ABSTRACT
In the recent years, yeasts have evolved as potent bioremediative candidates for the detoxification of xenobiotic compounds found in the natural environment. Candida sp. are well-studied apart from Saccharomyces sp. in heavy metal detoxification mechanisms. In the current study, Candida parapsilosis strain ODBG2, Candida sp. strain BANG3, and Candida viswanathii strain ODBG4 were isolated from industrial effluents and contaminated ground water, and were studied for their metal tolerance. Among these three isolates, the metal tolerance was found to be more towards Lead (Pb 2 mM), followed by Cadmium (Cd 1.5 mM) and Chromium [Cr(VI), 1 mM]. On further exploring the involvement of primary defensive enzymes in these isolates towards metal tolerance, the anti-oxidative enzyme superoxide dismutase was found to be prominently high (25% with respect to the control) during first 24 h of metal-isolate interaction. The Catalase enzyme assay was observed to have increased enzyme activity at 48 h. It also triggered the activity of peroxidases, which lead to the increase in reduced glutathione in the organism by 0.87-1.9-fold as a metal chelator and also as a second-line defensive molecule. The exoproteome profile showed the early involvement (exponential growth phase) of secreted proteins (low-molecular-weight) of about ~ 40-45 kDa under Cd and Pb stress (0.5 mM). The exoproteome profiling under heavy metal stress in Candida parapsilosis strain ODBG2 and Candida viswanathii strain ODBG4 is the first report.
Subject(s)
Metals, Heavy , Saccharomyces cerevisiae , Cadmium/toxicity , Chromium , Metals, Heavy/toxicity , Oxidative StressABSTRACT
BACKGROUND: Altered expression of N-glycans such as polylactosamine is observed in colon cancer. AHL, a polylactosamine specific lectin from Adenia hondala from a medicinal plant from the Passifloraceae family has been reported earlier. OBJECTIVE: The aim of the present study is to study the interaction of AHL with human colon cancer epithelial HT-29 cells and colon cancer tissues. METHODS: Cell viability was determined by MTT [3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] assay, while cell surface binding, apoptosis by Annexin-V-PI assay and ROS production using DCFDA [2',7' - dichlorofluorescindiacetate] kit method were analysed by flowcytometry, immunohistochemistry was performed using biotinylated AHL, protein purification by affinity chromatography using asialofetuin-coupled Sepharose -4B column. RESULTS: AHL strongly binds to HT-29 cells with a Mean Fluorescence Intensity of 12.4, which could be blocked by competing glycoprotein asialofetuin. AHL inhibits HT-29 cell growth in a dose and time-dependent manner with IC50 of 2.5 µg/mL and differentially binds to human normal and cancerous tissues. AHL induces apoptosis and slight necrosis in HT-29 cells with an increase in the early apoptotic population of 25.1 and 36% for 24 h and 48 h respectively and necrotic population of 1.5 and 4.6% at 24 h and 48 h respectively as revealed by Annexin-V-PI assay. AHL induces the release of Reactive Oxygen Species in HT-29 cells in a dose-dependent manner. CONCLUSION: To the best of knowledge, this is the first report on lectin from Adenia hondala which is not a RIP with apoptotic and necrotic effects. These findings support the promising potential of AHL in cancer research.
Subject(s)
Amino Sugars/chemistry , Colonic Neoplasms/drug therapy , Lectins/chemistry , Necrosis/drug therapy , Passifloraceae/chemistry , Plant Extracts/chemistry , Polysaccharides/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , HT29 Cells , Humans , Lectins/pharmacology , Plant Extracts/pharmacology , Reactive Oxygen SpeciesABSTRACT
BACKGROUND: Lectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. METHODS AND RESULTS: Here we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49â¯kDa and 17.4â¯kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29â¯cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29â¯cells with IC50 of 3.75â¯â¯µg/ml at 48â¯h. LCL has potent antibacterial and anti fungal activity. CONCLUSION: LCL can be explored for its clinical potential.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Lantana/chemistry , Plant Lectins/pharmacology , Anti-Bacterial Agents/isolation & purification , Antifungal Agents/isolation & purification , Antineoplastic Agents/isolation & purification , Chitin/chemistry , Chitin/metabolism , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Fungi/drug effects , Fungi/growth & development , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/growth & development , HT29 Cells , Humans , Melibiose/chemistry , Melibiose/metabolism , Methylgalactosides/chemistry , Methylgalactosides/metabolism , Microbial Sensitivity Tests , Mucins/chemistry , Mucins/metabolism , Plant Extracts/chemistry , Plant Leaves/chemistry , Plant Lectins/isolation & purification , Plants, Medicinal , Protein BindingABSTRACT
Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.
