Neha Nair

γ-Glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region.

Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70°C. It had K(m), V(max) of 0.045 mM, 4.35 μmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

Synthesis, Biological Evaluation, Docking and QSAR Studies of SomeNovel Naphthalimide Dithiocarbamate Analogs as Antitumor and Anti-Inflammatory Agents

A series of novel naphthalimide dithiocarbamate 4a-f, 5a-f were efficiently synthesized via introduce dithiocarbamate and dithioate side chain onto the naphthalic anhydride core. The structures of the synthesized analogs were elucidated by spectroscopic methods, including IR,1H and 13C NMR, and (ESIHRMS) techniques. The anti-cancer activities of the generated naphthalimide derivatives 4c, 4d, 4e, 4f, and 5d were evaluated against 21 tumour cell lines; inculding 10 tumor subpanels using MTT assay. Analogue 4c offer antitumor activity with an IC50 of 10.54 μM against SKBR3 breast cancer cells. Compound 4d showed varying degrees of antitumor activities towards several tumour cell lines ranging from 21.1 to 71.7 μM. In addition to the antitumor activities; the synthesized compounds were evaluated for their In vitro anti-inflammatory activity. Compounds 4c and 4d revealed potent anti-inflammatory properties in comparison with the reference drug celecoxib. Molecular docking studies provided complementary theoretical support for experimental biological data.

Microbial Keratinases: Diversity and Applications

Keratinases are unique proteases that are capable of degrading recalcitrant, “hard-to-degrade” keratin residues. Diverse microorganisms that belong to Eukarya, bacteria, and Archaea produce these enzymes. A large number of Bacilli, Actinobacteria, and fungi are reported to produce keratinases. Microbial keratinases present great diversity in their biochemical and biophysical properties. They are robust enzymes with wide temperature and pH activity range. They are optimally active at neutral to alkaline and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Studies with specific substrates and inhibitors indicated that keratinases preferentially act on hydrophobic and aromatic residues at P1 position. Keratinases have several current and potential applications in agro-industrial, pharmaceutical, and biomedical fields. These enzymes are useful in processes related with the bioconversion of keratin waste into feed and fertilizers. Other promising applications are enzymatic dehairing for leather and cosmetic industry, detergent industry, and development of biopolymers from keratin fibers. The use of keratinases to enhance drug delivery in some tissues and hydrolysis of prion proteins arises as novel outstanding applications. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling. Looking into their biotechnological impetus, they are being cloned and expressed in a variety of heterologous hosts.

Abstract LB-278: Cancer stem cells as the novel origin of cancer-associated fibroblast-like cells

Cancer associated fibroblast (CAF) is an indispensible component of stromal microenvironment mediating cancer progression notably in breast, prostate and pancreatic carcinoma. CAF heterogeneity is attributed to its possible multiple origin majorly from tumor resident fibroblast, mesenchymal stem cells, epithelial cells. The present work analyses the possibility of cancer stem cells (CSC) as the source of CAF like cells. For this, we have used the model of CSC established in our laboratory derived from mouse induced pluripotent cells (miPSCs) by culturing with conditioned medium from cancer cell lines mimicking tumor microenvironment. In the present study, human breast cancer derived T47D and BT549 cells have been cultured to collect the conditioned medium. Since the green fluorescent protein (GFP) gene was integrated under the control of Nanog promoter in the miPSCs used in this study, the resulting CSCs was assessed by the expression of GFP for the undifferentiated state. The differentiation of CSCs was tracked by the attenuation of GFP. The CSC population was multiplied by serial subcutaneous and orthotropic transplantation in nude mice. This population was further enriched by sphere formation. Then these spheres were allowed to differentiate into adhesive cells. The CAF-like cells were separated from the differentiated population. The expression of prominent CAF markers fibroblast specific protein (FSP), fibroblast activation protein (FAP), vimentin, collagen type1 α1, and smooth muscle actin (SMA) were assessed by real time PCR. Immuno-cytochemistry of the cells for the expression of FSP, FAP and SMA was further confirmed for the evidence that cancer stem cells should be a possible source of CAF. This model should elaborate the functional heterogeneity amongst CAFs and help design therapeutic interventions to inhibit such conversions. Citation Format: Neha Nair, Arun Vaidyanath, Kenta Hoshikawa, Anna Sanchez Calle, Tomonari Kasai, Masaharu Seno. Cancer stem cells as the novel origin of cancer-associated fibroblast-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-278.