[Show abstract][Hide abstract] ABSTRACT: The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.
[Show abstract][Hide abstract] ABSTRACT: On the basis of our previous studies, we investigated the possible role of focal adhesion kinase (FAK) in the development of insulin resistance in skeletal muscle, a major organ responsible for insulin-stimulated glucose uptake.
Insulin-resistant C2C12 skeletal muscle cells were transfected with FAK wild-type or FAK mutant plasmids, knocked down using small interfering RNA (siRNA), and their effects on the levels and activities of insulin-signalling molecules and on glucose uptake were determined.
A significant decrease in tyrosine phosphorylation of FAK in insulin-resistant C2C12 cells was observed. A similar decrease was observed in skeletal muscle obtained from insulin-resistant Sprague-Dawley rats fed a high-fat diet. Increased levels of FAK in insulin-resistant C2C12 skeletal muscle cells increased insulin sensitivity and glucose uptake. These effects were reversed by an increase in the level of kinase activity mutant FAK or suppression of endogenous FAK by siRNA. FAK was also found to interact downstream with insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase C and glycogen synthase kinase 3beta, leading to translocation of glucose transporter 4 and resulting in the regulation of glucose uptake.
The present study provides strong evidence that the modulation of FAK level regulates the insulin sensitivity of skeletal muscle cells. The results demonstrate a direct role of FAK in insulin-resistant skeletal muscle cells for the first time.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily known to regulate adipocyte differentiation. However, its role in skeletal muscle differentiation is not known. To investigate possible involvement of PPARgamma in skeletal muscle differentiation, we modulated its expression in C2C12 mouse skeletal muscle cells by stable transfection with sense or antisense plasmid constructs of PPARgamma cDNA. Phenotypic observations and biochemical analysis of different myogenic markers showed that altered expression of PPARgamma inhibited the formation of myotubes, as well as expression of muscle-specific myogenic proteins including myogenin, MyoD and creatine kinase activity. Together, we show that critical expression of PPARgamma is required for skeletal muscle cells differentiation.
[Show abstract][Hide abstract] ABSTRACT: The n-hexane, ethyl acetate, methanol, and
acetone extracts of Piper cubeba Linn. and P. retrofractum
Vahl. (Piperaceae) were evaluated in vitro against promastigotes
of Leishmania donovani, and all exhibited significant
in vitro activity at 100 lg/ml. Two lignans,
cubebin and hinokinin, were isolated from the hexane extract
of P. cubeba; and one bis-epoxy lignan, (–)-sesamin,
and two amides, pellitorine and piplartine, were isolated
from the hexane and methanol extracts of P. retrofractum.
Cubebin and piplartine showed significant antileishmanial
activity in vitro at 100 lM and were further tested in vivo
in a hamster model of visceral leishmaniasis. Piplartine
showed activity at 30 mg/kg dose. This is the first report of
antileishmanial activity of these two plants and their isolated
[Show abstract][Hide abstract] ABSTRACT: Miltefosine, the first oral anti-leishmanial drug, is reported to inhibit phosphatidylinositol 3-kinase (PI3K)/Akt activity in carcinoma cell lines. Inhibition of the PI3K/Akt pathway is known to result in insulin resistance. Therefore, we investigated whether miltefosine has any deleterious effect(s) on insulin sensitivity in L6E9 skeletal muscle cells.
L6E9 myotubes were treated with miltefosine and its effect was observed on insulin-signalling proteins such as Akt, PI3K, insulin receptor-beta, IRS-1, c-Jun N-terminal kinase, p38 and glycogen synthase kinase beta, as well as on glucose uptake.
Miltefosine caused skeletal muscle insulin resistance in vitro by interfering with the insulin-signalling pathway and inhibiting insulin-stimulated glucose uptake.
Miltefosine may contribute to the risk of type 2 diabetes and needs further clinical exploration.
[Show abstract][Hide abstract] ABSTRACT: In this study the anti-leishmanial activity and anti-microtubule effects of paclitaxel, trifluralin and a combination of paclitaxel and trifluralin have been tested in a wild type and sodium arsenite-resistant strain of Leishmania donovani. Both paclitaxel and trifluralin have been shown to be effective in limiting parasite growth. Specific alterations in morphology, tubulin polymerization dynamics, post-translational modifications and cellular distribution of the tubulins have been confirmed to be a part of the intracellular anti-microtubule-events that occur in arsenite-resistant L. donovani in response to these agents, ultimately leading to death of the parasite. DNA analyses of the drug-treated wild type and arsenite-resistant strains revealed an apoptosis-like death in response to paclitaxel and the combination but not to trifluralin. Data provide valuable information for further development of chemotherapeutic strategies based on anti-microtubule agents against drug resistant Leishmania parasites.
[Show abstract][Hide abstract] ABSTRACT: Leishmaniasis affects millions of people worldwide every year. Lack of effective vaccination, co-infection with other dreaded diseases like AIDS and generation of drug resistant strains demand immediate attention into this neglected area of research. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which shows a 13-fold increase in resistance compared with wild type. Here we report that the arsenite resistant strain of L. donovani promastigotes shows cross-resistance to novobiocin, a catalytic inhibitor of topoisomerase II, with IC50 value of 320 microg ml-1 as compared with 242 microg ml-1 for wild type L. donovani. Leishmanicidal action of novobiocin induces dose- and time-dependent increase in cell death. Treatment with IC50 of novobiocin caused morphological and biochemical changes which lead to induction of cell death exhibiting characteristic features of metazoan apoptosis. Phosphatidylserine externalization, cytochrome C release to cytoplasm, activation of caspases, oligonucleosomal DNA fragmentation and in situ labelling of condensed and fragmented nuclei in both wild type and arsenite resistant L. donovani promastigotes strongly suggest the apoptosis-like mode of cell death. Cross-resistance to novobiocin in arsenite resistant strain has been correlated to over-expression of topoisomerase II and substantiated by differential inhibition of enzyme activity in wild type and arsenite resistant L. donovani.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression is very low in skeletal muscle cells, which is one of the most important target tissues for insulin and plays a predominant role in glucose homeostasis. It has recently been shown that muscle-specific PPAR-γ deletion in mouse causes insulin resistance. However, it is likely that the observed effects might be due to secondary interaction in whole animal.
The aim of the study was to explore the role of muscle PPAR-γ in insulin sensitivity. We stably transfected C2C12 skeletal muscle cells with plasmids containing sense or antisense constructs of PPAR-γ and examined the effect of modulation of PPAR-γ expression in terms of glucose uptake. Effect was also examined in insulin-resistant C2C12 skeletal muscle cells.
In transfected C2C12 cell line, the inhibition of PPAR-γ expression (23.0±0.005%) was observed to induce insulin resistance as determined by functional assessment of 2-deoxyglucose incorporation.
Overexpression of PPAR-γ (28.5±0.008%) produced an additional effect on insulin (100 nM) and Pioglitazone (50 μM), resulting in 42.7±3.5% increase in glucose uptake as against 29.2±2.8% in wild-type C2C12 skeletal muscle cells differentiated under normal (2% horse serum) condition. Under similar treatment, PPAR-γ overexpressing cells resistant to insulin exhibited enhanced glucose uptake upto 60.7±4.08%, as compared to 23.8±5.1% observed in wild-type C2C12 skeletal muscle cells.
These data demonstrate a direct involvement of PPAR-γ in insulin sensitization of TZD action on skeletal muscle cells, and suggest that pharmacological overexpression of muscle PPAR-γ gene in skeletal muscle might be a useful strategy for the treatment of insulin resistance.
British Journal of Pharmacology (2004) 143, 1006–1013. doi:10.1038/sj.bjp.0706002
British Journal of Pharmacology 01/2005; 143(8):1006-13. DOI:10.1038/sj.bjp.0706002 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA interference, part of a complicated network of interconnected pathways for cellular defence, RNA surveillance and development, has become a powerful tool for the experimental manipulation of gene expression. It is the process by which double-stranded (dsRNA) silences specific gene expression through homology-dependent degradation of cognate mRNA. The dsRNA is converted into 21nt small interfering RNAs (siRNAs), which directs a complex ribonuclease system to substrate mRNA targets. The degradation of the target mRNA is initiated with the cleavage at a position corresponding to the centre of the siRNA. Dissecting individual cellular pathways to reveal the function of numerous proteins is an approach to drug discovery. Interfering RNA (RNAi) serves as a rapid and convenient tool, which works in various organisms. RNAi technology has the potential to facilitate our understanding of biological processes and potentially lead to exciting new drugs. Here we review various experimental approaches adopted with RNAi and possible therapeutic applications.
Journal of Clinical Pharmacy and Therapeutics 11/2004; 29(5):395-404. DOI:10.1111/j.1365-2710.2004.00579.x · 1.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients on total parenteral nutrition without Cr supplementation develop symptoms similar to those of diabetes. Zn has been implicated in diabetes because of its antioxidant properties and interaction with insulin. To study the effect of these metal ions on insulin signaling proteins, cultured mouse skeletal muscle cells was used as an in vitro model, as the tissue accounts for more than 80% of insulin-stimulated glucose disposal in the body. In the present study, it has been observed that both Cr and Zn, upon prolonged exposure, could stimulate tyrosine phosphorylation of insulin receptor (IR) even in the absence of insulin. Insulin-mediated IR tyrosine phosphorylation was enhanced by the treatment with both of the metal ions. Both Cr and Zn could phosphorylate insulin receptor substrate-1 (IRS-1). Phosphorylation of IRS-1 induced by metal ions was higher than that induced by insulin. Hence, both Cr and Zn were found to have insulin mimetic activity. Both of the metal ions were also found to potentiate insulin-mediated activation of IRS-1. The basal level of glucose uptake was also increased by prolonged treatment of the cells with the metal ions. The ions could also enhance the insulin-stimulated glucose uptake into the cells. Therefore, both Zn and Cr seem to have a positive effect on insulin signaling leading to glucose uptake.
Biological Trace Element Research 11/2004; 101(1):19-36. DOI:10.1385/BTER:101:1:19 · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra- and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.
[Show abstract][Hide abstract] ABSTRACT: Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.
International Journal for Parasitology 08/2004; 34(8):915-25. DOI:10.1016/j.ijpara.2004.03.009 · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transdermal iontophoresis is a physical enhancement strategy primarily for charged molecules and offers a number of advantages for the delivery of peptides and proteins. The singular advantage of iontophoresis lies in the precise control of dose by manipulating the current protocol. The objective of the present investigation was to understand the role of electronic parameters on iontophoretic transport of large peptides using insulin as a model peptide. Ex vivo permeation experiments were conducted using excised rat skin and the influence of varying current strengths, duration, on/off ratios and switching iontophoresis on insulin permeation were studied. High performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) were used to assess the electrochemical stability of insulin; while Fourier transform infra-red (FT-IR) spectroscopy and thermogravimetric analysis (TGA) were used to understand the biophysical changes in skin during iontophoresis. The permeation of insulin was found to increase as a function of current strength and duration of current application. Skin barrier integrity and electrochemical stability of insulin was dependent on the charge applied during iontophoresis. FT-IR spectroscopy and TGA studies showed that the skin hydration increased with increase in the charge applied and thus facilitated the transport of insulin. Periodic iontophoresis did not show any significant difference in insulin permeation compared with continuous current application; 1:1 on/off ratio resulted in higher amount of insulin permeation, while flux was highest with mixed duty cycle. Switching iontophoresis was useful in reducing the pH shift and in improving the electrochemical stability of insulin at pH 3.6 and 7.4, respectively. The electroosmotic flow was influenced by the pH of the donor medium, as well as by the electrode polarity during switching and non-switching iontophoresis. Overall, the study demonstrates the issues related to the optimization of electronic parameters for the iontophoretic delivery of a large peptide.
Methods and Findings in Experimental and Clinical Pharmacology 07/2004; 26(6):399-408. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.
[Show abstract][Hide abstract] ABSTRACT: We examined the effect of combination of thiazolidinediones (TZDs) and metformin on insulin-resistant skeletal muscle cells. The combined use of TZDs and metformin resulted in maximum tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) at 12.5 microM of TZDs and 100 microM of metformin as compared to the maximum tyrosine phosphorylation of IR and IRS-1 achieved at 50 microM of TZDs or 400 microM of metformin. The glucose uptake was significantly high at the combination of lower concentration (12.5 microM of TZDs and 100 microM of metformin) as compared to the combination of higher concentration (50 microM of TZDs and 400 microM of metformin). Results demonstrated that (1) Additive effect on insulin sensitization can be achieved by a combination of TZDs and metformin at lower concentration; (2) combination of TZDs and metformin act on insulin signaling molecules in insulin resistance; (3) in vitro system has the potentiality to determine possible target molecule(s) and mechanism of action of drugs.
Life Sciences 03/2004; 74(15):1877-88. DOI:10.1016/j.lfs.2003.08.040 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies on skeletal muscle differentiation showed that myogenesis is regulated by extracellular signal-regulated kinases (ERK-1/-2) and p38 mitogen activated kinase (MAPK) pathways. Present study shows that c-Jun NH2-terminal protein kinase (JNK) activities were up regulated during skeletal muscle differentiation in rat skeletal muscle L6E9 cells, as determined by Western immunoblot of differentiating cells probed with anti-phospho-JNK antibody. Inhibition of JNK activities by JNK inhibitor II drastically inhibited differentiation as determined by decreased myosin, myogenin expression and creatine kinase activity. The inhibition of the differentiation was regulated by apoptosis as determined by the detection of poly(ADP-ribose) polymerase (PARP) cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells when JNK activities were inhibited. Apoptosis was accompanied by marked expression and activation of c-Jun and p53 transcription factors. Taken together, our results indicate that basal JNK activities are essential for regulating skeletal muscle differentiation, and inhibition of JNK activation affects myogenesis by apoptosis dependent on c-Jun and p53 transcription factors.
Journal of Muscle Research and Cell Motility 02/2004; 25(8):645-55. DOI:10.1007/s10974-004-7099-1 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the issues associated with the use of platinum electrodes for transdermal iontophoretic delivery of peptides, using insulin as a model peptide. Insulin permeation was studied using full-thickness rat skin by varying the donor solution pH as a function of electrode polarity. The stability of insulin under the iontophoretic conditions was studied using TLC, SDS-polyacrylamide gel electrophoresis and HPLC. Large pH shifts were observed during anodal iontophoresis (AI), when the donor solution pH was above the isoelectric point of insulin and in cathodal iontophoresis (CI), when the donor solution pH was below the isoelectric point of insulin. The direction and magnitude of electroosmotic flow was influenced by pH of the donor solution and the electrode polarity. On the other hand, the buffer used to maintain the pH governed the contribution of electrorepulsion to the overall transport of insulin. Electrochemical degradation of insulin was significant during AI at pH 7.4. Among the pH investigated, AI of insulin at pH 3.6 and CI at pH 8.35 were better, as the pH shift was relatively less and electrochemically more stable during iontophoresis as compared with other pH. In summary, the pH shift caused by platinum electrodes had a significant influence on the permeation and stability of insulin.
Journal of Pharmacy and Pharmacology 12/2003; 55(11):1505-13. DOI:10.1211/0022357022197 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to possess functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route. Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux. Broad substrate recognition by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors. In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest. Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination. Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors. In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.
Pharmacological Research 11/2003; 48(4):347-59. DOI:10.1016/S1043-6618(03)00158-0 · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mitogen-activated protein kinase (MAPK) family is involved in the regulation of cellular proliferation, differentiation and stress signals. In this study, we investigated the role of MAPK in response to H2O2, an oxidative stress, in neuronal cells. Activation of c-Jun-N-terminal kinase (JNK) was sustained, where as extracellular signal-regulated kinase (ERK) and p38 MAPK were transiently activated in response to H2O2 treatment. Inhibition of ERK and p38 resulted in higher activation of JNK in response to H2O2 treatment. The treatment with H2O2 led to apoptosis. The results implicate potential role of MAP kinases in neurodegenerative disorders mediated by oxidative stress.
Neuroscience Research Communications 07/2003; 33(1):17-29. DOI:10.1002/nrc.10077
[Show abstract][Hide abstract] ABSTRACT: Western immunoblot analyses of whole cell lysates probed with a human specific monoclonal anti-topoisomerase IIalpha antibody identified a 190 kDa protein over expressed in the arsenite resistant Leishmania donovani strain. The crude nuclear extract of the resistant strain showed higher topoisomerase II-like enzyme activity. suggesting a possible regulatory role of putative topoisomerase II in arsenite resistant Leishmania.
Microbiological Research 02/2003; 158(1):55-8. DOI:10.1078/0944-5013-00177 · 2.56 Impact Factor