Nandini Rangaraj

Curcumin mediated apoptosis in AK-5 tumor cells involves the production of reactive oxygen intermediates.

Curcumin, the active ingredient of the rhizome of Curcuma longa has anti‐inflammatory, antioxidant and antiproliferative activities. Although its precise mode of action remains elusive, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis in cancer cells. Curcumin was shown to be responsible for the inhibition of AK‐5 tumor (a rat histiocytoma) growth by inducing apoptosis in AK‐5 tumor cells via caspase activation. This study was designed to investigate the mechanism leading to the induction of apoptosis in AK‐5 tumor cells. Curcumin treatment resulted in the hyperproduction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (Δψm) and cytochrome c release to the cytosol, with the concomitant exposure of phosphatidylserine (PS) residues on the cell surface. This study suggests redox signalling and caspase activation as the mechanisms responsible for the induction of curcumin mediated apoptosis in AK‐5 tumor cells.

Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Antifungal Activities of Human Beta-Defensins HBD-1 to HBD-3 and Their C-Terminal Analogs Phd1 to Phd3

ABSTRACT The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3 against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3, although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the fungal membrane.

Design and Synthesis of C3-Pyrazole/Chalcone-Linked Beta-Carboline Hybrids: Antitopoisomerase I, DNA-Interactive, and Apoptosis-Inducing Anticancer Agents

A series of β‐carboline hybrids bearing a substituted phenyl and a chalcone/(N‐acetyl)‐pyrazole moiety at the C1 and C3 positions, respectively, was designed, synthesized, and evaluated for anticancer activity. These new hybrid molecules showed significant cytotoxic activity, with IC50 values ranging from <2.0 μM to 80 μM, and the structure–activity relationships (SAR) associated with substitutions at positions 1 and 3 of these hybrids was clearly addressed. Further, induction of apoptosis was confirmed by Annexin V‐FITC, Hoechst staining, and DNA fragmentation analysis. In addition, DNA photocleavage studies proved that two of the hybrids, (E)‐1‐(furan‐2‐yl)‐3‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)prop‐2‐en‐1‐one (7 d) and 1‐(3‐(furan‐2‐yl)‐5‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethanone (8 d) could effectively cleave pBR322 plasmid DNA upon irradiation with UV light. Active hybrid 8 d inhibited DNA topoisomerase I activity efficiently and preserved DNA in the supercoiled form. To further corroborate the biological activities, as well as to understand the nature of the interaction of these hybrids with DNA, spectroscopic studies were also performed. Unlike simple β‐carboline alkaloids, the binding mode of these new hybrid molecules with DNA was not similar, and both biophysical as well as molecular docking studies speculated a combilexin‐type of interaction with DNA. Further, an in silico study of these β‐carboline hybrids revealed their drug‐like properties.

Recombinant fusion proteins TAT‐Mu, Mu and Mu‐Mu mediate efficient non‐viral gene delivery

The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide‐based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA‐binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA‐binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in‐frame with TAT of sequence YGRKKRRQRRR to generate TAT‐Mu, while the other two constructs, Mu and Mu‐Mu, harbor a single copy or two copies of the Mu moiety.

Activity of Pyruvate Dehydrogenase A (PDHA) in Hamster Spermatozoa Correlates Positively with Hyperactivation and Is Associated with Sperm Capacitation

Abstract Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.

Selective cancer targeting via aberrant behavior of cancer cell-associated glucocorticoid receptor.

Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell-associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgenes in the nucleus, a phenomenon that remained unobserved in normal cells. The targeted lipoplex (i) showed GR-targeted transfections in all cancer cells experimented (P < 0.01), (ii) significantly diminished transfection in cancer cells when GR is downregulated (P < 0.01), and (iii) elicited specific nuclear translocation of targeted lipoplex in cancer cells, followed by upregulated transactivation of glucocorticoid response element (GRE)- promoted gene. Using anticancer gene, targeted lipoplex induced significant tumor growth retardation in mice in comparison to different control groups (P < 0.05). Interestingly, cell surface-associated Hsp90 in cancer cells assisted the intracellular uptake of GR-targeted lipoplex. Moreover, selective inhibition of Hsp90 in noncancer cells resulted in cancer cell-like, aberrant, GR activation. The current study discovers a therapeutically important, unique property of cancer cell associated-GR that may be linked to a compromised role of Hsp90.Molecular Therapy (2009) 17 4, 623-631 doi:10.1038/mt.2009.4.

Quantitative aspects of endocytic activity in lipid-mediated transfections

Variation in transfection efficiency observed in different cell‐types is poorly understood. To investigate the influence of endocytic activity on lipid‐mediated transfections, we have monitored both the processes in 12 different cell‐types. The endocytic activity shows a strong positive correlation (P < 0.01), with transfection efficiency. Treatment with wortmannin resulted in cell‐type‐dependent inhibition of transfection. Studies on M‐phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell‐types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid‐mediated transgene expression.

Involvement of NKR-P2/NKG2D in DC-mediated killing of tumor targets: indicative of a common, innate, target-recognition paradigm?

DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein‐2 (NKR‐P2) on rat and mouse DC, and we show that NKR‐P2 gets reorganized upon antigen contact. DC activated with anti‐NKR‐P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR‐P2 and its ligand with rNKR‐P2 abrogated apoptotic killing, suggesting NKR‐P2 to function as an activating molecule on DC. In vivo injection of anti‐NKR‐P2 mAb augmented DC activity and delayed tumor progression. NKR‐P2 signaling involved Ca2+ influx, culminating in the expression of the apoptosis‐inducing molecule, TNF‐α. Taken together, these observations suggest that NKR‐P2 (the rat orthologue of human NKG2D) acts as a target‐recognition molecule on DC.

Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis

Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE−/− mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE−/− mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.