Sumita Sengupta

Direct amide bond formation from carboxylic acids and amines using activated alumina balls as a new, convenient, clean, reusable and low cost heterogeneous catalyst

For the first time, we have used activated alumina balls (3–5 mm diameter) for amide synthesis from carboxylic acids (unactivated) and amines (unactivated) under neat reaction conditions that produce no toxic by-products and has the advantages of being low-cost, easily available, heterogeneous, reusable and environmentally benign with no troublesome/hazardous disposal of the catalyst.

Stevioside Induced ROS-Mediated Apoptosis Through Mitochondrial Pathway in Human Breast Cancer Cell Line MCF-7

Stevioside is a diterpene glycoside found in the leaf of Stevia rebaudiana, a traditional oriental medicinal herb, which has been shown to have various biological and ethno-medicinal activities including antitumor activity. In this study, we investigated the effects of stevioside on the cytotoxicity, induction of apoptosis, and the putative pathways of its action in human breast cancer cells (MCF-7). For the analysis of apoptotic pathway, measurement of reactive oxygen species (ROS) and assessment of mitochondrial transmembrane potential (MTP) were achieved. We showed that stevioside was a potent inducer of apoptosis and it conveyed the apoptotic signal via intracellular ROS generation; thereby inducing change in MTP and induction of mitochondrial mediated apoptotic pathway. Taken together, our data indicated that stevioside induces the ROS-mediated mitochondrial permeability transition and results in the increased expression of apoptotic proteins such as Bax, Bcl-2 and Caspase-9. Effect of stevioside on stress-related transcription factors like NF-E2-related factor-2 opens up a new vista for further studies. This is the first report on the mechanism of the antibreast cancer (in vitro) activity of stevioside.

Role of Di-allyl Disulfide, a Garlic Component in NF-κB Mediated Transient G2-M Phase Arrest and Apoptosis in Human Leukemic Cell-lines

Diallyl disulfide (DADS), the major organosulfur component of processed garlic is very effective in chemoprevention of several types of cancers; however, its detailed mechanism is yet to be divulged. Present study shows antiproliferative activity of DADS against human leukemic cell-lines, mainly U937. DADS induced transient G2/M phase arrest, which is evident from FACS analysis. The results revealed that a significant transcriptional induction of p21 happened in early hours of treatment, which is due to increased nuclear translocation of NF-κB and its specific binding to p21 promoter. However, in the later hours, G2/M arrest is lost leading to apoptosis via intrinsic mitochondria-mediated pathway through generation of reactive oxygen species followed by changes in mitochondrial membrane potential. Western blots indicate release of cytochrome-c, activation of caspase-3, cleavage of PARP1, and finally decrease in bcl-2 levels. In addition, inactivation of NF-κB by its inhibitor BAY 11-7085 causes early onset of apoptosis without any transient G2/M arrest. Thus, in conclusion, DADS induces reversible G2/M arrest through NF-κB mediated pathway in human leukemic cell lines, like U937, K562, and Jurkat, lacking wild type p53. However, G2/M arrest is lost owing to the incapability of the damage repair system that leads to apoptosis.

Synthesis of 2-alkyl substituted benzimidazoles under microwave irradiation: Anti-proliferative effect of some representative compounds on human histiocytic lymphoma cell U937

The objective of this research was the synthesis of 2-alkyl substituted benzimidazoles under microwave irradiation using 62% aqueous hexafluorophosphoric acid (10 mol%) as catalyst. Some of the newly synthesised compounds were tested for their potential to inhibit proliferation of cancer cells (histiocytic lymphoma cell U937) by cell viability assay.

Role of diallyl disulfide-mediated cleavage of c-Myc and Sp-1 in the regulation of telomerase activity in human lymphoma cell line U937

OBJECTIVE Garlic (Allium sativum) has been considered a wonder herb for years with a reputation of disease prevention. Telomerase, a ribonucleoprotein enzyme responsible for telomere integrity, is strongly up-regulated in different types of cancers. The aim of this study was to reveal the role of diallyl disulfide (DADS), an organosulfur component of garlic, on telomerase activity in human lymphoma with an emphasis on key transcription factors c-Myc and Sp-1. METHODS Human lymphoma cell line U937 was used as model cell line. Telomerase activity was measured by telomerase repeat amplification protocol assay, levels of related proteins and mRNAs were measured by Western blot and reverse transcriptase polymerase chain reaction, respectively. Moreover, in vitro binding assay was performed using radiolabeled double-stranded DNA having specific sequences to detect involvement of transcription factors in DADS-dependent modulation of telomerase activity. RESULTS The present study demonstrated DADS-mediated decrease in telomerase activity in U937 cells with concomitant transcriptional down-regulation of human telomerase reverse transcriptase (hTERT) that is caused by reduced binding of c-Myc and Sp-1 to their respective binding sites on hTERT promoter. Lowering of DNA-binding activity of c-Myc and Sp-1 due to DADS treatment is caused by the deactivation of these transcription factors due to cleavage. Additionally, Mad1-the repressor protein of hTERT expression-is also overexpressed in DADS-treated U937 cells. CONCLUSIONS These findings strongly suggest that DADS down-regulate telomerase activity through c-Myc-, Sp-1-, and Mad1-dependent transcriptional down-regulation of hTERT.

Withaferin A induced impaired autophagy and unfolded protein response in human breast cancer cell-lines MCF-7 and MDA-MB-231

The autophagy-lysosome pathway and the ubiquitin-proteasome systems are the two major routes for eukaryotic intracellular protein clearance. Cancerous cells often display elevated protein synthesis and byproduct disposal, thus, inhibition of the protein degradation pathways became an emerging approach for cancer therapy. The present study revealed that withaferin-A (WA), the biologically active withanolide derived from Withania somnifera, initially induced formation of autophagosomes in human breast cancer cell-lines, MCF-7 and MDA-MB-231. WA treatment elevated the levels of autophagic substrate p62/SQSTM1 (p62) and both LC3-II and LC3-I (microtubule-associated protein 2 light chain 3) and simultaneously reduced the upstream autophagy markers like beclin-1 and ATG5-ATG12 complex, which indicate accumulation of autophagosomes in the cells. WA induced disruption of microtubular network through inhibition of tubulin polymerization and its hyper-acetylation, thus prevent the formation of autolysosome (by merging of autophagosomes with lysosomes) and its recycling process, leading to incomplete autophagy. Further, WA caused ER (Endoplasmic Reticulum) stress, which is evident from the activation of ER-related caspase-4 and increased levels of ER stress marker proteins. Thus, these findings altogether indicate that WA mediated inhibition of proteasomal degradation system and perturbation of autophagy, i.e. suppression of both the intracellular degradation systems caused accumulation of ubiquitinated proteins, which in turn led to unfolded protein response and ER stress mediated proteotoxicity in human breast cancer cell-lines, MCF-7 and MDA-MB-231.

Phorbol-12-myristate-13-acetate (PMA) mediated transcriptional regulation of Oncostatin-M

Oncostatin-M (OSM), an IL-6 family cytokine, exhibits varied roles in different patho-physiological conditions. Differential expression of OSM in response to varying stimuli indicates importance of its regulation of expression. The present study illustrated transcriptional induction of osm on treatment with chemical inducer, phorbol-12-myristate-13-acetate (PMA). Following initial hours of PMA treatment, a nuclear protein C/EBP-β binds specifically to the CCAAT consensus sequence at the proximal end of the OSM promoter. Genistein (a specific Tyr phosphorylation inhibitor) leads to the interaction of CHOP (C/EBP Homologous Protein) with C/EBP-β, thus negatively regulating it. Knockdown of C/EBP-β also leads to inhibition of PMA-mediated OSM induction.

Synthesis of diallyl disulfide (DADS) induced gold nanoparticles: characterization and study of its biological activity in human leukemic cell-lines

Novel approaches to nanoparticle synthesis using herbal products and their potential application in treatments are now in the limelight of recent cancer research. Diallyl disulfide (DADS), a bioactive component of garlic has been used for centuries as an effective remedy for different ailments including cancer. In the present study, DADS is used to synthesize less toxic, eco-friendly gold nanoparticles having anti-proliferative effects against cancer cells. DADS induced gold nanoparticles (D-GNPs) display a characteristic surface plasmon band near 551 nm which is 22 nm red shifted compared to conventionally prepared gold nanoparticles (GNPs) using tri-sodium citrate. Moreover, the hydrodynamic diameter of D-GNP ranges from 70 to 77 nm and its zeta potential is −24.6 mV. A nearly spherical ultra-structure of D-GNPs was visualized under atomic force microscopy and transmission electron microscopy. FT-IR analysis confirms the association of the sulfur group of DADS with these nanoparticles. D-GNPs show dose dependent cytotoxicity in human leukemic cell-lines U937 and K562. Cellular uptake of D-GNPs in U937 leading to nuclear fragmentation and DNA ladder formation are other insightful findings. This report therefore details the synthesis of stable gold nanoparticles using DADS and reveals D-GNPs to be a highly effective anti-proliferative agent showing apoptosis in human leukemia cell-lines.

Establishment of twist-1 and TGFBR2 as direct targets of microRNA-20a in mesenchymal to epithelial transition of breast cancer cell-line MDA-MB-231

Abstract Messenchymal to epithelial transition (MET) is a significant physiological phenomenon involved in embryogenesis and cancer. This study aims at investigating the mechanism of microRNA‐20a (miR‐20a) mediated regulation of mesenchymal to epithelial transition and identification of its direct target genes in breast cancer cell‐line, MDA‐MB‐231. Reduced migratory and invasive property, altered cellular morphology along with reduced capability for attachment to basement membrane was acquired by over‐expression of miR‐20a in invasive MDA‐MB‐231 cell‐line initially expressing low level of this micro‐RNA, indicating direct correlation between abundance of miR‐20a and metastatic property. The switch from mesenchymal to epithelial cells mediated by miR‐20a involved post‐transcriptional down‐regulation of twist1, which in turn controls downstream epithelial markers like E‐cadherin, claudin and mesenchymal markers like N‐cadherin, fibronectin, the crucial players of mesenchymal to epithelial transition (MET). Furthermore, another key component, TGF‐&bgr; and one of its receptors (TGFBR2) were found to be down‐regulated by miR‐20a. Additionally, reporter assay established that post‐transcriptional down‐regulation of TGFBR2 occurred through direct binding of miR‐20a to its 3′UTR, thus abrogating the TGF‐&bgr; signaling pathway resulting in inhibition of MET. Delineating the underlying molecular mechanism of miR‐20a‐mediated MET and defining the target genes will help us to introduce a miRNA‐mediated effective therapeutic strategy against breast cancer. HighlightsMetastatic property and miR‐20a level is directly correlated in MDA‐MB‐231 cell‐line.Over‐expression of miR‐20a down‐regulates expression of twist1.twist1 upregulates crucial epithelial markers and down‐regulates mesenchymal markers.TGF‐&bgr; and its receptor, TGFBR2 were found to be targets of miR‐20a.

Abstract C218: Novel therapeutic potential in targeting micro tubules by naturally occurring anti cancer agent withaferin A in human cancers.

Background: Chemical compounds from natural sources continue to serve as important pharmacological tools for the understanding of biological pathways and also provide chemical platform for new drug discovery. The utility of various anti-microtubule agents from natural origins are the best examples that continuously serve these two purposes. In this study, we have identified a novel tubulin-targeting activity of a natural product Withaferin-A. Though several groups have already shown that Withaferin-A is a potent anti-cancer agent, can kill variety of carcinomas by inducing apoptosis, but not much is known about its molecular targets inside the cell. This study is to evaluate the therapeutic efficacy of WA towards human cancer, in relation to its ability to directly targeting microtubule dynamics within cell. Methods: The IC-50 value of WA was determined by MTT assay, apoptosis and cell cycle assay was performed by FACS analysis, wound healing assay was done to see cellular migration, immune fluorescent detection was done to check microtubular network of WA treated cells. Effect of WA on tubulin polymerization in cell free system was studied by light scattering assay, binding measurements of WA to tubulin was determined by flurometric assay and probable WA-tubulin interaction site was proposed by molecular modeling method. Results: We have identified WA as a novel anti-microtubule agent that inhibits proliferation, migration and cellular progression by affecting mitosis in HeLa and MCF-7 cells. Brief incubation with nontoxic concentrations of WA induced significant depolymerization of interphase and mitotic spindle microtubules. It inhibits tubulin polymerization, induces conformational changes in tubulin by directly binding to purified tubulin. Both in silico and in-vitro studies indicated that WA preferably binds to a novel site on tubulin. Conclusion: It is evident that WA suppresses microtubule dynamics by directly binding to tubulin which sheds light on mechanisms behind its anti-proliferative activity towards cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C218.