Purification and Characterization of a Mitogenic Lectin from Cephalosporium, a Pathogenic Fungus Causing Mycotic Keratitis

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent

Rhizoctonia bataticola lectin (RBL) induces caspase-8-mediated apoptosis in human T-cell leukemia cell lines but not in normal CD3 and CD34 positive cells.

We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins--Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia

RBL induces apoptosis of Molt-4 and Jurkat cells.

<p>(A) Molt-4 cells were treated with RBL (5 µg/ml) for 6, 12 and 24 h followed by Annexin- V-FITC and PI staining. The X-axis depicts Annexin-V positive cells and Y- axis depicts PI positive cells. The numbers in each quadrant represent percent positive cells. Dot plots are representative of three similar experiments. (B) The graph represents % apoptosis (Annexin-V-positive cells) mean±SE of three independent experiments. *p<0.05 indicates significant difference between treated and untreated cells. (C) Whole cell lysates of Molt-4 and Jurkat cells were exposed to RBL (1.25 to 5 µg/ml) for 24 h and probed with anti-PARP antibody. The blot shows total and cleaved PARP. The data is representative of two similar experiments.</p

Binding of RBL and inhibition with sugars.

<p>The binding of FITC- labeled RBL to Molt-4 (A) and Jurkat (B) cells was determined by flow cytometry analysis. The histoplots depict the profiles of unstained cells (shadow) and cells stained with FITC-RBL (green). The binding of RBL to cell membrane of Molt-4 (A inset) and Jurkat (B inset) was visualized by confocal laser scanning microscopy. Original magnification 60×. (C) Molt-4 cells were stained with FITC-labeled RBL alone or RBL pre-incubated with different sugars and flow cytometry analysis was performed. X-axis represents fluorescence intensity, Y-axis represents cell number. The overlay shows profiles of the unstained cells (shadow), RBL stained cells (green) and cells stained with RBL +Mucin (red), +asialofetuin (orange), +GalNAc (blue), +glucose (pink), and sucrose +(yellow). (D) The graph represents mean MFI±SE from three independent experiments. *p<0.05 difference in MFI compared to cells stained with RBL alone.</p

Effect of RBL on proliferation and viability of leukemic cell lines-.

<p>Molt-4 (A) and Jurkat (B) cells were exposed to serial concentrations of RBL for 72 h and proliferation was determined by tritiated thymidine incorporation assay. The percentage cell proliferation was calculated by considering the counts per minute of untreated control cells as 100. Molt-4(C) and Jurkat (D) cell lines were exposed to serial concentrations of RBL for different time periods and cell viability was assessed by MTT assay. The absorbance value of untreated cells was considered as 100 to calculate percent viable cell number. The values presented in the graph are mean±SE of three independent experiments done in triplicates. *p<0.05 difference compared to untreated cells.</p

Effect of RBL on HUT-78 cells.

<p>(A) HUT-78 cells were exposed to RBL (5 µg/ml) and cell viability was assessed by MTT assay. The absorbance of untreated cells was considered as 100 to calculate percent viable cell number. The values presented in the graph are mean±SE of three independent experiments done in triplicates. *p<0.05 difference compared to untreated cells. (B) Cells treated with RBL were stained with PI and cell cycle analysis was done with data acquired on FL2-A channel. The X-axis represents the DNA content of the cells and the Y-axis represents the cell number. (C) The graph depicts the percentage of cells in different phases of cell cycle. The graph represents mean ±SE of three independent experiments. *p<0.05 significant difference between untreated and RBL-treated cells.</p

Effect of RBL on activation of caspases.

<p>Molt-4 cells were treated with 5 µg/ml RBL for different time periods and activities of caspase-8 (A) and caspase-9 (B) were assessed by colorimetric assay using specific substrates Ac-IETD-pNa and Ac-LEHD-pNa respectively. The graph depicts fold increase in caspase activity with respect to 0 h controls (activity at 0 h was considered as 1). The data is mean±SE values from three independent experiments. *p value <0.05 in comparison with 0 h controls. (C) Molt-4 cells were treated with RBL (5 µg/ml) for 12 h and activity of caspase-3 was determined by flow cytometry analysis using FITC tagged caspase–3 inhibitor (blue line). Cells pretreated with caspase-3 inhibitor ZVAD-FMK (red line) and untreated cells (green line) were used as controls. The histoplot is representative of three similar experiments.</p

Effect of caspase-8 inhibition on RBL-induced apoptosis.

<p>Molt-4 (A) and Jurkat cells (B) were treated with RBL(5 µg/ml) for 12 h in the presence or absence of caspase inhibitors(40 µM) zVAD-FMK (pan caspase inhibitor), zIETD-FMK (caspase-8 inhibitor), or zLEHD-FMK (caspase-9 inhibitor) and viability was assessed by MTT assay. The graphs depict mean±SE values from three independent experiments. *p value <0.05 in comparison with untreated controls. Molt-4 (C) and Jurkat (D) cells were treated with RBL (5 µg/ml) for 12 h in the presence or absence of caspase-8 inhibitor (zIETD-FMK) and caspase-3 activation was assessed using flow cytometry. The overlay depicts profile of untreated cells (black line), cells pretreated with caspase-3 inhibitor followed by RBL exposure (red line) and cells treated with RBL alone (blue line).</p