Shajrul Amin

Oxidative stress mediated Ca2+ release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells

BACKGROUND Exposure of skin to ultraviolet (UV) radiation, an environmental stressor induces number of adverse biological effects (photodamage), including cancer. The damage induced by UV-irradiation in skin cells is initiated by the photochemical generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress and consequent activation of unfolded protein response (UPR). OBJECTIVE To decipher cellular and molecular events responsible for UV-B mediated ER stress and UPR activation in skin cells. METHODS The study was performed on human skin fibroblast (Hs68) and keratinocyte (HaCaT) cells exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analyzed using Western-blotting, microscopic studies and flow cytometry. RESULTS Our results depicted that UV-B induces an immediate ROS generation that resulted in emptying of ER Ca(2+) stores inducing ER stress and activation of PERK-peIF2α-CHOP pathway. Quenching ROS generation by anti-oxidants prevented Ca(2+) release and subsequent induction of ER stress and UPR activation. UV-B irradiation induced PERK dependent G2/M phase cell cycle arrest in Hs68 and G1/S phase cell cycle arrest in HaCaT. Also our study reflects that UV-B exposure leads to loss of mitochondrial membrane potential, activation of apoptotic cascade as evident by AnnexinV/PI staining, decreased expression of Bcl-2 and increased cleavage of PARP-1 protein. CONCLUSION UV-B induced Ca(2+) deficit within ER lumen was mediated by immediate ROS generation. Insufficient Ca(2+) concentration within ER lumen developed ER stress leading to UPR activation. These changes were reversed by use of anti-oxidants which quench ROS.

In Vitro Antioxidant and Cytotoxic Activities of Arnebia benthamii (Wall ex. G. Don): A Critically Endangered Medicinal Plant of Kashmir Valley

Arnebia benthamii is a major ingredient of the commercial drug available under the name Gaozaban, which has antibacterial, antifungal, anti-inflammatory, and wound-healing properties. In the present study, in vitro antioxidant and anticancer activity of different extracts of Arnebia benthamii were investigated. Antioxidant potential of plant extracts was evaluated by means of total phenolics, DPPH, reducing power, microsomal lipid peroxidation, and hydroxyl radical scavenging activity. The highest phenolic content (TPC) of 780 mg GAE/g was observed in ethyl acetate, while the lowest TPC of 462 mg GAE/g was achieved in aqueous extract. At concentration of 700 µg/mL, DPPH radical scavenging activity was found to be highest in ethyl acetate extract (87.99%) and lowest in aqueous extract (73%). The reducing power of extracts increased in a concentration dependent manner. We also observed its inhibition on Fe2+/ascorbic acid-induced lipid peroxidation (LPO) on rat liver microsomes in vitro. In addition, Arnebia benthamii extracts exhibited antioxidant effects on Calf thymus DNA damage induced by Fenton reaction. Cytotoxicity of the extracts (10–100 µg/mL) was tested on five human cancer cell lines (lung, prostate, leukemia, colon, and pancreatic cell lines) using the Sulphorhodamine B assay.

ROLE OF HEMATOLOGICAL PARAMETERS IN DIAGNOSIS AND PROGNOSIS OF GASTRI C CARCINOMA IN KASHMIR , INDIA

Gastric cancer is the second leading cause of death in the world . More than two - thirds of the patients are dia gnosed at an advanced stage. Metastatic gastric cancer has poor prognosis with a median 5 years - survival rate of 7 %. Hematological parameters including leukocyte count, platelet count and their ratios have been used as prognostic indicators in several tum or types. The aim of the study was to examine an association of the difference in haematological parameters between gastric cancer patients and normal controls of Kashmir valley. We enrolled 210 subjects of which 110 were newly diagnosed gastric cancer ca ses and 100 were healthy controls. Participants were re cruited from hospitals, clinics and radiology department of Sh ri Maharaja Hari Singh ( SMHS) hospital Srinagar, India from May 2011 to Apr 2012 . After informed consent, all patient s were interviewed and examined and demographic and clinical information was collected. Blood samples were drawn for examination of hematological measures and for measurement of carcino embryonic antigen (CEA) . W e found the hematological parameters like: Hb (10 ± 2, P = 0.004 ) , MCV (84.25 ± 5, p = 0.01), Granulocyte % (70.04 ± 10.63, p = 0.001), Lymphocyte % (26.12 ± 10.7, p < 0.0001) , RDW (48 ± 10, p = 0.004 ) in gastric cancer patients and these were found to be highly decreased as compared to normal healthy controls where hema tological paramete rs was in normal range . Our study was statistically significant (p < 0.005). The study suggests that the hematological parameters like HB, MCV, Granuloc yte %, Lymphocyte % and RDW are de creased in gastric cancer patients and acts as a n ea rly marker in the prognosis and diagnosis of gastric cancer.

Antiproliferative Activity of Lavatera cashmeriana- Protease Inhibitors towards Human Cancer Cells

BACKGROUND Proteases play a regulatory role in a variety of pathologies including cancer, pancreatitis, thromboembolic disorders, viral infections and many others. One of the possible strategies to combat these pathologies seems to be the use of protease inhibitors. LC-pi I, II, III and IV (Lavatera cashmerian-protease inhibitors) have been found in vitro to strongly inhibit trypsin, chymotrypsin and elastase, proteases contributing to tumour invasion and metastasis, indicated possible anticancer effects. The purpose of this study was to check in vitro anticancer activity of these four inhibitors on human lung cancer cell lines. MATERIAL AND METHODS In order to assess whether these inhibitors induced in vitro cytoxicity, SRB assay was conducted with THP-1 (leukemia), NCIH322 (lung) and Colo205, HCT-116 (colon) lines. RESULTS LC-pi I significantly inhibited the cell proliferation of all cells tested and also LC-pi II was active in all except HCT-116. Inhibition of cell growth by LC-pi III and IV was negligible. IC50 values of LC-pi I and II for NCIH322, were less compared to other cell lines suggesting that lung cancer cells are more inhibited. CONCLUSION These investigations might point to future preventive as well as curative solutions using plant protease inhibitors for various cancers, especially in the lung, hence warranting their further investigation.

Type 2 diabetes and metabolic syndrome – adipokine levels and effect of drugs

Abstract Type 2 diabetes mellitus (T2DM) is a consequence of complex interactions among multiple genetic variants and environmental risk factors. This complex disorder is also characterized by changes in various adipokines. In this study, our objective was to estimate the levels of adiponectin, leptin, and resistin (ALR) in T2DM patients, besides studying the effect of various drugs on their levels. Study participants included 400 diabetic and 300 normal patients from the Department of Endocrinology and Department of Biochemistry, Govt Medical College Srinagar. Subjects were categorized under various groups, i.e., Group 1 (metformin treated) and Group 2 (glimepiride treated), and cases were also categorized as obese with T2DM (Group A), obese without T2DM (Group B), and T2DM only (Group C). The serum ALR levels were estimated by ELISA (Alere), and biochemical parameters were also evaluated before and after treatment. Adiponectin levels were found to be significantly lower in T2DM cases as compared to controls (12 ± 5.5 versus 22.5 ± 7.9 μg/ml), while leptin and resistin levels were found to be significantly higher than controls (14.3 ± 7.4 versus 7.36 ± 3.73 ng/ml) (13.4 ± 1.56 versus 7.236 ± 2.129 pg/ml). Taking the effect of drugs into consideration, the effect on adiponectin and resistin levels was found to be highly significant in Group 2 before and after treatment (11 ± 5 versus 19.2 ± 4.5 μg/ml) (13.6 ± 2.5 versus 7.3 ± 2.9 pg/ml), while more effect was observed in leptin among Group 1 (metformin)-treated cases (27 ± 15 ng/ml versus 15 ± 15 ng/ml). Further the adiponectin levels were found to be significantly lower in Group B, while leptin and resistin levels were found to be significantly higher among obese cases when compared to T2DM cases only. Glimepiride also shows more effect on FBG, HbA1c% levels, while metformin shows more effect on Lipid profile levels. From the study, it can be concluded that ALR levels are affected by use of antidiabetic drugs among which glimepiride shows more effect on adiponectin and resistin levels, while leptin gets affected more by metformin. It can also be proposed that ALR levels are not affected by diabetes only, suggesting that their alterations in T2DM may be due to obesity as we observed more ALR changes in obese cases when compared to T2DM cases, and so there might be an important link between adiposity and insulin resistance.

Pr oteases as Targets in Anticancer Therapy Using Their Inhibitors

Abstract Proteases, also known as proteolytic enzymes, are enzymes that catalyze the breakdown of proteins by hydrolysis of peptide bonds. Earlier proteases were considered as only protein degrading enzymes, however now dramatically the view has changed. Proteases are extremely important signalling molecules that are involved in numerous vital processes like apoptosis, cell growth and activation, adhesion, invasion, cell migration and metastasis, protein secretion, cellular interactions and signal transduction, phagocytosis and angiogenesis. Thus, show complete anticancer mechanism. And proteases from all six classes have been found to be involved in tumor growth and progression. Inhibitors of such proteases are emerging with promising therapeutic uses in the treatment of cancer. Protease inhibitor suppression of carcinogenesis is related to ability to effect the expression of certain oncogens and the levels of certain types of proteolytic activities. Protease inhibitors being found to be as special agents in anticancer therapy have been isolated from plants, bacteria and prepared synthetically. Under the same trend different protease inhibitors have been used and are being in clinical and pre-clinical stage. Thus, studying PIs as anticancer agents open a new field for treatment of cancer.

Inflammation: A Multidimensional Insight on Natural Anti-Inflammatory Therapeutic Compounds

Derailed inflammation causes severe damage to the normal tissues resulting in various pathological conditions such as auto-inflammatory disorders, neurodegenerative diseases and cancer. Cure of inflammatory diseases is a big challenge. Medicinal herbs used traditionally represent the best option for obtaining effective anti-inflammatory therapeutics. Present review provides a thorough insight about various pathways, consequences and therapeutic strategies of inflammation with prime focus to expose indigenous anti-inflammatory herbal compounds along with their structures and diverse range of mechanisms of action. Over hundred medicinal plants with scientifically reported anti-inflammatory properties were reviewed. Different parts of the plants like roots, stem, bark, leaves, flowers and seeds contain active compounds with potential anti-inflammatory properties. Such compounds act at multiple targets in the inflammatory response pathways and regulate multitude of chemical mediators, enzymes, genes or cellular functions to alleviate inflammation. Although a large number of antiinflammatory herbal compounds have been isolated but the mechanism of action of bulk of compounds has not been elucidated comprehensively. Besides there is need for conducting well designed clinical trials so that the promising compounds could be used as effective antiinflammatory therapeutic agents in future.

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, •OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl4)-treated animals. It was observed that CCl4-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.

Efficacy of Aqueous and Methanolic Extracts of Rheum Spiciformis against Pathogenic Bacterial and Fungal Strains.

INTRODUCTION Rheum spiciformis is a newly identified edible medicinal plant of genus Rheum. The plant is used to treat various diseases on traditional levels in Kashmir Valley, India. AIM To evaluate the phytochemical screening, antibacterial and antifungal potential of aqueous and methanolic extracts of Rheum spiciformis, a traditionally used edible medicinal plant. MATERIALS AND METHODS Methanolic and aqueous extracts of Rheum spiciformis were tested for their antimicrobial activities against six bacterial strains namely Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia coli and four fungal strains Penicillium chrysogenum, Aspergillus fumigatus, Candida albicans and Saccharomyces cerevisiae. The susceptibility of microbial strains to the two extracts was determined using agar well diffusion method. Phytochemical screening was carried out by using various standard procedures. RESULTS Methanolic extract showed potent antimicrobial activity as compared to aqueous extract at the concentrations of 10, 30, 50, 80 and 100mg/ml. The most susceptible bacterial strains were Staphylococcus aureus with zone of inhibition (25±0.10mm), Klebsiella pneumonia (23±0.25mm), Proteus vulgaris (22±0.10mm) at the concentration of 100mg/ml. Aqueous extracts at the higher concentration were found effective against Proteus vulgaris and Bacillus subtilis with zone of inhibition (17±0.24mm) and (17±0.10mm), respectively. Among fungal strains the most susceptible were Aspergillus fumigatus (21±0.10mm), Saccharomyces cerevisiae (20±0.20mm) and Penicillium Chrysogenum (17±0.15mm) at the concentration of 100mg/ml methanol extract. The zone of inhibition for aqueous extract against fungal strains ranged between 14±0.13mm to 16±0.19mm at the highest concentration of plant extract. Phytochemical analysis revealed the presence of various secondary metabolites like flavonoids, saponins, volatile oils, phenols, steroids, terpenoids and alkaloids. CONCLUSION Our results indicate that this plant has enough potential to serve as an excellent candidate for obtaining antimicrobial compounds to combat bacterial and fungal infections.

Plant-Derived Protease Inhibitors LC-pi (Lavatera cashmeriana) Inhibit Human Lung Cancer Cell Proliferation In Vitro

The objective of this study was to check the anticancer activity of purified protease inhibitors of Lavatera cashmeriana viz LC-pi I, II, III, and IV (Lavatera cashmeriana protease inhibitors) on A549 (lung) cell. It was found that LC-pi I and II significantly inhibited the proliferation of A549 cells with IC50 value of 54 μg/ml and 38μg/ml, respectively, whereas inhibition by LC-pi III and IV was negligible. LC-pi I and II were further found to inhibit formation of colonies in a dose-dependent manner. Also, both inhibitors were found to induce apoptosis causing chromatin condensation and DNA fragmentation, without loss of mitochondrial membrane potential. Cell cycle revealed a significant increase of subG0/G1 phase cells that are apoptotic cells. We also demonstrated a dose-dependent decrease in migration of A549 cells on cell migration assay by both inhibitors. Taken together, we demonstrate that LC-pi I and II inhibited proliferation through arresting cells before apoptosis, inducing apoptosis and inhibiting cell migration in human lung cancer cells, but the study warrants further investigation. Our results support the notion that plant protease inhibitors may have the potential to advance as chemopreventive agents.