ABSTRACT
Pancreatic stellate cells (PSCs) secrete various factors, which can influence the ß-cell function. The identification of stellate cell infiltration into the islets in pancreatic diseases suggests possible existence of cross-talk between these cells. To elucidate the influence of PSCs on ß-cell function, mouse PSCs were cocultured with Min6 cells using the Transwell inserts. Glucose-stimulated insulin secretion from Min6 cells in response to PSCs was quantified by enzyme-linked immunosorbent assay and insulin gene expression was measured by quantitative polymerase chain reaction. Upon cytometric identification of IL6 in PSC culture supernatants, Min6 cells were cultured with IL6 to assess its influence on the insulin secretion and gene expression. PLC-IP3 pathway inhibitors were added in the cocultures, to determine the influence of PSC-secreted IL6 on Glucose-stimulated insulin secretion from Min6 cells. Increased insulin secretion with a concomitant decrease in total insulin content was noticed in PSC-cocultured Min6 cells. Although increased GSIS was noted from IL6-treated Min6 cells, no change in the total insulin content was noted. Coculture of Min6 cells with PSCs or their exposure to IL6 did not alter either the expression of ß-cell-specific genes or that of miRNA-375. PSC-cocultured Min6 cells, in the presence of PLC-IP3 pathway inhibitors (U73122, Neomycin, and Xestospongin C), did not revoke the observed increase in GSIS. In conclusion, the obtained results indicate that augmented insulin secretion from Min6 cells in response to PSC secretions is independent of IL6-mediated PLC-IP3 pathway.
Subject(s)
Glucose/pharmacology , Insulin Secretion/drug effects , Insulin-Secreting Cells/metabolism , Insulinoma/metabolism , Interleukin-6/metabolism , Pancreatic Stellate Cells/metabolism , Animals , Cells, Cultured , Coculture Techniques , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Insulinoma/pathology , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Pancreatic Stellate Cells/cytology , Pancreatic Stellate Cells/drug effects , Sweetening Agents/pharmacologyABSTRACT
This article describes the synthesis and characterization of three new Ru(II) polypyridyl complexes including [Ru(phen)2(dpphz)]2+ (1), [Ru(bpy)2(dpphz)]2+ (2) and [Ru(dmb)2(dpphz)]2+ (3) where dpphz = dipyrido[3,2-a:2',3'-c] phenazine-11-hydrazide, phen =1,10-phenanthroline, bpy = 2,2'-bipyridine and dmb = 4,4'-dimethyl2,2'-bipyridine. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were explored by spectroscopic titrations, viscosity measurements. Results suggest that these complexes can bind to CT-DNA through intercalation. However, their binding strength differs from each other; this may be attributed to difference in the ancillary ligand. The cytotoxicity of 1-3 was evaluated by MTT assay; results indicated that all complexes have significant dose dependent cytotoxicity with HeLa tumor cell line. All complexes exhibited efficient photocleavage of pBR322 DNA upon irradiation. The DNA binding ability of 1-3 was also studied by docking the complexes into B-DNA using docking program.
Subject(s)
Apoptosis/drug effects , Coordination Complexes/pharmacology , Cytotoxins/pharmacology , DNA Cleavage/drug effects , DNA/metabolism , Organometallic Compounds/pharmacology , Ruthenium/chemistry , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Bacteria/drug effects , Binding Sites , Cattle , Cell Cycle/drug effects , Coordination Complexes/chemistry , Cytotoxins/chemistry , DNA Cleavage/radiation effects , HeLa Cells , Humans , Intercalating Agents/chemistry , Intercalating Agents/pharmacology , Ligands , Microbial Sensitivity Tests , Organometallic Compounds/chemistryABSTRACT
Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Chitosan/analogs & derivatives , Delayed-Action Preparations/chemistry , Doxorubicin/administration & dosage , Gold/chemistry , Nanoparticles/chemistry , Cell Line, Tumor , Chitosan/chemistry , Chitosan/metabolism , Delayed-Action Preparations/metabolism , Drug Delivery Systems , Gold/metabolism , Humans , Nanoparticles/metabolism , Neoplasms/drug therapy , Particle SizeABSTRACT
OBJECTIVES: Diabetes secondary to chronic pancreatitis (CP) presents clinical challenges due to insulin secretory defects and associated metabolic alterations. Owing to lack of molecular understanding, no pharmacotherapies to treat insulin secretory defects have been approved to date. We aimed to delineate the molecular mechanism of ß-cell dysfunction in CP. METHODS: Transcriptomic analysis was conducted to identify endocrine specific receptor expression in mice and human CP on microarray. The identified receptor (NR4A1) was overexpressed in MIN6 cells using PEI linear transfection. RNA-Seq analysis on NovaSeq 6000 of NR4A1 overexpressed (OE) MIN6 cells was performed to identify aberrant metabolic pathways. Upstream trigger for NR4A1OE was studied by InBio Discover and cytokine exposure. Downstream effect of NR4A1OE was examined by Fura2 AM based fluorometric and imaging studies of intracellular calcium. Mice with CP were treated with IFN-γ neutralizing monoclonal antibodies to assess NR4A1 expression and insulin secretion. RESULTS: Increased expression of NR4A1 associated with decreased insulin secretion in islets (humans: controls 9 ± 0.2, CP 3.7 ± 0.2, mice: controls 8.5 ± 0.2, CP 2.1 ± 0.1 µg/L). NR4A1OE in MIN6 cells (13.2 ± 0.1) showed reduction in insulin secretion (13 ± 5 to 0.2 ± 0.1 µg/mg protein/minute, p = 0.001) and downregulation of calcium and cAMP signaling pathways. IFN-γ was identified as upstream signal for NR4A1OE in MIN6. Mice treated with IFN-γ neutralizing antibodies showed decreased NR4A1 expression 3.4 ± 0.11-fold (p = 0.03), improved insulin secretion (4.4 ± 0.2-fold, p = 0.01), associated with increased Ca2+ levels (2.39 ± 0.06-fold, p = 0.009). CONCLUSIONS: Modulating NR4A1 expression can be a promising therapeutic strategy to improve insulin secretion in CP.
ABSTRACT
Neurolathyrism is past history in India since Lathyrus sativus (khesari dal) is no longer used as a staple. A consensus has evolved that khesari dal is harmless as part of a normal diet. L-ODAP (ß-N-oxalyl-l-α-diamino propionic acid) the neurotoxic amino acid, from this pulse, is detoxified in humans but not in animals but still no laboratory animal is susceptible to it under acceptable feeding regimens. L-ODAP is an activator of protein kinase C and consequential crucial downstream effects such as stabilization of hypoxia inducible factor-1 (HIF-1) could be extremely conducive to humans under a variety of situations. ODAP is gradually finding a place in several patents for this reason. Homoarginine the second amino acid from L. sativus can be a better substrate for endogenous generation of nitric oxide, a crucial signaling molecule associated with the cardiovasculature and control of hypertension. These features could make L. sativus a prized commodity as a functional food for the general cardiovasculature and overcome hypoxic events and is set to change the entire perception of this pulse and neurolathyrism.
Subject(s)
Lathyrism/physiopathology , Lathyrus/toxicity , Amino Acids, Diamino/toxicity , Animals , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , India/epidemiology , Lathyrism/epidemiology , Protein Kinase C/metabolismABSTRACT
In this study, we evaluated the ability of negatively charged bio-degradable nanoparticles, ONP- 302, to inhibit tumor growth. Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. Examination of ONP-302 effects on components of the tumor microenvironment (TME) were explored. ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Together, these data support further development of ONP-302 as a novel first-in- class anti-cancer therapeutic that can be used as a single-agent as well as in combination therapies for the treatment of solid tumors due to its ability to modulate the TME.
ABSTRACT
ß-N-Oxalyl-L-α,ß-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.
Subject(s)
Amino Acids, Diamino/physiology , Down-Regulation/physiology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/physiology , Phosphatidylethanolamine Binding Protein/antagonists & inhibitors , Phosphatidylethanolamine Binding Protein/metabolism , Animals , Cell Line, Tumor , Chickens , Humans , MAP Kinase Signaling System/drug effects , Male , RatsABSTRACT
The present study was undertaken to develop an animal model to study neurolathyrism. For this purpose 24 goat (Capra hircus) kids (new born, 15 days old) were divided into four groups. Group I Control, Group II Low toxin (0.17 g% ß-ODAP containing grass pea), Group III high toxin (0.96 g% ß-ODAP containing grass pea) and Group IV high toxin (0.96 g% ß-ODAP containing grass pea flour (powder) fortified with 5 mg% pure ß-ODAP). The experiment was continued for 3 months. Clinical examination was carried out weekly. Muscle conduction velocity (MCV), nerve conduction velocity (NCV), blood and urinary ß-ODAP, nitrite in blood and cerebrospinal fluid (CSF) examination were performed by standard methods. Clinical examination showed neurolathyrism symptoms in three kids. The abnormal MCV and NCV were observed in all the experimental animals. Blood nitrite, blood and urine ß-ODAP levels were significantly increased in experimental groups. Three kids were affected with neurolathyrism due to consumption of grass pea irrespective of its ß-ODAP content and kid may serve as a neurolathyrism model.
Subject(s)
Disease Models, Animal , Goat Diseases/etiology , Lathyrism/veterinary , Lathyrus/poisoning , Neurotoxicity Syndromes/veterinary , Animals , Goats , Lathyrism/etiology , Neurotoxicity Syndromes/etiologyABSTRACT
A silkworm excretory red fluorescent protein (SE-RFP) having light-dependent activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified. Light was observed to be essential also for the SE-RFP synthesis as it was produced only when silkworms were reared in light. SE-RFP has exhibited a high fluorescence quantum yield of 0.86. The apparent mass of native SE-RFP was about 1100 kDa as analysed by gel filtration chromatography. Two photochromic moieties associated with the SE-RFP, namely tetrapyrrole-I (TP-I) and tetrapyrrole-II (TP-II), were isolated by employing TLC and HPTLC techniques. The purified tetrapyrroles were characterized by UV-absorption, fluorescence, atomic absorption and FT-IR spectral analyses. The molecular masses of TP-I and TP-II were 535 and 870 Da, respectively, as determined by ESI-MS and MALDI-TOF-MS. The molar ratio of TP-I to TP-II was 1.14 : 1.00, and a total of 7.251 micromol tetrapyrroles (TP-I + TP-II) were found to be present per mg of SE-RFP. TP-I and TP-II were identified as chlorophyll derivatives, namely, pyropheophorbide a and pheophytin a, respectively. Hence, the SE-RFP was concluded to be a unique insect red fluorescent protein having two photochromic moieties and potent photobiological activity.
Subject(s)
Bombyx/chemistry , Insect Proteins/chemistry , Insect Proteins/pharmacology , Luminescent Proteins/chemistry , Luminescent Proteins/pharmacology , Amino Acid Sequence , Animals , Biological Assay , Bombyx/physiology , Bombyx/virology , Chlorophyllides/metabolism , Insect Proteins/administration & dosage , Insect Proteins/isolation & purification , Light , Luminescent Proteins/administration & dosage , Luminescent Proteins/isolation & purification , Nucleopolyhedroviruses/drug effects , Survival Rate , Red Fluorescent ProteinABSTRACT
BACKGROUND: ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ODAP) is a non-protein amino acid with haemostatic property present in Lathyrus sativus. It is considered to be the causative agent of neurolathyrism that occurs upon prolonged overconsumption of Lathyrus sativus seeds. L-ODAP is used as a haemostatic drug in surgical dressings. We previously reported that it can stabilize hypoxia inducible factor (HIF)-1α in normoxic conditions. HYPOTHESIS: We hypothesised that L-ODAP might affect wound healing by modulating cellular proliferation, migration and angiogenesis via HIF-1α stabilization. STUDY DESIGN: We performed in vitro assays to evaluate wound healing activity of L-ODAP. Further, we prepared pharmaceutical gel containing L-ODAP and checked its effect on healing of full thickness excision wounds using Wistar albino rats. METHODS: Effect of L-ODAP on HT1080 cell line proliferation, migration and invasion was investigated. Further, gel containing L-ODAP was applied on full thickness excision wounds of Wistar rats. Western blot and zymography were performed with wound tissue extracts obtained 2 days post-wounding and histological and immunohistochemical analysis with regenerated tissue obtained 10 days post-wounding. Evaluation was made based on wound contraction percentage, histological analysis and protein expression levels. RESULTS: L-ODAP significantly (Pâ¯<â¯0.05) affected wound healing both in vitro and in vivo. At non-toxic concentrations, it induced cell proliferation, migration, invasion and MMP-2 & -9 expressions. L-ODAP treated wounds healed faster than vehicle treated ones. Significantly higher expression level of HIF-1α, VEGF-A, PDGF-A and matrix metalloproteases were observed in L-ODAP treated wounds. CONCLUSION: The present investigation explores potential of L-ODAP as a wound healing agent. L-ODAP positively affected wound healing both in vitro and in vivo and thus could be considered a natural wound healing agent.
Subject(s)
Amino Acids, Diamino/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Wound Healing/drug effects , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Lathyrus/chemistry , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Platelet-Derived Growth Factor/metabolism , Rats, Wistar , Seeds/chemistry , Vascular Endothelial Growth Factor A/metabolism , Wound Healing/physiologyABSTRACT
BACKGROUND: Resveratrol (RV) and its analogues Aza-stilbenes were found effective in exhibiting anticancer activity. OBJECTIVE: The present study mainly focused on the green synthesis of novel imine stilbene analogues and evaluation of their anticancer activity besides their influence on hypoxia-induced gene expression in cancer cells. METHOD: Novel imine stilbenes, differing in number and/or position of hydroxyl and methoxy functional groups, have been synthesized using green chemistry mediated condensation reaction between aldehydes and amines in the ethanolic extract of Psoralea corylifolia hairy roots and tested for their anticancer potential. RESULTS: Ethanol containing 1% hairy root extract facilitated instant reaction and yielded more than 99% product( s). MTT assay on HeLa cells treated with imine stilbene analogues revealed an increase in the inhibition of cell proliferation as compared to RV. Treatment of nontumor HEK293 cells with these compounds disclosed minimal toxicity implying the selective advantage of these compounds for cervical cancer therapy. Scratch assay on HeLa cells displayed inhibition of directional cell motility by these analogues and compound 3e [4-((E)-(4- hydroxyphenylimino)methyl)-2-methoxyphenol] recorded maximum inhibition. In reporter assay, as compared to untreated N-(2-Methoxy-2-oxoacetyl) glycine methyl ester (DMOG) induced cells, hypoxia response element- directed transcriptional activity has been significantly reduced in DMOG induced cells treated with imine stilbene analogues. CONCLUSION: Overall results indicated that four of the five imine stilbene analogues exhibited enhanced anticancer activity than that of the RV. As such, the novel synthetic compounds 3d, 3e and 3b endowed with potent anticancer activity than RV can serve as drug lead molecules.
Subject(s)
Antineoplastic Agents/pharmacology , Imines/chemistry , Imines/pharmacology , Stilbenes/chemistry , Stilbenes/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biological Availability , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HEK293 Cells , HeLa Cells , Humans , Imines/chemical synthesis , Molecular Structure , Stilbenes/chemical synthesis , Structure-Activity RelationshipABSTRACT
Hypoxia inducible factor (HIF)-1α, a subunit of HIF transcription factor, regulates cellular response to hypoxia. In normoxic conditions, it is hydroxylated by prolyl hydroxylase (PHD)-2 and targeted for proteosomal degradation. Drugs which inhibit PHD-2 have implications in conditions arising from insufficient blood supply. ß-ODAP (ß-N- oxalyl-L-α, ß- diaminopropionic acid), a non-protein excitatory amino acid present in Lathyrus sativus, is an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor agonist known to activate conventional protein kinase C and stabilize HIF-1α under normoxic conditions. However, the mechanism of HIF-1α stabilization by this compound is unknown. In silico approach was used to understand the mechanism of stabilization of HIF-1α which revealed ß-ODAP interacts with key amino acid residues and Fe2+ at the catalytic site of PHD-2. These results were further corroborated with luciferase HRE (hypoxia response element) reporter system in HeLa cells. Different chemical modulators of PHD-2 activity and HIF-1α levels were included in the study for comparison. Results obtained indicate that ß-ODAP inhibits PHD-2 and facilitates HIF dependent HRE expression and hence, might be helpful in conditions arising from hypoxia.
Subject(s)
Amino Acids, Diamino/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Oxygen/metabolism , Response Elements/genetics , Catalytic Domain , DNA/metabolism , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Molecular Docking Simulation , Protein Stability/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.
Subject(s)
Actins/metabolism , Contractile Proteins/metabolism , Microfilament Proteins/metabolism , Peptides/metabolism , Animals , Blood Platelets/metabolism , Cattle , Chromones/pharmacology , Contractile Proteins/isolation & purification , Microfilament Proteins/isolation & purification , Morpholines/pharmacology , Phorbol 12,13-Dibutyrate/pharmacology , Phosphatidylinositols/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Profilins , Protein Binding/drug effects , Signal Transduction/drug effects , Thrombin/pharmacologyABSTRACT
We investigated the effect of UV-B irradiation on PI 3-kinase activity in human fetal hepatocytes. When cells were exposed to monochromatic (304nm) UV-B light, a significant increase in intracellular PI 3-kinase activity was observed in a dose dependent manner with maximal activity upon 1500 Jm(-2) irradiation. At 1500 Jm(-2) dose PI 3-kinse activity increased by 80% in membrane fraction of fetal hepatocytes of 25 weeks gestation. PI 3-kinse inhibitors wortmannin and LY294002 specifically inhibited the UV-B induced lipid kinase activity and blocked significantly the UV-B induced cell viability. The data suggests a correlation between cell survival and elevated levels of PI 3-kinase and suggest that UV-B irradiation at a dose of 1500 Jm(-2) is ideal for fetal hepatocyte transplantation. Also, PI 3-kinase levels could be a representative marker for viable UV-B irradiated fetal hepatocytes for transplantation.
ABSTRACT
Three new ruthenium(II) polypyridyl complexes [Ru(phen)2BrIPC](2+) (1), [Ru(bpy)2 BrIPC](2+) (2) and [Ru(dmb)2BrIPC](2+) (3) where, BrIPC = (6-bromo-3-(1H-imidazo[4,5-f] [1,10]-phenanthroline, phen = 1,10-phenanthroline, bpy = 2,2' bipyridine, dmb = 4,4'-dimethyl 2,2' bipyridine, were synthesised and characterised. DNA-binding nature was investigated by spectroscopic titrations and mode of binding was assessed by viscosity measurements. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be in the order of 10(5). Experimental results showed that these complexes interact with CT-DNA by intercalative mode. Photocleavage and antimicrobial activities were complex concentration dependent, at high concentration, high activity and vice versa. MTT assay was performed on HeLa cell lines, IC50 values of complexes in the order of 3 > 2 > 1 > cisplatin. From comet assay, cellular uptake studies, we observed that complexes could enter into the cell membrane and accumulate inside the nucleus. Molecular docking studies support the DNA binding affinity with hydrogen bonding and van der Waals attractions between base pairs and phosphate backbone of DNA with metal complexes.
Subject(s)
Antineoplastic Agents/chemical synthesis , Coordination Complexes/chemical synthesis , Ruthenium/chemistry , 2,2'-Dipyridyl/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Binding Sites , Comet Assay , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , DNA/chemistry , DNA/metabolism , DNA Cleavage/drug effects , HeLa Cells , Humans , Hydrogen Bonding , Kinetics , Molecular Docking Simulation , Nucleic Acid Conformation , Phenanthrolines/chemistry , Static ElectricityABSTRACT
Three new compounds, [Ru(Hdpa)2PyIP](ClO4)2·2H2O (1) [Ru(Hdpa)2FyIP](ClO4)2·2H2O (2) and [Ru(Hdpa)2IIP](ClO4)2·2H2O (3) have been synthesized and characterized by spectroscopic techniques such as elemental analysis, UV/Vis, FT-IR, (1)H NMR, (13)C NMR and mass spectra. The CT-DNA binding properties of 1-3 have been investigated by absorption, emission spectroscopy and viscosity measurements. Experimental results suggested that they can interact with DNA through intercalative mode with different binding strengths. These were found to promote the cleavage of plasmid DNA. Cell viability results indicated that all compounds showed significant dose dependent cytotoxicity in selected cell lines and 1 shown higher cytotoxicity than cisplatin on HeLa cells. Cellular uptake studies were studied by flow cytometry and confocal microscopy.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA/drug effects , Organometallic Compounds/pharmacology , Rubidium/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Binding Sites/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Models, Molecular , Molecular Conformation , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: Profilins are actin-modulating proteins regulating many intracellular functions based on their multiple and diverse ligand interactions. They have been implicated to play a role in many pathological conditions such as allergies, cardiovascular diseases, muscular atrophy, diabetes, dementia and cancer. Post-translational modifications of profilin 1 can alter its properties and subsequently its function in a cell. In the present study, we identify the importance of phosphorylation of profilin 1 at serine 137 (S137) residue in breast cancer progression. METHODS/PRINCIPAL FINDINGS: We found elevated profilin 1 (PFN) in human breast cancer tissues when compared to adjacent normal tissues. Overexpression of wild-type profilin 1 (PFN-WT) in breast cancer MCF7 cells made them more migratory, invasive and adherent independent in comparison to empty vector transfected cells. Mutation in serine phosphorylation site (S137) of profilin 1 (PFN-S137A) significantly abrogated these properties. Mutation affecting actin-binding ability (PFN-R74E) of profilin 1 enhanced its tumorigenic function whereas mutation affecting its poly-L-proline binding function (PFN-H133S) alleviated these mechanisms in breast cancer cells. PFN-WT was found to activate matrix metalloproteinases by zymography, MMP2 and MMP9 in presence of PDBu (phorbol 12, 13 dibutyrate, PI3K agonist) to enhance migration and invasion in MCF7 cells while PFN-S137A did not. Phosphorylation increased migration and invasion in other mutants of profilin 1. Nuclear profilin levels also increased in the presence of PDBu. CONCLUSIONS: Previous studies show that profilin could be executing a dual role in cancer by either suppressing or promoting tumorigenesis in a context dependent manner. In this study we demonstrate for the first time that phosphorylation of profilin 1 at serine 137 enhances oncogenic properties in breast cancer cells. Inhibitors targeting profilin 1 phosphorylation directly or indirectly through inhibition of kinases that phosphorylate profilin could be valuable therapeutic agents that can alter its activity and thereby control the progression of cancer.
Subject(s)
Breast Neoplasms/metabolism , Profilins/metabolism , Blotting, Western , Breast Neoplasms/genetics , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Female , Flow Cytometry , Humans , In Vitro Techniques , Phosphorylation/genetics , Phosphorylation/physiology , Profilins/genetics , Wound Healing/genetics , Wound Healing/physiologyABSTRACT
Three new mononuclear [Ru (phen)2 ptip](2+) (1), [Ru (bpy)2 ptip](2+) (2) and [Ru (dmb)2 ptip](2+) (3) [ptip=(2-(5-phenylthiophen-2-yl)-1H-imidazo[4, 5-f][1,10 phenanthroline, phen=1, 10 phenanthroline, bpy=2, 2' bipyridine, dmb=4, 4'-dimethyl 2, 2' bipyridine] complexes were synthesized and characterised by elemental analysis, IR, NMR and Mass spectra. The DNA-binding behaviours were investigated by electronic absorption titration, luminescence spectra, viscosity measurements and photo-activated cleavage. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be 7.0 (± 0.06)× 10(5), 3.87 (± 0.04) × 10(5), 2.79 (±0.07) × 10(5) respectively. The results showed that these complexes interact with CT-DNA by intercalative mode. Cell viability experiments indicated that the Ru(II) complex showed significant dose-dependent cytotoxicity to HeLa tumour cell lines. Further flow cytometry experiments showed that the cytotoxic Ru(II) complex induced apoptosis of HeLa tumour cell lines. Our data demonstrated that the Ru(II) polypyridyl complex binds to DNA and thereby induces apoptosis in tumor cells, suggesting that anti-tumor activity of the Ru(II) complex could be related to its interaction with DNA. The molecular dynamic simulations and docking methods were used to predict the DNA binding affinity of ruthenium complexes and with good visualisation images supporting with experimental results.
Subject(s)
Coordination Complexes/toxicity , DNA Cleavage/drug effects , DNA/chemistry , Ruthenium/chemistry , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Apoptosis/drug effects , Binding Sites , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Crystallography, X-Ray , DNA/metabolism , Escherichia coli/drug effects , HeLa Cells , Humans , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Intercalating Agents/toxicity , Microbial Sensitivity Tests , Molecular Conformation , Molecular Dynamics Simulation , Nucleic Acid Conformation , Staphylococcus aureus/drug effects , ViscosityABSTRACT
In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as evidenced by UV-visible, luminescence, emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP](2+) (1), [Ru(Hdpa)2PIP](2+) (2), [Ru(Hdpa)24HEPIP](2+) (3) clearly demonstrated that they can bind to DNA through the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated. The complexes were docked into DNA-base-pairs using the 'GOLD' (Genetic Optimization for Ligand Docking), docking program.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA/drug effects , Organometallic Compounds/pharmacology , Ruthenium/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cattle , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Humans , MCF-7 Cells , Models, Molecular , Molecular Structure , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Nickel and cobalt are obligate nutrients for the gammaproteobacteria but when present at high concentrations they display toxic effects. These two metals are present in the environment, their origin being either from natural sources or from industrial use. In this study, the effect of inhibitory concentrations of Ni or Co was assessed on the soil bacterium Pseudomonas putida KT2440 using a proteomic approach. The identification of more than 400 spots resulted in the quantification of 160 proteins that underwent significant variations in cells exposed to Co and Ni. This analysis allowed us to depict the cellular response of P. putida cells toward metallic stress. More precisely, the parallel comparison of the two proteomes showed distinct responses of P. putida to Ni or Co toxicity. The most striking effect of Co was revealed by the accumulation of several proteins involved in the defense against oxidative damage, which include proteins involved in the detoxification of the reactive oxygen species, superoxides and peroxides. The up-regulation of the genes encoding these enzymes was confirmed using qRT-PCR. Interestingly, in the Ni-treated samples, sodB, encoding superoxide dismutase, was up-regulated, indicating the apparition of superoxide radicals due to the presence of Ni. However, the most striking effect of Ni was the accumulation of several proteins involved in the synthesis of amino acids. The measurement of the amount of amino acids in Ni-treated cells revealed a strong accumulation of glutamate.