Manoj Kumar Bhat

Synthesis and antifungal activity of 1,2,3-triazole containing fluconazole analogues

Fluconazole based novel mimics containing 1,2,3-triazole were designed and synthesized as antifungal agents. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 12, 15, and 16 were found to be more potent against Candida fungal pathogens than control drugs fluconazole and amphotericin B. The studies presented here provide structural modification of fluconazole to give 1,2,3-trazole containing molecules. Furthermore, these molecules were evaluated in vivo against Candida albicans intravenous challenge in Swiss mice and antiproliferative activities were tested against human hepatocellular carcinoma Hep3B and human epithelial carcinoma A431. It was found that compound 12 resulted in 97.4% reduction in fungal load in mice and did not show any profound proliferative effect at lower dose (0.001 mg/ml).

The hypoglycaemic activity of fenugreek seed extract is mediated through the stimulation of an insulin signalling pathway

The in vivo hypoglycaemic activity of a dialysed fenugreek seed extract (FSE) was studied in alloxan (AXN)‐induced diabetic mice and found to be comparable to that of insulin (1.5 U kg−1). FSE also improved intraperitoneal glucose tolerance in normal mice. The mechanism by which FSE attenuated hyperglycaemia was investigated in vitro. FSE stimulated glucose uptake in CHO‐HIRc‐mycGLUT4eGFP cells in a dose‐dependent manner. This effect was shown to be mediated by the translocation of glucose transporter 4 (GLUT4) from the intracellular space to the plasma membrane. These effects of FSE on GLUT4 translocation and glucose uptake were inhibited by wortmannin, a phosphatidylinositol 3‐kinase (PI3‐K) inhibitor, and bisindolylmaleimide 1, a protein kinase C (PKC)‐specific inhibitor. In vitro phosphorylation analysis revealed that, like insulin, FSE also induces tyrosine phosphorylation of a number of proteins including the insulin receptor, insulin receptor substrate 1 and p85 subunit of PI3‐K, in both 3T3‐L1 adipocytes and human hepatoma cells, HepG2. However, unlike insulin, FSE had no effect on protein kinase B (Akt) activation. These results suggest that in vivo the hypoglycaemic effect of FSE is mediated, at least in part, by the activation of an insulin signalling pathway in adipocytes and liver cells.

Direct interaction with and activation of p53 by SMAR1 retards cell-cycle progression at G2/M phase and delays tumor growth in mice.

The tumor‐suppressor p53 is a multifunctional protein mainly responsible for maintaining genomic integrity. p53 induces its tumor‐suppressor activity by either causing cell‐cycle arrest (G1/S or G2/M) or inducing cells to undergo apoptosis. This function of wild‐type p53 as “guardian of the genome” is presumably achieved by forming molecular complexes with different DNA targets as well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45, p21, 14‐3‐3σ, Bax and Apaf‐1. Upon activation, p53 activates p21, which in turn controls the cell cycle by regulating G1 or G2 checkpoints. Here, we report SMAR1 as one such p53‐interacting protein that is involved in delaying tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a newly identified MARBP involved in chromatin‐mediated gene regulation. The SMAR1 gene encodes at least 2 alternatively spliced variants: SMAR1L (the full‐length form) and SMAR1S (the shorter form). We report that expression of SMAR1S, but not of SMAR1L, mRNA was decreased in most of the human cell lines examined, suggesting selective silencing of SMAR1S. Overexpression of SMAR1S in mouse melanoma cells (B16F1) and their subsequent injection in C57BL/6 mice delays tumor growth. Exogenous SMAR1S causes significant retardation of B16F1 cells in the G2/M phase of the cell cycle compared to SMAR1L. SMAR1S activates p53‐mediated reporter gene expression in mouse melanoma cells, breast cancer cells (MCF‐7) and p53 null cells (K562), followed by activation of its downstream effector, p21. We further demonstrate that SMAR1 physically interacts and colocalizes with p53. These data together suggest that SMAR1 is the only known MARBP that delays tumor progression via direct activation and interaction with tumor‐suppressor p53.

Diet-induced obesity increases melanoma progression: Involvement of Cav-1 and FASN

Recent population‐based epidemiological studies strongly hint towards a link between obesity and its occurrence as well as progression of several cancers including melanoma. Although effects of obesity on breast, colon and liver cancers have been extensively investigated, the links between obesity and melanoma remain largely unexplored. Present study aimed to understand the effect of high fat diet‐induced weight gain on susceptibility of C57BL/6J mice to melanoma. For this, mice routinely were fed on high fat diet for 6 months (HFD mice). Subsequently, mouse melanoma cells were injected subcutaneously in control as well as HFD mice and followed for tumor initiation and progression. We provide strong evidence that diet‐induced obesity leads to increased melanoma progression in male C57BL/6J mice. We observed that increased melanoma progression is associated with enhanced Cav‐1 and FASN expression in tumors from HFD mice. Cav‐1 and FASN are co‐ordinately regulated and Cav‐1 interacts with FASN in melanoma cells. Enhanced levels of Cav‐1, FASN and pAkt control melanoma cell proliferation. Our study establishes a causative relationship between diet‐induced obesity and melanoma progression as well as demonstrates that obesity affects important tumorigenic pathways in melanoma.

Synthesis, characterization, plasmid cleavage and cytotoxicity of cancer cells by a copper(II) complex of anthracenyl-terpyridine

Metallo-organic compounds are interesting to study for their antitumor activity and related applications. This paper deals with the syntheses, characterization, structure determination of a copper complex of anthracenyl terpyridine (1) and its plasmid cleavage and cytotoxicity towards different cancer cell lines. The complex binds CT-DNA through partial intercalation mode. The plasmid cleavage studies carried out using pBR322 and pUC18 resulted in the formation of all the three forms of the plasmid DNA. Plasmid cleavage studies carried out with a non-redoxable Zn(2+) complex (2) supported the role of the redox activity of copper in 1. The complex 1 showed remarkable antiproliferative activity against cancer cell lines, viz., cervical (HeLa, SiHa, CaSki), breast (MCF-7), liver (HepG2) and lung (H1299). A considerable lowering was observed in the IC(50) values of HPV-infected (viz., HeLa, SiHa, CaSki) vs. non-HPV-infected cell lines (MCF-7, HepG2, H1299). Antiproliferative activity of 1 was found to be much higher than the carboplatin when treated with the same cell lines. Incubation of the cells with 1 results in granular structures only with the HPV-infected cells and not with others as studied by phase contrast and fluorescence microscopy. The lower IC(50) value observed in case of 1 with HPV-infected cell lines may be correlated with the involvement of HPV oncoprotein. The role of HPV has been further augmented by transfecting the MCF-7 cells (originally not possessing HPV copy) with e6 oncoprotein cDNA. To our knowledge this is the first copper complex that causes cell death by interacting with HPV oncoprotein followed by exhibition of remarkable antiproliferative activity.

Inherent and Acquired Resistance to Paclitaxel in Hepatocellular Carcinoma: Molecular Events Involved

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and is a major cause of cancer related deaths worldwide. Only 10 to 20% of HCC can be surgically excised. Therefore, chemotherapeutic intervention and treatment is essential for achieving favorable prognosis. However, therapeutic outcome of chemotherapy is generally poor owing to inherent resistance of cancer cells to the treatment or due to development of acquired resistance. To differentiate and delineate the molecular events, we developed drug resistant Hep3B cells (DRC) by treating cells with the increasing concentration of paclitaxel. We also developed a unique single cell clone of Hep3B cells (SCC) by selecting single cell colonies and screening them for resistant phenotype. Interestingly, both DRC and SCC were resistant to paclitaxel in comparison to parental Hep3B cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, level of P-glycoprotein (P-gp) was elevated in DRC. In addition, Caveolin-1 (Cav-1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) protein levels were elevated in DRC whereas in SCC, FASN and CYP450 levels were elevated. Downregulation of these molecules by respective siRNAs and/or by specific pharmacological inhibitors resensitized cells to paclitaxel. Interestingly, these drug resistant cells were also less sensitive to vinblastine, doxorubicin and methotrexate with the exception of cisplatin. Our results suggested that differential levels of P-gp, Cav-1 and FASN play a major role in acquired resistant phenotype whereas FASN level was associated with the presentation of inherent resistant phenotype in HCC.

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

Backgroundp53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV) E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5) is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment.ResultsTo determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well.ConclusionPresent study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

Hyperglycemia regulates MDR‐1, drug accumulation and ROS levels causing increased toxicity of carboplatin and 5‐fluorouracil in MCF‐7 cells

There is constant increase in number of diabetic cases thereby giving it status of a serious epidemic. Diabetes increases the risk of occurrence of several cancers including breast cancer and may also have a serious impact on the outcome of cancer treatment. In the present study we investigated effect of hyperglycemia on cytotoxic efficacy of carboplatin and 5‐fluorouracil in MCF‐7 cells. MCF‐7 cells were grown in 5.5 or 25 mM glucose chronically. We show that hyperglycemia favors proliferation of MCF‐7 cells and increases expression of cell cycle regulatory proteins cyclin E and cdk‐2. Hyperglycemia enhances cytotoxicity of carboplatin and 5‐fluorouracil in MCF‐7 cells by approximately 30% and decreases their IC50 by 1.5‐ and 1.3‐folds, respectively. Hyperglycemia reduces expression of P‐glycoprotein and promotes cell killing by increasing drug accumulation. Treatment with 40 µM verapamil, an inhibitor of P‐gp activity specifically increases killing of MCF‐7 cells cultured in 5.5 mM glucose. Further, this effect is synergized by elevated reactive oxygen species and treatment with N‐Acetylcysteine, an inhibitor of ROS, increases survival by 30 and 18% in carboplatin‐ and 5‐fluorouracil‐treated cells cultured in high glucose, respectively. Cytotoxicity of these drugs is associated with reduced activation of Akt and decreased transcriptional activation of NF‐κB. In conclusion, hyperglycemia potentiates cytotoxicity of drugs by reducing P‐gp expression and, increased ROS levels may partially or completely contribute to enhanced toxicity. J. Cell. Biochem. 112: 2942–2952, 2011.

Inhibition of hsp27 and hsp40 potentiates 5-fluorouracil and carboplatin mediated cell killing in hepatoma cells

Heat shock proteins (Hsps) modulate several cellular functions and are ubiquitously present in cell. Here, we investigated alterations in the expression of Hsps and explored functional consequences of the same. Moreover, effect of quercetin (Qctn), an inhibitor of Hsps, on chemotherapeutic drugs treatment in hepatoma cells Hep3B and HepG2 was investigated. We for the first time report that 5-fluorouracil (5-FU) and carboplatin specifically induce expression of Hsp40 in addition to Hsp27 in Hep3B and HepG2 cells. Induction of Hsps following exposure to sub lethal dose of drugs is a cellular challenge to survival. However, under lethal environmental conditions with reduced cell viability, cells fail to sustain the induction of survival proteins, Hsp27 and Hsp40. Though Qctn itself, to certain extent is cytotoxic to cells, it potentiates the pro-apoptotic action of 5- FU and carboplatin, by inhibiting expression of Hsps. The increased cell killing correlates with decreased levels of procaspase-3. Furthermore, siRNA mediated knockdown of Hsp27 and Hsp40 diminishes survival of drugs exposed cells. Altogether, our data provides clear evidence that Hsp27 and 40 promote cell survival and inhibition of their expression does not allow cells to adapt to drug exposure and survive. Collectively, our novel findings on compelling action of 5-FU or carboplatin following knockdown of Hsp40 and that of Hsp27 highlights their strategic implications towards an effective therapy against HCC.

Cytotoxic activity of 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India). AIM OF THE STUDY To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells. MATERIALS AND METHODS The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents-chloroform-methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including (1)H NMR, (13)C NMR, NOESY, COSY, LC-MS and IR. RESULTS The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 microg/ml concentration. CONCLUSION 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer.