Parasuraman Jaisankar

Mitochondria-Dependent Reactive Oxygen Species-Mediated Programmed Cell Death Induced by 3,3′-Diindolylmethane through Inhibition of F0F1-ATP Synthase in Unicellular Protozoan Parasite Leishmania donovani

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3′-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (ΔΨm). Because ΔΨm is the driving force for mitochondrial ATP synthesis, loss of ΔΨm results in depletion of cellular ATP level. The loss of ΔΨm causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ΔΨm leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl+ CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl(+) cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl(+) cells.

Nanoemulsion strategy for olmesartan medoxomil improves oral absorption and extended antihypertensive activity in hypertensive rats.

Olmesartan medoxomil (OM) is hydrolyzed to its active metabolite olmesartan by the action of aryl esterase to exert its antihypertensive actions by selectively blocking angiotensin II-AT1 receptor. Poor aqueous solubility and uncontrolled enzymatic conversion of OM to its poorly permeable olmesartan limits its oral bioavailability. The aim of the current study was to formulate a novel nanoemulsion of OM to improve its pharmacokinetics and therapeutic efficacy. The oil-in-water (o/w) nanoemulsion of OM was developed using lipoid purified soybean oil 700, sefsol 218 and solutol HS 15. We have characterized the nanoemulsions by considering their thermodynamic stability, morphology, droplet size, zeta potential and viscosity and in vitro drug release characteristics in fasting state simulated gastric fluid (pH 1.2) and intestinal fluid (pH 6.5). The thermodynamically stable nanoemulsions comprises of spherical nanometer sized droplets (<50 nm) with low polydispersity index showed enhanced permeability through the Caco-2 cell monolayer. The concentration of active olmesartan in rat plasma following oral absorption study was determined by our validated LC-MS/MS method. The result of the pharmacokinetic study showed 2.8-fold increased in area under the curve (AUC0-27) of olmesartan upon oral administration of OM nanoemulsion and sustained release profile. Subsequent, in vivo studies with nanoemulsion demonstrated better and prolonged control of experimentally induced hypertension with 3-fold reduction in conventional dose. By analysing the findings of the present investigations based on stability study, Caco-2 permeability, pharmacokinetic profile and pharmacodynamic evaluation indicated that the nanoemulsion of OM (OMF6) could significantly enhance the oral bioavailability of relatively insoluble OM contributing to improved clinical application.

Binding of the 9-O-N-aryl/arylalkyl Amino Carbonyl Methyl Substituted Berberine Analogs to tRNAphe

Background Three new analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNAphe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. Methodology/Principal Findings Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNAphe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNAphe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNAphe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process. Conclusions/Significance The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNAphe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNAphe.

The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice.

Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti‐leishmanial agent. The compound induces topoisomerase I‐mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topoisomerase of L. donovani and acts as a non‐competitive inhibitor interacting with both subunits of the enzyme. Niranthin interacts with DNA–protein binary complexes and thus stabilizes the ‘cleavable complex’ formation and subsequently inhibits the religation of cleaved strand. The compound inhibits the proliferation of Leishmania amastigotes in infected cultured murine macrophages with limited cytotoxicity to the host cells and is effective against antimony‐resistant Leishmania parasites by modulating upregulated P‐glycoprotein on host macrophages. Importantly, besides its in vitro efficacy, niranthin treatment leads to a switch from a Th2‐ to a Th1‐type immune response in infected BALB/c mice. The immune response causes production of nitric oxide, which results in almost complete clearance of the liver and splenic parasite burden after intraperitoneal or intramuscular administration of the drug. These findings can be exploited to develop niranthin as a new drug candidate against drug‐resistant leishmaniasis.

9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogues induce self-structure in polyadenylic acid

This manuscript describes the interaction of 9-O-substituted analogues of the plant alkaloid berberine with single stranded (ss) poly(A). Three new analogues of berberine with aryl/arylalkyl amino carbonyl methyl substituents at the 9-position of the isoquinoline chromophore were evaluated for their binding to ss poly(A) by a wide variety of biophysical techniques. The results indicated that these analogues exhibited several-fold higher binding affinities than berberine. The Scatchard binding isotherms revealed that all the analogues retained the cooperative binding mode of berberine. Circular dichroism and FTIR studies unequivocally established the strong interaction potential of the analogues to ss poly(A). Optical melting and the unique dilution experiments revealed that these analogues induced self-structure formation in poly(A) and the nature of the substituent was important for the ease of formation of self-structure. Energetics of the binding suggested an entropy driven binding for the analogues in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process. The study also revealed that the spacer length at the 9-position had an important role in the self-structure induction.

Versatile Reagent for Reduction of Azides to Amines

Abstract Triphenylphosphine (TPP) in refluxing methanol effectively reduces a variety of azides 1a–k to amines 2a–k in very good yields.

Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

Alcoholic extract of Piperbetle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr‐Abl with imatinib resistance phenotype. Hydroxychavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti‐CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr‐Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria‐derived reactive oxygen species. Reactive oxygen species‐dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase‐mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly‐adenosine diphosphate‐ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr‐Abl, without showing significant bodyweight loss. Our data describe the role of JNK‐dependent endothelial nitric oxide synthase‐mediated nitric oxide for anti‐CML activity of HCH and this molecule merits further testing in pre‐clinical and clinical settings. (Cancer Sci 2012; 103: 88–99)

Microwave Assisted Pictet–Spengler and Bischler–Napieralski Reactions

Abstract Pictet–Spengler and Bischler–Napieralski reaction products have been prepared–using microwave irradiation on silicagel support under solvent free condition. Microwave assisted reactions have resulted in better yields of the desired products than prepared under conventional conditions.

Synergistic Apoptosis of CML Cells by Buthionine Sulfoximine and Hydroxychavicol Correlates with Activation of AIF and GSH-ROS-JNK-ERK-iNOS Pathway

Background Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress –induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism. Materials and Methods Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexinV-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. Results Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells. Conclusion/Significance BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.