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A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins, hemoglobin and myoglobin
No full textThe binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21 ± .05) × 105 M−1, while that to myoglobin was three times higher (3.59 ± .15) × 105 M−1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms
of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in
understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridin
Lysophosphatidic Acid Promotes Epithelial to Mesenchymal Transition in Ovarian Cancer Cells by Repressing SIRT1
Full text linkEpithelial-to-mesenchymal transition (EMT) plays an essential role in the transition from early to invasive phenotype, however the underlying mechanisms still remain
elusive. Herein, we propose a mechanism through which the class-III deacetylase SIRT1 regulates EMT in ovarian cancer (OC) cells. Methods: Expression analysis was performed using Q-PCR, western blot, immunofluorescence and fluorescence-IHC study. Matrigel invasion assay was
used for the invasion study. Morphological alterations were observed by phalloidin-staining. Co-immunoprecipitation study was performed to analyze protein-protein interaction. Results: Overexpression of SIRT1-WT as well as Resveratrol-mediated SIRT1 activation antagonized the invasion of OC cells by suppressing EMT. SIRT1 deacetylates HIF1α, to inactivate its transcriptional activity. To further validate HIF1α inactivation, its target gene, i.e. ZEB1, an EMTinducing factor was found to attenuate upon SIRT1 activation. To uncover the regulatory factor
governing SIRT1 expression, lysophosphatidic acid (LPA), a highly enriched oncolipid in ascites/ serum of OC patients, was found to down-regulate SIRT1 expression. Importantly, LPA was found to induce the mesenchymal switch in OC cells through suppression of SIRT1. Decreased level of SIRT1 was further validated in ovarian tissue samples of OC patients. Conclusion: We have identified a mechanism that relates SIRT1 down-regulation to LPA-induced EMT in OC
cells and may open new arenas on developing novel anti-cancer therapeutics
Methyl Jasmonate Regulates Podophyllotoxin Accumulation in Podophyllum hexandrum by Altering the ROS-Responsive Podophyllotoxin Pathway Gene Expression Additionally through the Down Regulation of Few Interfering miRNAs
Full text linkPodophylloxin (ptox), primarily obtained from Podophyllum hexandrum, is the precursor for semi-synthetic anticancer drugs viz. etoposide, etopophos, and teniposide. Previous
studies established that methyl jasmonate (MeJA) treated cell culture of P. hexandrum accumulate ptox significantly. However, the molecular mechanism of MeJA induced ptox
accumulation is yet to be explored. Here, we demonstrate that MeJA induces reactive oxygen species (ROS) production, which stimulates ptox accumulation significantly and
up regulates three ROS-responsive ptox biosynthetic genes, namely, PhCAD3, PhCAD4 (cinnamyl alcohol dehydrogenase), and NAC3 by increasing their mRNA stability. Classic
uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone, as well as H2O2 treatment induced the ROS generation and consequently, enhanced the
ptox production. However, when the ROS was inhibited with NADPH oxidase inhibitor diphenylene iodonium and Superoxide dismutase inhibitor diethyldithio-carbamic acid,
the ROS inhibiting agent, the ptox production was decreased significantly. We also noted that, MeJA up regulated other ptox biosynthetic pathway genes which are not affected by the MeJA induced ROS. Further, these ROS non-responsive genes were controlled by MeJA through the down regulation of five secondary metabolites biosynthesis specific miRNAs viz. miR172i, miR035, miR1438, miR2275, and miR8291.
Finally, this study suggested two possible mechanisms through which MeJA modulates the ptox biosynthesis: primarily by increasing the mRNA stability of ROS-responsive
genes and secondly, by the up regulation of ROS non-responsive genes through the down regulation of some ROS non-responsive miRNA
Expedient Synthesis of Phenanthro-Imidazo-Pyridine Fused Heteropolynuclear Framework via CDC coupling: A New Class of Luminophores
Full text linkAll the reagents and solvents used in this study, were purchased from Sigma Aldrich, Thermo Fischer Scientific and TCI chemicals respectively. Open capillary was used to measure all the melting points. IR spectrum of the solid sample was recorded in the range 500 to 3500 cm-1
in an FT-IR spectrometer in KBr cell. Bruker 600 MHz spectrometer was used to record all 1H and 13C
NMR spectra. EI mass spectral analysis was done using JEOL The Mstation JMS-700 instrument. All the UV & Fluorescence data were collected using Jasco & Cary Eclipse
spectrophotometer respectively. Bruker Kappa Apex II X-raycrystallography machine was used to solve the crystal structure. Singlet (s), doublet (d), triplet (t) & multiplet (m) were designated as 1H NMR multiplicity patterns. Silica gel (60-120 mesh) and (100-200 mesh) were used for column chromatographic separations
Pathway-based expression profiling of benign prostatic hyperplasia and prostate cancer delineates an immunophilin molecule associated with cancer progression
Full text linkAberrant restoration of AR activity is linked with prostate tumor growth, therapeutic failures and development of castrate-resistant prostate cancer. Understanding the processes leading to ARreactivation should provide the foundation for novel avenues of drug discovery. A differential gene expression study was conducted using biopsies from CaP and BPH patients to identify the components putatively responsible for reinstating AR activity in CaP. From the set of genes upregulated in CaP,
FKBP52, an AR co-chaperone, was selected for further analysis. Expression of FKBP52 was positively correlated with that of c-Myc. The functional cross-talk between c-Myc and FKBP52 was established using c-Myc specific-siRNA to LNCaP cells that resulted in reduction of FKBP52. A non-canonical E-box sequence housing a putative c-Myc binding site was detected on the FKBP4 promoter using in silico
search. LNCaP cells transfected with the FKBP52 promoter cloned in pGL3 basic showed increased luciferase activity which declined considerably when the promoter-construct was co-transfected with c-Myc specific-siRNA. ChIP-PCR confirmed the binding of c-Myc with the conserved E-box located in the
FKBP52 promoter. c-Myc downregulation concomitantly affected expression of FGF8. Since expression of FGF8 is controlled by AR, our study unveiled a novel functional axis between c-Myc, AR and FGF8 operating through FKBP52
Role of TRB3 in Neuronal Cell Death in Alzheimer’s Disease Model
No full textNeurodegeneration is one of the pathognomonic features in Alzheimer’s disease (AD). Among several death modalities, autophagy and apoptosis play important roles in the death of neurons. The general mechanism which governs these two phenomena mutually is still not understood fully. Amyloid beta (Aβ) induced neuron death is considered pivotal in the pathogenesis of AD. This study aims to understand the complex mechanism by which Aβ1-42 induces neuronal death in AD. Unravelling these mechanisms may prove to be pivotal for development of therapeutics for AD. We report that Tribbles3 (Trib3/TRB3 a mammalian ortholog of the Drosophila Trib3), is upregulated in neurons, both in vivo and in vitro upon Aβ treatment. Increased levels of Trib3 inhibit the activity of Akt by interacting with it. As a result, FoxO1, a member of Fork-head box transcription factors that is negatively regulated by Akt, gets activated, translocates to the nucleus, and induces an apoptotic gene Bim. Conversely, FoxO1 also binds with Trib3 and enhances its expression upon Aβ insult. This establishes a feed forward loop between Trib3 and FoxO1 in Aβ-treated neurons. On the other hand, our investigations reveal that upon Aβ insult, Trib3 also leads to induction of autophagy via the Akt/m-TOR/ULK1 pathway. Further downstream this leads to the augmentation of autophagosome formation as confirmed by presence of LC3-II and reduced autophagy flux as evident by increase in p62 levels. Thus, we find that Trib3 is required for autophagosome formation and impaired autophagy flux. Most importantly, silencing endogenous Trib3 strongly protects neurons from Aβ insult. We find that inhibition of autophagy leads to better survival of neuronal cells upon Aβ treatment. Further studies into the cross talk between autophagy and apoptosis reveals that autophagy induced Beclin1 is cleaved by active caspases which may thwart further autophagy and induces apoptosis. Moreover downregulating Beclin1 leads to increased survival of neurons treated with Aβ. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt and FoxO1 in Aβ-treated neurons leads to induction of both apoptosis and autophagy that culminates in neuron death. Thus, Trib3 may serve as potential therapeutic target for AD
STUDIES ON THE INTERACTION OF PHENAZINIUM AND PHENOTHIAZINIUM DYES WITH NUCLEIC ACIDS AND PROTEINS
No full textBioisosteric modification of known fucosidase inhibitors to discover a novel inhibitor of a-L-fucosidase
No full textBioisosteric modification of known fucosidase inhibitors A and B, resulted in three new types of molecules, 4b, 5c and 6a (belonging to furopyridinedione, thiohydantoin and hydantoin chemotypes) that could potentially bind to a-L-fucosidase (bovine kidney origin). Molecular docking revealed and compared the putative binding interaction between 4b, 5c and 6a with A and B against the active site of a homology model of a-L-fucosidase. Based on this initial investigation, design and synthesis of a library of small molecules based on furopyridinedione, thiohydantoin and hydantoin, followed by their in vitro screening
against a-L-fucosidase (bovine kidney origin) generated a potent inhibitor (compound 4e) with IC50 of �0.7 mM. Compound 4e possessed no cytotoxic properties when tested against healthy mammalian COS-1 cells. Reaction kinetics study suggested it to be a mixed inhibitor. Finally compounds 4a, b, e and f, bearing the furopyridinedione motif also exhibited substantial inhibition of the proliferation of MCF 7 breast cancer cells
Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice
No full textLipid generates reactive oxygen species (ROS) in consequence to mitochondrial fission followed by inflammation in propagating hepatic fibrosis. The interaction of SIRT1/Mitofusin2 is critical for maintaining mitochondrial integrity and functioning,
which is disrupted upon excess lipid infiltration during the progression of steatohepatitis.The complex interplay between hepatic stellate cells and steatotic hepatocytes is critically regulated by extracellular factors including increased circulating free fatty acids during fibrogenesis. Melatonin, a potent antioxidant, protects against lipidmediated mitochondrial ROS generation. Lipotoxicity induces disruption of SIRT1 and Mitofusin2 interaction leading to mitochondrial morphological disintegration in hepatocytes. Further, fragmented mitochondria leads to mitochondrial permeability
transition pore opening, cell cycle arrest and apoptosis and melatonin protects against all these lipotoxicity-mediated dysfunctions. These impaired mitochondrial dynamics also enhances the cellular glycolytic flux and reduces mitochondrial oxygen consumption rate that potentiates ROS production. High glycolytic flux generates metabolically unfavorable milieu in hepatocytes leading to inflammation, which is abrogated by melatonin. The melatonin-mediated protection against mitochondrial
dysfunction was also observed in high-fat diet (HFD)-fed mice through restoration of enzymatic activities associated with respiratory chain and TCA cycle. Subsequently, melatonin reduces hepatic fat deposition and inflammation in HFD-fed mice. Thus, melatonin disrupts the interaction between steatotic hepatocyte and stellate cells, leading to the activation of the latter to abrogate collagen deposition. Altogether, the results of the current study document that the pharmacological intervention with low dose of melatonin could abrogate lipotoxicity-mediated hepatic stellate cell activation
and prevent the fibrosis progressio
Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B
Full text linkThe present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B.
Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress,
generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2 − (mitosox) were
measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that
KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further,study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also
observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host
macrophag