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

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

111-118<span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension in to subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted sc in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, <span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (2 1 %) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as <span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation. </span

N-glycans and metastasis in galectin-3 transgenic mice

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3 (wild type), gal-3 (hemizygous) and gal-3 (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3 mice which showe

Understanding the Role of Keratins 8 and 18 in Neoplastic Potential of Breast Cancer Derived Cell Lines

<div><h3>Background</h3><p>Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood.</p> <h3>Methodology/Principal Findings</h3><p>In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, <em>in-vitro</em> invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion <em>in-vitro</em>, while MCF10A did not show any changes in cell transformation assays.</p> <h3>Conclusions/Significance</h3><p>These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.</p> </div

Analysis of change in tumorigenicity and metastatic potential on K8 up−/down- regulated clones of MDA MB 435 and MCF10A cell lines.

<p>(<b>A</b>) Representative images of NOD-SCID mice bearing tumors of vector control (K8Vc) and K8 over-expressed (K8C1) clones, 7 weeks after the injection in mammary fat pad. (<b>B</b>) Tumor growth was plotted against time (*p<0.05, **p<0.01 by student’s t-test). Results are mean of ± SE for five animals injected for each clone. (<b>C</b>) Representative images of excised lungs of animals injected with vector control (K8Vc) and K8 over-expressed (K8C1) clones. (<b>D</b>) H&E stained lung sections of animals injected with MDA MB 435 vector control clone (K8Vc) showing metastatic foci throughout the lungs and K8 over-expressed clone (K8C1) showing no metastasis. (<b>E</b>) Representative images of NOD-SCID mice injected with MCF10A vector control (MVc) and K8 down-regulated (MShC1) clones, 7 weeks after the injection in mammary fat pad. <b>Note:</b> Lungs of animals injected with MDA MB 435 vector control clone showing metastatic nodules and lungs of animals injected with K8 over-expressed MDAMB 435 clone (K8C1) showing no visible metastatic nodules.</p

Analysis of K7 expression in K8 (over-expressed) MDA-MB-435 and K8 (down-regulated) MDA-MB-468 and MCF10A clones.

<p>Western blot analysis of K7 using mAb to K7 (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1, C2 and C3) and vector control (K8Vc) clones. (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>C</b>) MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones. β-actin was taken as loading control. <b>Note:</b> K7 up-regulation in K8 down-regulated MDA MB 468 clones. B).</p

Analysis of change in <i>in-vitro</i> invasion on K8 up−/down-regulation.

<p>Representative 10X images of H&E stained membrane showing invaded cells. (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of invaded cells of MDA MB 435 K8 overexpressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of invaded cells of MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) K8 down-regulated MCF10A (MShC1) and vector control (MVc) clones. Histogram showing number of invaded cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Decreased invasion in K8 over-expressed MDA MB 435 clones (K8C1 and C2) and increased invasion in K8 down-regulated MDA MB 468 clones (ShC1, C2, and C3) as compared to their respective vector controls (K8Vc) and (Vc).</p

Analysis of changes in soft agar colony forming potential and cell proliferation on K8 up−/down-regulation.

<p>Representative phase contrast images (10X) of colonies formed in soft agar (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of colonies of MDA MB 435 K8 overexpressed (K8C1, and C2) and vector control (K8Vc) clones (right hand side). (<b>C</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of colonies of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (right hand side). (<b>E</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clones. Histogram showing number of colonies of MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones (right hand side). <b>Note:</b> Increase in soft agar colonies formed in K8 down-regulated MDA MB 468 clones (*p<0.05 by students t-test). Cell proliferation curves of (<b>B</b>) MDA MB 435 K8 overexpressed (K8C1, C2 and C3) and vector control (K8Vc) clones. (<b>D</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>F</b>) MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones. Cell proliferation was plotted against time. Results are mean ± SE of three independent experiments performed in triplicate. <b>Note:</b> Decrease in proliferation in K8 over-expressed clones of MDA MB 435 (*p<0.05, **p<0.01 by students t-test).</p

Analysis of change in motility on K8 up−/down-regulation.

<p>Representative 10X Phase contrast images of time lapse microscopy at 0 hour and 20 hours showing wound healing (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing % wound closure at the end of 20 hours of MDA MB 435 K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing % wound closure at the end of 20 hours of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing % wound closure at the end of 20 hours of MCF10A K8 down-regulated (MShC1, C2and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed. Migration rate was calculated by AxioVision software. <b>Note:</b> Significant Increase in motility in K8 down-regulated MDA MB 468 clones as compared to vector control. Motility of K8 up−/down-regulated clones by transwell assay: Representative images of H &E stained migrated cells (<b>D</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 435K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>E</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 468 K8 knockdown clones (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>F</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing number of migrated cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Significant decrease in motility in K8 over-expressed MDA MB 435 clones and significant increase in motility in K8 down-regulated MDA MB 468 clones (**p<0.01 by students t-test).</p