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1.
Arch Microbiol ; 204(11): 673, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36255493

RESUMO

A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non-reducing conditions. PCL is a blood group non-specific lectin and has highest affinity towards chitin, mucin, asialomucin, fetuin with a MIC of 0.15 µg/mL and also recognizes L-fucose, galactose, lactose, N-acetyl galactosamine, hyaluronic acid. PCL is stable up to 60 °C and within the pH range 4-8. To understand its role in pathogenesis, effect of PCL was evaluated on human corneal epithelial cells (HCECs). PCL showed strong glycan mediated binding to HCECs and PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.


Assuntos
Antígenos de Grupos Sanguíneos , Ceratite , Humanos , Lectinas , Fucose , Galactose , Lactose , Sulfato de Amônio/metabolismo , Sefarose , Ácido Hialurônico , Interleucina-6 , Ceratite/microbiologia , Quitina/metabolismo , Fetuínas , Mucinas/metabolismo , Misturas Complexas , Galactosamina
2.
Glycoconj J ; 38(6): 669-688, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34748163

RESUMO

A N-glycan specific lectin from Rhizoctonia bataticola [RBL] was shown to induce growth inhibitory and apoptotic effect in human ovarian, colon and leukemic cells but mitogenic effect on normal PBMCs as reported earlier, revealing its clinical potential. RBL has unique specificity for high mannose tri and tetra antennary N-glycans, expressed in ovarian cancer and also recognizes glycans which are part of CA 125 antigen, a well known ovarian cancer marker. Hence, in the present study diagnostic and therapeutic potential of RBL was investigated using human ovarian epithelial cancer SKOV3 and OVCAR3 cells known for differentially expressing CA 125. RBL binds differentially to human ovarian normal, cyst and cancer tissues. Flow cytometry, western blot analysis of membrane proteins showed the competitive binding of RBL and CA 125 antibody for the same binding sites on SKOV3 and OVCAR3 cells. RBL has strong binding to both SKOV3 and OVCAR3 cells with MFI of 173 and 155 respectively. RBL shows dose and time dependent growth inhibitory effect with IC50 of 2.5 and 8 µg/mL respectively for SKOV3 and OVCAR3 cells. RBL induces reproductive cell death, morphological changes, nuclear degradation and increased release of ROS in SKOV3 and OVCAR3 cells leading to cell death. This is also supported by increase in hypodiploid population, altered MMP leading to apoptosis possibly involving intrinsic pathway. Adhesion, wound healing, invasion and migration assays demonstrated anti-metastasis effect of RBL apart from its growth inhibitory effect. These results show the promising potential of RBL both as a diagnostic and therapeutic agent.


Assuntos
Antígeno Ca-125 , Neoplasias Ovarianas , Apoptose , Ascomicetos , Antígeno Ca-125/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Lectinas/metabolismo , Lectinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia
3.
Protein Pept Lett ; 28(10): 1108-1114, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34137358

RESUMO

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.


Assuntos
Amino Açúcares/química , Neoplasias do Colo/tratamento farmacológico , Lectinas/química , Necrose/tratamento farmacológico , Passifloraceae/química , Extratos Vegetais/química , Polissacarídeos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Humanos , Lectinas/farmacologia , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio
4.
Glycoconj J ; 38(4): 509-516, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34146213

RESUMO

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.


Assuntos
Acremonium/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/citologia , Ceratite/microbiologia , Lectinas/toxicidade , Apoptose , Linhagem Celular , Proliferação de Células , Humanos , Ceratite/patologia , Lectinas/imunologia , Fator 88 de Diferenciação Mieloide
5.
Cell Biochem Funct ; 39(3): 401-412, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33527486

RESUMO

The L-fucose-specific lectin from Aspergillus niger (ANL), isolated from the corneal smears of a keratitis patient was reported earlier. Here, we examined the interaction of ANL with human hepatocellular and colon cancer cells, evaluated its anti-cancer activity and diagnostic potential to detect aberrantly glycosylated tumour-associated serum glycoproteins such as alpha-fetoprotein (AFP). We observed that ANL strongly bound to both HepG2 and HT-29 cell-lines and this interaction was effectively blocked with L-fucose and mucin in a dose and time-dependent manner with an IC50 of 1.25 and 5 µg/mL for HepG2 and HT-29 cells respectively at 48 hours. ANL treatment increased hypodiploidy and decreased the number of HepG2 cell in G0 -G1 phase at both 24 and 48 hours. Furthermore, ANL increased the level of apoptosis in both HepG2 and HT-29 cells in a time-dependent manner via enhanced production of reactive oxygen species and altered mitochondrial membrane potential, indicative of intrinsic apoptotis pathway activation. Immunoblot analysis confirmed the time-dependent elevation of levels of cytochrome c, initiator caspase-9 and activation of caspase-3. ANL immunohistochemistry on colon cancer tissue and quantification of AFP in HCC patient serum samples by developing an ANL-anti-AFP antibody sandwich enzyme-linked immunosorbent assay confirmed the diagnostic potential of ANL. Here, interaction of ANL with AFP could be effectively blocked in the presence of competing fucose-bearing glycans. We found ANL to be more sensitive than Lens culinaris lectin, a well-known fucose-specific lectin and currently used diagnostic agent. ANL can be further explored as a diagnostic and anti-cancer agent.


Assuntos
Antineoplásicos , Aspergillus niger/química , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Proteínas Fúngicas , Lectinas , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Células HT29 , Células Hep G2 , Humanos , Lectinas/química , Lectinas/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia
6.
Int J Biol Macromol ; 165(Pt B): 2089-2095, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33045300

RESUMO

An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.


Assuntos
Apoptose/efeitos dos fármacos , Aspergillus niger/química , Células Epiteliais/patologia , Epitélio Corneano/patologia , Lectinas/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Fucose/metabolismo , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucinas/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais
7.
Glycoconj J ; 37(4): 435-444, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32367479

RESUMO

Cephalosporium curvulum lectin (CSL), a lectin from pathogenic fungus has exquisite specificity towards α1-6 linkage of core fucosylated glycans, expressed in hepatocellular and pancreatic cancer. Interaction and effect of CSL and other fucose specific lectins LCA and AOL on HepG2 and PANC-1 cells was investigated. CSL, LCA and AOL exhibited strong binding to PANC-1 cells which could be effectively blocked by competing glycoprotein mucin. Effect of CSL, LCA and AOL on PANC-1 and HepG2 cells was determined by MTT assay and all the three lectins inhibited the cell growth which could be blocked by mucin, cell cycle analysis revealed that CSL increased hypodiploid HepG2 cell population indicating cellular apoptosis. CSL induced apoptosis in HepG2 cells was confirmed by Annexin V/PI assay. CSL induced increase in early apoptotic HepG2 cell population, a time dependent increase in the expression of caspases-3, 9 and cytochrome-c was observed by western blotting suggesting the possible involvement of intrinsic caspase dependent apoptosis. Increase in ROS and decrease in MMP demonstrated involvement of intrinsic caspase dependent apoptosis. Quantification of AFP in HCC patients using CSL lectin-antibody sandwich ELISA, supports diagnostic potential of CSL.


Assuntos
Acremonium/química , Ensaio de Imunoadsorção Enzimática/métodos , Lectinas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , alfa-Fetoproteínas/análise , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fucose/metabolismo , Células Hep G2 , Humanos , Lectinas/química , Lectinas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo
8.
Glycoconj J ; 37(2): 251-261, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31900725

RESUMO

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 µg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 µg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.


Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Antineoplásicos/farmacologia , Basidiomycota/química , Proteínas Fúngicas/farmacologia , Lectinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Células Jurkat , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/fisiologia , Ligação Proteica
9.
Protein Expr Purif ; 170: 105574, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31978534

RESUMO

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 14.49 kDa and 17.4 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 HT29 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 HT29 cells with IC50 of 3.75  µg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. CONCLUSION: LCL can be explored for its clinical potential.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Lantana/química , Lectinas de Plantas/farmacologia , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Quitina/química , Quitina/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Células HT29 , Humanos , Melibiose/química , Melibiose/metabolismo , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Testes de Sensibilidade Microbiana , Mucinas/química , Mucinas/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Lectinas de Plantas/isolamento & purificação , Plantas Medicinais , Ligação Proteica
10.
Int J Biol Macromol ; 134: 487-497, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31051203

RESUMO

An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30 kDa by SDS-PAGE and 31.6 kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50 °C and over a pH range of 7-10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125 µg for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25 µg/ml at 48 h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85 µg/ml at 48 h revealing promising clinical potential of ANL.


Assuntos
Aspergilose/microbiologia , Aspergillus niger/química , Fucose/metabolismo , Ceratite/microbiologia , Lectinas/isolamento & purificação , Lectinas/metabolismo , Animais , Linhagem Celular Tumoral , Eritrócitos , Humanos , Concentração de Íons de Hidrogênio , Lectinas/química , Camundongos , Peso Molecular , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura
11.
Nutr Cancer ; 71(4): 634-642, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30672325

RESUMO

TF antigen binding lectins from dietary sources PNA, ACA, ABL, JAC, and SRL from Sclerotium rolfsii have been reported to induce diverse effects on cancer cell proliferation by different mechanisms. This study aimed to compare effects of these lectins on growth and cell cycle progression in colon cancer HT29 and SW620 cells. As reported SRL, ABL, and JAC inhibited while PNA and ACA increased cell proliferation. ABL and JAC treated HT29 cells showed increased cell population in G0/G1 phase. PNA, ACA, ABL, and JAC increased SW620 cell population in S and decreased in G2/M phase. In contrast, SRL and JAC increased hypodiploid population in both the cells. PNA and ACA reduced whereas SRL and ABL diminished cell cyclin D1 expression. SRL, PNA, and ACA also reduced cellular cyclin D3 level while SRL, ABL, and JAC reduced cyclin E levels. ABL decreased CDK5 levels while SRL and ACA completely abolished CDK5 expression. All the lectins completely abolished cyclin D2 expression. These results not only confirms growth regulatory effects of TF-binding lectins but also indicates different effects of these lectins on cell growth is associated with regulation on expression of cell cycle associated proteins in G1-S phase and on cell cycle progression.


Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Lectinas/farmacologia , Amaranthus/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Arachis/química , Basidiomycota/química , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Ciclina D3/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Células HT29 , Humanos , Lectinas/isolamento & purificação , Lectinas/metabolismo
12.
Glycoconj J ; 35(6): 511-523, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30306293

RESUMO

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.


Assuntos
Acetilgalactosamina/metabolismo , Lectinas/isolamento & purificação , Passifloraceae/química , Açúcares/metabolismo , Acetilgalactosamina/química , Animais , Desoxirribonucleases/metabolismo , Haptenos/metabolismo , Hemaglutinação , Células Hep G2 , Humanos , Lectinas/química , Peso Molecular , Monossacarídeos/análise , Raízes de Plantas/química , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
13.
Chem Biol Drug Des ; 92(2): 1488-1496, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29675931

RESUMO

Expression of altered glycans such as TF, Tn, and sTn antigens has been observed in a number of carcinomas which are targeted in cancer therapy. Sclerotium rolfsii lectin (SRL) is known to recognize TF and its substituted forms. Clinical potential of SRL has been demonstrated by studying its interaction with different types of cancer cells. Here we report, in vitro studies of SRL on breast cancer MDA-MB-468 cells and in vivo studies with MCF-7 xenografts. In vitro growth inhibitory studies of SRL on metastatic triple negative breast cancer MDA-MB-468 cells were performed by MTT assay, flow cytometry, adhesion, and CAM assay. In vivo efficacy studies of SRL were performed using NOD SCID mice bearing MCF-7 xenografts. SRL has strong binding to MDA-MB-468 cells with MFI of 85.5 and has growth inhibitory effect with IC50 of 32 µg/ml at 48 hr. SRL has antiangiogenesis effect and also anti adhesive effect with fibronectin and collagen at 20 µg/ml by 36% and 42%, respectively. In vivo efficacy studies of SRL on NOD SCID mice bearing MCF-7 xenogratfs revealed 61.77% and 75.71% tumor regressing effect, respectively, at 20 and 30 mg/kg body weight without any toxicity. All these results substantiate clinical potential of SRL on breast cancer.


Assuntos
Basidiomycota/metabolismo , Neoplasias da Mama/tratamento farmacológico , Lectinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Rim/efeitos dos fármacos , Rim/patologia , Lectinas/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Fisiológica/efeitos dos fármacos , Transplante Heterólogo
14.
J Cell Biochem ; 119(7): 5632-5645, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29384227

RESUMO

The correlation between colorectal cancer (CRC) progression and altered expression of N-glycans can be considered in search for new biomarkers and anticancer agents to control CRC. Earlier N-glycan specific mitogenic lectin from Rhizoctonia bataticola (RBL) has been reported which has growth inhibitory and apoptotic effect on human ovarian and leukemic cells, but mitogenic effect on normal PBMCs revealing its clinical potential. Here, we report the effect of RBL on human colon cancer HT 29, SW480, and SW620 cell growth and its differential binding to human normal colon and cancer tissues. RBL has strong binding to both primary and metastatic colon cancer cells with MFI of 403, 404, and 192, respectively for HT 29, SW480, and SW620 cells. RBL shows dose and time dependent growth inhibitory effect with IC50 of 5, 6.4, and 6.8 µg/mL, respectively for HT 29, SW480, and SW620 cells. RBL inhibited the clonogenicity of colon carcinoma cells. RBL arrests metastatic SW620 cell growth at S phase, increased hypodiploid population by 6.1%, 14.3%, and 23.2%, respectively at 12, 24, and 36 h. Further, RBL induces SW620 cell apoptosis in time dependent manner, showed increased release of ROS and nuclear degradation compared to lectin untreated control. Adhesion, wound healing, invasion, and migration assays demonstrated anti-metastasis effect of RBL in SW620 cells apart from its growth inhibitory effect. Anti angiogenic effect of RBL was demonstrated by CAM assay. All these results show the promising potential of RBL both as diagnostic and therapeutic agent.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Lectinas/farmacologia , Mitógenos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Rhizoctonia/metabolismo , Animais , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Humanos , Metástase Neoplásica , Células Tumorais Cultivadas
15.
Biomed Pharmacother ; 93: 654-665, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28686979

RESUMO

Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells.


Assuntos
Antineoplásicos/farmacologia , Araceae/química , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Lectina de Ligação a Manose/farmacologia , Invasividade Neoplásica/patologia , Polissacarídeos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Galinhas , Feminino , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
16.
Int J Biol Macromol ; 102: 1146-1155, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28472687

RESUMO

Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research. SDS-PAGE showed that lectin is a monomer of Mr 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC50 of 110µg, 9.8µg, 40µg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.


Assuntos
Dioscorea , Lectina de Ligação a Manose/isolamento & purificação , Lectina de Ligação a Manose/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Células HT29 , Haptenos/metabolismo , Hemaglutinação/efeitos dos fármacos , Humanos , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/metabolismo , Peso Molecular , Coelhos , Especificidade por Substrato
17.
Oncol Rep ; 37(5): 2803-2810, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28394001

RESUMO

Sclerotium rolfsii lectin (SRL) is a lectin isolated from the fungus Sclerotium rolfsii and has exquisite binding specificity towards the oncofetal Thomsen-Friedenreich antigen (TF-Ag; Galß1-3GalNAcα-O-Ser/Thr) and its derivatives. Previous studies have shown that SRL inhibits the proliferation of human colon, breast and ovarian cancer cells in vitro and suppresses tumour growth in mice when introduced intratumourally. The present study assessed the effect of SRL on tumour growth when introduced intraperitoneally in BALB/c nude mice and investigated the pharmacokinetics and biodistribution of SRL in Swiss albino mice. When 9 doses of SRL (30 mg/kg body weight/mice) was administered to BALB/c nude mice bearing human colon cancer HT-29 xenografts, a substantial reduction in tumour size was observed. A 35.8% reduction in tumour size was noted in the treated animals after 17 days. SRL treatment also inhibited angiogenesis, and the tumours from the treated animals were observed to carry fewer blood vessels and express less angiogenesis marker protein CD31, than that from the control animals. Pharmacokinetics and biodistribution analysis revealed that SRL was detected in the serum after 1 h and its level peaked after 24 h. SRL was not detected in any of the organs apart from the kidney where a trace amount was detected after 24 h of SRL injection. No significant changes were observed in any of the biochemical parameters tested including SGOT, SGPT, LDH, CREAT and BUN in the SRL-treated mice compared to these levels in the controls. This suggests that SRL has good potential to be developed as a therapeutic agent for cancer treatment and warrant further investigations in vivo and subsequent clinical trials.


Assuntos
Antineoplásicos/administração & dosagem , Basidiomycota/metabolismo , Neoplasias do Colo/tratamento farmacológico , Lectinas/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/metabolismo , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/farmacocinética , Células HT29 , Humanos , Lectinas/farmacocinética , Camundongos , Camundongos Nus , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Glycoconj J ; 33(1): 19-28, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26514868

RESUMO

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.


Assuntos
Acremonium/química , Fucose/análogos & derivados , Glucanos/metabolismo , Lectinas/metabolismo , Sequência de Carboidratos , Glucanos/química , Células HT29 , Células Hep G2 , Humanos , Dados de Sequência Molecular , Ligação Proteica
19.
Glycobiology ; 25(12): 1375-91, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26347523

RESUMO

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galß1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.


Assuntos
Antineoplásicos/farmacologia , Proteínas Fúngicas/farmacologia , Lectinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transcriptoma , Agaricales/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Humanos
20.
Molecules ; 20(6): 10848-65, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26076107

RESUMO

SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galß1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.


Assuntos
Basidiomycota/genética , Clonagem Molecular , Variação Genética , Lectinas/química , Lectinas/genética , Modelos Moleculares , Sequência de Aminoácidos , Basidiomycota/metabolismo , Metabolismo dos Carboidratos , Carboidratos/química , Ligação de Hidrogênio , Lectinas/isolamento & purificação , Lectinas/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato
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