A NANOG-pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics.

The self-renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine-mediated ERK signaling in Nanog-high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self-perpetuating reciprocal NANOG-pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog-high cells to establish a NANOG-pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells.

Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

Synthesis of DNA interactive C3-trans-cinnamide linked ß-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors.

A series of new C3-trans-cinnamide linked ß-carboline conjugates has been synthesized by coupling between various ß-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC50 values 13-45 nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05 nM and 13.84 nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.

BODIPY-based Ru(II) and Ir(III) organometallic complexes of avobenzone, a sunscreen material: Potent anticancer agents.

The use of organic compounds with known medicinal properties in the synthesis of metal-based complexes is an important alternative to improve the biological activity of metal-based drugs. The reaction of [M(arene)Cl2]2 (M = Ru, arene = p-cymene and M = Ir, arene = pentamethylcyclopentadienyl, cp*) with avobenzone (1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3-dione, AVBH) and KOH in methanol leads to the formation of the neutral complexes [Ru(p-cymene)(AVB)Cl] 1 and [Ir(cp*)(AVB)Cl] 2 (cp* = pentamethylcyclopentadienyl). Subsequent reaction of 1 and 2 with pyridyl derivative-BODIPY ligands, BDP and BDPCC (BODIPY = boron dipyrromethene, BDP = 4-dipyridine boron dipyrromethene, BDPCC = 4-ethynylpyridine boron dipyrromethene) in methanol gives a series of four new dicationic supramolecules: [Ru2(p-cymene)2(AVB)2BDP][2CF3SO3] 3, [Ir2(cp*)2(AVB)2BDP][2CF3SO3] 4, [Ru2(p-cymene)2(AVB)2BDPCC][2CF3SO3] 5 and [Ir2(cp*)2(AVB)2BDPCC][2CF3SO3] 6. The synthesized complexes are fully characterized using multiple analytical techniques, including elemental analysis, 1H NMR, 13C NMR, 19F NMR (NMR = Nuclear Magnetic Resonance), Infrared Radiation (IR), Electrospray Ionization-Mass Spectrometry (ESI-MS), Ultraviolet-visible (UV-Vis) and fluorescence spectroscopy. The structures of these complexes are further rationalized using density functional theory (DFT) calculations. The antiproliferative activity of the neutral and dinuclear cationic complexes is evaluated in vitro in different human cancer cell lines. These complexes are found to be active against different cancer cell lines with half maximal inhibitory concentration (IC50) values between 1 and 5 µM. Complexes 5 and 6 displayed the lowest IC50 values in all the cell lines studied. The activity of 5 and 6 is comparable to that of the well-known chemotherapy drug doxorubicin. Detailed biophysical studies indicate that complexes 5 and 6 exhibit very good Deoxyribonucleic acid (DNA) binding properties, causing the unwinding of the double helix, which is a probable reason for their high cytotoxicity.

Protective Role of Hypothermia Against Heat Stress in Differentiated and Undifferentiated Human Neural Precursor Cells: A Differential Approach for the Treatment of Traumatic Brain Injury.

INTRODUCTION: The present study aimed to explore protective mechanisms of hypothermia against mild cold and heat stress on highly proliferative homogeneous human Neural Precursor Cells (NPCs) derived from Subventricular Zone (SVZ) of human fetal brain. METHODS: CD133+ve enriched undifferentiated and differentiated human NPCs were exposed to heat stress at 42°C. Then, Western-blot quantification was performed using Hsp-70 (70 kilodalton heat shock proteins) recombinant protein. Finally, changes in pluripotency and Hsp-70 expression were measured using immunofluorescence staining and RT-qPCR (Quantitative reverse transcription PCR) analysis, respectively. RESULTS: Heat stress resulted in abnormal neurospheres development. The apoptosis rate was enhanced during long-term in vitro culture of neurospheres. Neurogenic differentiation reduced and showed aberrent phenotypes during heat stress. After hypothermia treatment significant improvement in neurospheres and neuronal cell morphology was observed. CONCLUSION: Mild-hypothermia treatment induces attenuated heat shock response against heat stress resulting in induced HSP-70 expression that significantly improves structure and function of both undifferentiated human NPCs and differentiated neurons.

In vitro quantitative and relative gene expression analysis of pancreatic transcription factors Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1 in trans-differentiated human hepatic progenitors.

AIMS/INTRODUCTION: Diabetes is a major health concern throughout the world because of its increasing prevalence in epidemic proportions. ß-Cell deterioration in the pancreas is a crucial factor for the progression of diabetes mellitus. Therefore, the restoration of ß-cell mass and its function is of vital importance for the development of effective therapeutic strategies and most accessible cell sources for the treatment of diabetes mellitus. MATERIALS AND METHODS: Human fetuses (12-20 weeks gestation age) were used to isolate human hepatic progenitor cells (hHPCs) from fetal liver using a two-step collagenase digestion method. Epithelial cell adhesion molecule-positive (EpCAM+ve)-enriched hHPCs were cultured in vitro and induced with 5-30 mmol/L concentration of glucose for 0-32 h. Pdx-1 expression and insulin secretion was analyzed using immunophenotypic and chemifluorescence assays, respectively. Relative gene expression was quantified in induced hHPCs, and compared with uninduced and pancreatic cells to identify the activated transcription factors (Pdx-1, Ngn-3, Isl-1, Pax-4, Pax-6 and Nkx-6.1) involved in ß-cell production. RESULTS: EpCAM+ve cells derived from human fetal liver showed high in vitro trans-differentiation potential towards the ß-cell phenotype with 23 mmol/L glucose induction after 24 h. The transcription factors showed eminent expression in induced cells. The expression level of transcription factors was found significantly high in 23 mmol/L-induced hHPCs as compared with the uninduced cells. CONCLUSIONS: The present study has shown an exciting new insight into ß-cell development from hHPCs trans-differentiation. Relative quantification of gene expression in trans-differentiated cells offers vast possibility for the production of a maximum number of functionally active pancreatic ß-cells for a future cure of diabetes.

Biological studies of chalcogenolato-bridged dinuclear half-sandwich complexes.

A series of cationic chalcogenolato-bridged diruthenium complexes [(η(6)-p-MeC6H4Pr(i))2Ru2(µ-EC6H5)3](+) (E = S, 1; E = Se, 2; E = Te, 3) has been obtained in ethanol from the reaction of (η(6)-p-MeC6H4Pr(i))2Ru2(µ-Cl)2Cl2 with benzenethiol, benzeneselenol, and sodium tellurophenolate, respectively. The thiolato and selenolato derivatives are isolated in good yield as the chloride salts, while the tellurolato analogue is isolated as the hexafluorophosphate salt. Similarly, the dinuclear pentamethylcyclopentadienyl (C5Me5) rhodium and iridium complexes (η(5)-C5Me5)2M2(µ-Cl)2Cl2 react with benzenethiol, benzeneselenol, and sodium tellurophenolate in ethanol to give the corresponding cationic dinuclear complexes of the general formula [(η(5)-C5Me5)2M2(µ-EC6H5)3](+) (M = Rh, E = S, 4; E = Se, 5; E = Te, 6; M = Ir, E = S, 7; E = Se, 8; E = Te, 9). In addition, cationic dinuclear complexes with mixed thiolato-selenolato and thiolato-tellurolato bridges have been prepared, [(η(6)-p-MeC6H4Pr(i))2Ru2(µ-EC6H5)(µ-SCH2C6H4-p-Bu(t))2](+) (E = Se, 10; E = Te, 11) and [(η(5)-C5Me5)2M2(µ-EC6H5)(µ-SCH2C6H5)2](+) (M = Rh, E = Se, 12; E = Te, 13; M = Ir, E = Se, 14; E = Te, 15), starting from the neutral dinuclear complexes (η(6)-p-MeC6H4Pr(i))2Ru2Cl2(µ-SCH2C6H4-p-Bu(t))2 and (η(5)-C5Me5)2M2Cl2(µ-SCH2C6H5)2. All complexes are highly cytotoxic showing activity in the submicromolar range. The nature of the chalcogenolato bridges seems to have an impact on the activity, while the nature of the metal center plays a minor role. Among the complexes tested, the dinuclear complexes 1, 4, and 7 with the thiolato bridges show the highest activity on cancer cells and the best affinity for CT-DNA as demonstrated by cell biology and biophysical experiments.

Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.

Cholesterol biosynthesis and homeostasis in regulation of the cell cycle.

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Repercussion of Mitochondria Deformity Induced by Anti-Hsp90 Drug 17AAG in Human Tumor Cells.

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity.

Mitochondrial dysfunction and genetic heterogeneity in chronic periodontitis.

We performed an extensive study on mitochondrial dysfunction in chronic periodontitis (CP). Electron microscopic analysis of gingival cells revealed abnormal mitochondria in 60% of the patients. Mitochondrial membrane potential and oxygen consumption of gingival cells were reduced by 4 fold and 5.8 fold, respectively; whereas ROS production was increased by 18%. The genetic analysis by complete mitochondrial DNA sequencing revealed the identification of 14 novel mutations only in periodontal tissues but not in the blood, suggesting a role of oxidative stress on periodontal tissues. Thus, our functional and genetic analysis provided an evidence for the mitochondrial dysfunction in CP.

Cationic amphiphile with shikimic acid headgroup shows more systemic promise than its mannosyl analogue as DNA vaccine carrier in dendritic cell based genetic immunization.

Mannosylated cationic vectors have been previously used for delivering DNA vaccines to antigen presenting cells (APCs) via mannose receptors expressed on the cell surface of APCs. Here we show that cationic amphiphiles containing mannose-mimicking quinic acid and shikimic acid headgroups deliver genes to APCs via mannose receptor. Cationic amphiphile with shikimic acid headgroup was more efficacious than its mannosyl counterpart in combating mouse tumor growth by dendritic cell (the most professional APC) based genetic immunization.

Lipopeptide with a RGDK tetrapeptide sequence can selectively target genes to proangiogenic alpha5beta1 integrin receptor and mouse tumor vasculature.

Integrins, the major class of alphabeta heterodimeric transmembrane glycoprotein receptors, play crucial roles in mediating tumor angiogenesis. Genetic ablation experiments combined with use of antibodies/peptide ligands for blocking either alpha(5) or beta(1) integrins have convincingly demonstrated alpha(5)beta(1) integrin to be unquestionably proangiogenic among the 24 known integrin receptors. Herein, we report on a novel RGDK-lipopeptide 1 that targets selectively alpha(5)beta(1) integrin and is capable of targeting genes to mouse tumor vasculatures.