells are more sensitive than nontransformed cells to apoptotic induction by some HDACi. Although the mechanistic basis for this sensitivity is unclear, yet HDACi have emerged as important epigenetic target for single and combinatorial chemotherapy. HDACi derived from diverse sources such as microbial, dietary, and synthetic increase acetylation level of cells and bring about anti-proliferative and apoptotic effects specific to cancer cells by way of their role in cell cycle regulation and expression of epigenetically silenced genes.

PI3K target based novel cyano derivative of betulinic acid induces its signalling inhibition by down-regulation of pGSK3β and cyclin D1 and potentially checks cancer cell proliferation

A new series of diverse triazoles linked through the hydroxyl group of lactone ring opened osthol (1) were synthesized using click chemistry approach. All the derivatives were subjected to 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) cytotoxicity screening against a panel of seven different human cancer cell lines viz. colon (colo-205), colon (HCT-116), breast (T47D), lung (NCI-H322), lung (A549), prostate (PC-3) and Skin (A-431) to check their cytotoxic potential. Interestingly, among the tested molecules, most of the analogs displayed better cytotoxic activity than the parent osthol (1). Of the synthesized triazoles, compounds 8 showed the best activity with IC50 of 1.3, 4.9, 3.6, 41.0, 35.2, 26.4 and 7.2 μM against colon (Colo-205 and HCT-116), breast (T47D), lung (NCI-H322 and A549), prostate (PC-3) and Skin (A-431) cancer lines respectively. Compound 8 induced potent apoptotic effects in Colo-205 cells. The population of apoptotic cells increased from 11.4% in case of negative control to 24.1% at 25 μM of 8. Compound 8 also induced a remarkable decrease in mitochondrial membrane potential (ΛΨm) leading to apoptosis of cancer cells used. The present study resulted in identification of broad spectrum cytotoxic activity of analogs bearing electron withdrawing substituents, besides the enhanced selective activity of analogs with electron donating moieties.

Quinazoline based small molecule exerts potent tumour suppressive properties by inhibiting PI3K/Akt/FoxO3a signalling in experimental colon cancer

Cancer cells compared to their normal counterparts reveal different metabolic needs and this differential requirement of metabolic intermediates and their subsequent consequences require an elaborate understanding of cancer cell metabolism and increased energy production in these cells. Nevertheless these metabolic differences have provided opportunities for developing novel therapeutic approaches for the cancer diagnosis and treatment. In addition enhanced proliferative capacities of tumor cells associated with aberrations of many signal transduction pathways resulting from genetic or epigenetic alterations has made it possible to develop countless targeted therapeutics for several types of malignancies. However at present most of our understanding about the dysregulated cancer cell metabolism is at physiological stages. With advancement in technology development, we may eventually be able to differentiate the metabolic differences between normal cells and cancerous at the single-tumor level that may influence the development of personalized cancer medicine. In this review, the focal point will be the recent developments in understanding the crucial role of metabolic enzymes, oncogenes and tumor suppressor genes in progression of cancer and their targeting to establish the most appropriate therapeutic strategies for better clinical outcome.

Modulation of glycolysis and lipogenesis by novel PI3K selective molecule represses tumor angiogenesis and decreases colorectal cancer growth.

In spite of the Betulinic acid (BA) being recognized as anticancerous source; its further use in clinical development is greatly hampered because of its poor pharmacokinetic properties. To circumvent these limitations, we synthesized a PI3K target based library of 18 triazole based derivatives and we identified a C‐3 cyano analog of betulinic acid (CBA) with significant cell death effects with 5–7 fold higher potency than BA in various cancers. Importantly, no such report is available demonstrating the involvement of BA or its structural analogs in the modulation of PI3K pathway. Using, human leukemia HL‐60 cells as a model, we for the first time report that CBA decreased expression of PI3K p110α, p85α, and pAKT in HL‐60. Furthermore, we could find significant depletion of pGSK3β, cyclin D1 and increased expression of p21/cip, p27/Kip proteins. CBA induced G0/G1 cell cycle arrest, increased sub‐G0 DNA fraction and annexin V binding of the cells besides imparting the typical surface features of cell death. Also, this target specific inhibition was associated with mitochondrial apoptosis as was reflected by expression studies of various proteins together with reactive oxygen species generation and decline in mitochondrial trans membrane potential. The apoptotic effectors i.e., caspase 8 and caspase 9 were found to get upregulated besides PI3K associated DNA repair enzyme i.e., PARP cleavage was observed. Thus, our results elucidated that CBA or other BA based small molecules inhibit PI3K/AKT pathway with induction of subsequent cancer cell death which may be useful therapeutic strategy against leukemias and possibly other cancers.

Gene specific epigenetic regulation of hepatic folate transport system is responsible for perturbed cellular folate status during aging and exogenous modulation.

Deregulation of PI3K signalling pathway is strongly involved in pathology of cancer and development of resistance in tumour cells. Here, we report that pharmacologically active vasicinone analogue, RLX (7, 8, 9, 10-Tetrahydroazepino [2, 1-b] quinazolin-12-(6H)-on), exhibited potent anticancer activities both in vitro and in vivo. In this study, RLX treatment displayed strong inhibition of proliferation against various cancer cell lines. However, colon cancer cells were found to be the most sensitive towards RLX mediated inhibition of proliferation. The result showed that RLX treatment followed strong concentration dependent inhibition of HCT-116 cell proliferation and colony formation. RLX treatment to HCT-116 was observed to be associated with down-regulation of p110α and p85 subunits of PI3K thereby decreasing the expression of subsequent downstream effector proteins. Interestingly, silencing of PI3K gene by siRNA in combination with RLX confirmed the anti-proliferation effect of RLX against HCT-116 cells and is mediated by the PI3K pathway. We also found that RLX induced sub-G1 arrest and mitochondrial potential loss followed by pFoxO3a(Thr32) nuclear-cytoplasmic translocation inhibition. Moreover, RLX treatment in in vivo models substantially resulted in a tumour growth inhibition. Overall, our findings reveal the functional role of the PI3K/Akt/FoxO3a pathway that gets deregulated in cancer and suggests its simultaneous targeting by RLX thereby further identifying the compound as a potent inhibitor of the PI3K/Akt/FoxO3a pathway under in vitro and tumour regression in vivo.