Experimental and computational investigation on the binding of anticancer drug gemcitabine with bovine serum albumin.

This study reports the experimental and computational investigation on the binding of a common anticancer drug, gemcitabine, with the model plasma protein, bovine serum albumin (BSA). Several experimental and computational methods, such as intrinsic and synchronous fluorescence, UV-visible, and circular dichroism spectroscopies, consensus molecular docking and molecular dynamics simulation have been employed to elucidate the binding mechanism. Gemcitabine altered the UV-visible spectrum of BSA, which is a clear indication of the complex formation between them. The visual inspection of observed fluorescence quenching results at λex = 280 nm and 295 nm has shown the substantial involvement of tyrosine residue, even larger than tryptophan. However, after the correction of inner filter effect of the observed data, it became clear that tyrosine has a negligible role in quenching. A 20-fold decrease in quenching constant was found in the corrected data, as compared to the observed data at λex = 280 nm. There was a 1:1 weak binding between BSA and gemcitabine accompanied by dynamic quenching. The secondary structure of BSA remained almost intact in the presence of gemcitabine. The primary binding site of gemcitabine inside BSA was the drug binding site 2 or DS II, which is located in the subdomain 3 A. MD Simulation results suggested that gemcitabine doesn't affect or deviate the structure of BSA upon interaction throughout 100 ns time period. The dominating intermolecular forces were hydrophobic forces and hydrogen bonding. A small change in the frontier molecular orbitals of gemcitabine was also observed after its binding with BSA.Communicated by Ramaswamy H. Sarma.

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities.

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3ß,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5 mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5-125 and 250-1000 µg/mL respectively. The compound (50 and 100 mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90 min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100 mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4 h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.

Assessment of biological activity and UPLC-MS based chromatographic profiling of ethanolic extract of Ochradenus arabicus.

Natural products from wild and medicinal plants, either in the form of crude extracts or pure compounds provide unlimited opportunities for new drug leads owing to the unmatched availability of chemical diversity. In the present study, the cytotoxic potential of crude ethanolic extract of Ochradenus arabicus was analyzed by MTT cell viability assay in MCF-7 adenocarcinoma breast cancer cells. We further investigated its effect against oxidative stress induced by anticancer drug doxorubicin. In addition, Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) based chromatographic profiling of crude extract of O. arabicus was performed. The MTT assay data showed that the extract is moderately toxic to the MCF-7 cells. However, its treatment alone does not induce oxidative stress while doxorubicin increases the level of oxidative stress in MCF-7 cells. Whereas, simultaneous treatment of plant extract and doxorubicin significantly (p < 0.05) decreased the level of intracellular reactive oxygen species (ROS) and lipid peroxidation while an increase in the reduced glutathione and superoxide dismutase activity was observed in time and dose dependent manner. Hence, our finding confirmed cytotoxic and antioxidant potential of crude extract of O. arabicus in MCF-7 cells. However, further investigations on O. arabicus as a potential chemotherapeutic agent are needed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays such as chromatographic techniques is discussed.

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.

Molecular mechanism of bis(maltolato)oxovanadium(IV)-induced insulin signaling in 3T3-L1 and IM9 cells: impact of dexamethasone.

The organovanadium compound bis(maltolato)oxovanadium(IV) (BMOV) enhanced the tyr-phosphorylation of major upstream insulin signaling proteins including the vital site-specific phosphorylation of insulin receptor beta (IRbeta) in IM9 and 3T3-L1 cells in dose- and time-dependent manners more efficiently than insulin. Nevertheless, insulin in general had a synergistic impact on those phosphorylations in both cell lines, while its presence was obligatory to induce Tyr(972)-phosphorylation of IRbeta in IM9 cells at 18-h treatment with BMOV. However, prolonged exposure of cells to BMOV caused depletion in IR level and using IM9 cells we found that this event was counteracted by insulin, where monensin, a monocarboxylic acid ionophore made an additive impact, suggesting that a novel mechanism is being involved in the recycling of internalized IR in BMOV-treated cells. On the other hand, dexamethasone elevated the IR level in both cell lines. However, no correlation was found between the cellular content and the degree of phosphorylation of IRbeta in cells receiving combined treatment of BMOV, and dexamethasone with short insulin post-exposure. BMOV also induced the phosphorylation of Thr(308) and Ser(473) of Akt in both cell lines receiving insulin post-treatment, while dexamethasone decreased those phosphorylations. However, this activation/deactivation of Akt did not correlate with the phosphorylation status of Ser(9) and Ser(259) of glycogen synthase kinase (GSK)-3beta and Raf respectively. Taken together, it is conceivable that BMOV and/or dexamethasone modulate insulin signaling by acting differentially on the components of the insulin signaling network. We also consider that the observed dexamethasone-mediated modulation of insulin receptor kinase in BMOV-treated 3T3-L1 cells probably occurs through the activation/deactivation of some mechanism which needs further studies for proper characterization.

Evidence of apoptotic effects of 2,4-D and butachlor on walking catfish, Clarias batrachus, by transmission electron microscopy and DNA degradation studies.

Apoptosis or programmed cell death is characterized morphologically by chromatin condensation, cell shrinkage, fragmentation of the nucleus and cytoplasm, and consequently formation of apoptotic bodies. It has also been best characterized by the cleavage of DNA into nucleosomal size fragments of 180-200 bp or multiples of the same. Contrary to this, under extreme conditions, the cells were found to show adaptive response to apoptosis and unable to regulate their own death; necrosis is therefore predominantly observed. In the present study, we showed induction of apoptosis in Clarias batrachus due to sublethal concentration of 2,4-D and butachlor at multiple exposure time. The first phase of the study involved light microscopy (LM) and transmission electron microscopy (TEM) for ultrastructural abnormalities of the germinal tissues. While, in the second phase of the study, DNA degradation of blood and hepatic tissue was resolved on agarose gel electrophoresis. In histopathological studies, large numbers of stage II oocytes were noted for nuclear blebbing irrespective of the test chemical. Some of the butachlor-exposed oocytes showed vacuolation and electron dense cytoplasm along with thickened nuclear envelope, having close association with the lysosomes on the cytoplasmic side. Some oocytes undergo nuclear blebbing having inner dense core and translucent cytoplasm. Leydig cells were slightly hypertrophied and few appeared pycnotic, a process involving necrotic changes in which the cell nuclei were characterized by rounding up and condensation resulting in hyperchromatic staining or pycnosis. In testicular tissue, spermatogonial nuclei had irregular large clumps of heterochromatin adjoining the nuclear membrane indicating initial stage of apoptotic cell death. Electrophoretic separation resulted in a ladder pattern of blood DNA and smear like pattern of hepatic DNA. These results indicate that the above herbicides are able to induce apoptosis both at molecular as well as cytological level. A reference dose or safety factor approach to calculate risk of human exposure to both chemicals is still awaited.

Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests.

Neem (Azadirachta indica), an indigenous plant commonly grown in India and its sub-continent is a multipurpose plant well known for its insecticidal and biomedical properties, however, its antimutagenic effects in vertebrate organisms are lacking. The present work is therefore, focused on possible antimutagenic potential of ethanolic extract of neem leaves evaluated on the clastogenicity induced by Pentachlorophenol (PCP) and 2, 4-dichlorophenoxyacetic acid (2,4-D) in freshwater fish, Channa punctatus used as a vertebrate model, by cytogenetic endpoints: chromosome aberration (CA) and micronucleus (MN) test. In the first set of experiment, fish were exposed by medium treatment to a single treatment of each chemical (PCP, 0.6 ppm; 2,4-D, 75 ppm; neem extract, 3 ppm) along with the controls. The chromosome preparations were made after processing kidney cells and micronucleus slides were prepared from peripheral blood at multiple duration (48, 72 and 96 h). PCP and 2,4-D when used alone, induced significant CA and MN in a time dependent manner. Neem extract did not show genotoxic potential in both assays. The maximum frequency of CA were recorded as 18.58% and 15.17%, while frequency of MN reached to 8.08% and 4.62% by PCP and 2,4-D respectively, after 96 h exposure. In the second set of experiment, three concentrations of neem extract (1, 2 and 3 ppm) were run simultaneously with the same concentration of PCP (0.6 ppm) and 2,4-D (75 ppm) for antimutagenicity estimates. In mixed treatment, neem extract significantly reduced the frequency of CA and MN. The reduction in the frequency of CA ranged from 40-75% and 45.4-83.3% and similar values for MN were 40.2-75.3% and 44.1-65.8% for PCP and 2,4-D respectively. Although the reductions were significant but not dependent on concentration and time intervals employed. Results suggested that under present experimental conditions, neem extract exhibit strong antimutagenic activity in this fish model, which could further contribute to study its benefit in humans.

Analysis of glycated insulin by MALDI-TOF mass spectrometry.

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.

Studies on lethal concentrations and toxicity stress of some xenobiotics on aquatic organisms.

Three widely used xenobiotics pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (Butachlor) are evaluated for acute toxicity and stress behavior on freshwater fish (Heteropneustes fossilis, Clarias batrachus, Channa punctatus) and mosquito larvae (Culex pipiens fatigans). The experiment was carried out by medium treatment using intermittent flow-through system. Median lethal concentrations (LC50) were calculated by probit analysis. The LC50 values and 95% confidence intervals showed variable range for tested chemicals. Mosquito larvae generally appeared resistant than fish, while H. fossilis was found to be most sensitive. Stress signs in the form of behavioral changes are also observed. Both types of organisms are recommended as good bioindicator for the risk assessment of aquatic environment due to chemicals tested.

Evaluation of genotoxicity of PCP and 2,4-D by micronucleus test in freshwater fish Channa punctatus.

An in vivo study on the genotoxic effects of pentachlorophenol (PCP) and 2,4-dichlorophenoxyacetic acid (2,4-D) was carried out with freshwater air-breathing fish Channa punctatus. The fish were exposed to three sublethal doses of PCP (0.2, 0.4, and 0.6 ppm) and 2,4-D (25, 50, and 75 ppm) by medium treatment. Micronucleated erythrocytes were sampled at intervals of 48, 72, and 96 h post-treatment. Student's t test revealed significant increases in micronucleus (MN) frequency. Maximum incidence of MN was recorded at highest concentrations and durations for both chemicals. A time- and dose-dependent response of the MN frequency for both chemicals was confirmed. PCP-treated groups had 8-10 MN per cell, specifically at higher concentrations and duration. Echinocytes were recorded at higher concentrations of 2,4-D. The study confirmed the genotoxicity of PCP and 2,4-D in this organism. PCP was found to be more toxic than 2,4-D in terms of MN induction. This assay has further potential for detecting genotoxic compounds in aquatic ecosystems.

Computerized automated morphometric assay including frequency estimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis.

An in vivo study of the effects of pentachlorophenol was carried out with a pre-acclimatized fish species, Heteropneustes fossilis, using four sub-lethal concentrations, 0.1, 0.2, 0.3 and 0.4 ppm, and three sampling times, 48, 72 and 96 h. Cytogenetic preparations were stained by the haematoxylin-eosin technique. The incidence of micronuclei was scored by a manual and an automated method. Small-sized micronuclei appeared in the cytoplasm in addition to the main nucleus. The frequency of micronucleated erythrocytes peaked at 4 days (96 h) exposure. The percentage of single micronuclei increased with longer exposures. The Mann-Whitney U test showed all micronuclei frequencies were significantly different from control (P<0.05). No statistical difference was observed between scores obtained by the manual and automated methods. A linear relationship between the percentage of micronucleated erythrocytes and dose was confirmed at all levels. Computer image analysis of morphological variations of erythrocytes indicated a 1:5 ratio of micronuclei and main nucleus accompanied by a reduction in cell volume by 600 dot units. Pentachlorophenol-mediated genotoxicity was confirmed in this fish for the first time. Possible consequences of genotoxicity and cytotoxicity are discussed.