Two Chitotriose-Specific Lectins Show Anti-Angiogenesis, Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle.

The antiproliferative activity of two chito-specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI50 values were approximately 8.4 μg ml(-1) (0.247 μM) and 142 μg ml(-1) (14.8 μM) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G0/G1 arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway

Increase in intracellular [Ca²⁺]i release of lectin stimulated PANC-1 cells.

<p>PANC-1 cells were treated with lectins (<i>Bh</i>L: 0.247 and <i>Di</i>L9: 14.8 μM) for 4, 8 and 12 h, stained with Fluo-4/AM (4μM, green) and DAPI (blue). (A) Depicts the percentage of cells releasing calcium over a period of time. (B) Represents the overlay of confocal microscopy images of fluorescence intensity of cells bound with Fluo-4/AM (green) releasing calcium after 12 h of incubation. The analysis was carried out using LSCM, Magnification 20X (scale, 100 μm).</p

Alteration of mitochondrial transmembrane potential.

<p>Lectin (<i>Bh</i>L: 0.247 and <i>Di</i>L9: 14.8 μM) treated cells were incubated for 4, 8 and 12 h and stained with Mito Tracker Red (0.1 μmol l^-1) for 15 min at 37°C. (A) Quantification of average intensity of mitochondrial depolarized cells. Data are expressed as mean value ± SD, obtained from three independent experiments.(B) The overlay represents the fluorescence intensity of cells bound with Mitotracker Red, recorded by LSCM, Magnification 20X objective (scale, 100 μm). Loss in red intensity represents the loss in mitochondrial membrane potential.</p

<i>Bh</i>L and <i>Di</i>L9-induced apoptosis in PANC-1 cells.

<p>(A) Acridine orange staining of PANC-1 cells after incubation with lectins (<i>Bh</i>L: 0.247 and <i>Di</i>L9: 14.8 μM) for 24 h. The arrows indicate apoptotic bodies formed inside the lectin treated cells. Annexin V-FITC/PI staining of lectin stimulated PANC-1 cells incubated for indicated time. (B) The graph represents percentage of cells undergoing apoptosis, mean value ±SD of three independent experiments. (C) The overlay represents the cells that have undergone apoptosis (Annexin V-FITC positive, green) or necrosis (PI positive cells, red) after 72 h of incubation. The analysis was carried out using HCS 2.0 Cell Analysis Software.</p

Proposed model for mechanism of apoptosis induced by <i>Bh</i>L and <i>Di</i>L9 in PANC-1 cells.

<p>Lectins bind to the glycoprotein receptors of PANC-1 cells and triggers mitochondrial membrane depolarization cascade. The apoptotic signal was amplified by activation of caspase-9 and -3 leading to final cell death.</p

Inhibition of angiogenesis by <i>Bh</i>L and <i>Di</i>L9.

<p>HUVECs were plated on Matrigel (± LVES+VEGF) precoated 96-well plates as control. The cells were treated with lectins. The tubes were stained with rhodamine conjugated phalloidin (red) and nuclei with DAPI (blue). Anti-angiogenic activity is determined by the breakage in the tubule formation. Cell imaging was done on Cellomics’ ArrayScan HCS Reader.</p

Effect of <i>Bh</i>L and <i>Di</i>L9 on activation of caspases.

<p>The lectin (<i>Bh</i>L: 0.247 and <i>Di</i>L9: 14.8 μM) treated PANC-1, CFPAC-1 and MIA PaCa-2 cells were incubated for different time periods and activities of caspases were assessed by fluorimetric assay. (A) Caspase-8, (B) caspase-9 and (C) caspase-3 activity was measured with respect to untreated cells. This data is mean ± SD values from three independent experiments.</p

Effect of <i>Bh</i>L and <i>Di</i>L9 on different phases of cell cycle.

<p>The progressive cell cycle changes were observed with DAPI staining after 6, 12, 18 and 24 h on lectin (<i>Bh</i>L: 0.247 and <i>Di</i>L9: 14.8 μM) treatment and analyzed using HCS software. The darkened numbers indicate the percentage of cells arrested in different phases of cell cycle. The data is mean ± SD of three independent experiments.</p

Effect of <i>Bh</i>L and <i>Di</i>L9 on proliferation and cell viability.

<p>Primary cells and different cancer cell lines were treated with serial concentrations of <i>Bh</i>L and <i>Di</i>L9 and incubated for 48 h. The growth inhibition (%) was measured by MTT assay by considering untreated cells as 100%. (A) Effect of <i>Bh</i>L and (B) <i>Di</i>L9 treatment on various cell lines. The values presented in the graph are the mean ± SD of two independent experiments done in triplicates.</p