Over expression of hyperplasia suppressor gene inhibits the malignant phenotype of breast cancer cell / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of over expression of human hyperplasia suppressor gene (HSG) on proliferation, invasion, apoptosis and cell cycle of human breast cancer cells and to determine the relationship between HSG and Ras-dependent signaling pathway.</p><p><b>METHODS</b>Full length HSG coding sequences were cloned into plasmid pcDNA3.0. The recombinant plasmids were transfected into MDA-MB-231, a highly malignant breast cancer cell line. Vacant pcDNA3.0 was used as the control. MTT, Matrigel transwell assay and flow cytometric analysis were used to test for proliferation, invasion, cell cycle distribution and apoptosis of tumor cells after transient transfection of HSG.GST-pulldown and Western blotting assays were performed to investigate the activity of Ras protein.</p><p><b>RESULTS</b>HSG transfection inhibited proliferation of MDA-MB-231 cells, and significantly decreased the number of invading cells in Matrigel transwell assay compared with the vector/231 group (78.5 +/- 5.8 vs. 131.1 +/- 14.5) cells. FACS analyses demonstrated that compared with the vector/231 group, up-regulation of HSG promoted breast cancer cell apoptosis [(35.8 +/- 4.8)% vs. (25.6 +/- 3.5%)] and induced G(0)/G(1) phase arrest [(56.3 +/- 2.3)% vs. (50.4 +/- 1.9%)] after transfection for 18 hours. Furthermore, GST-pulldown assay showed that over-expression of HSG remarkably decreased the activity of Ras (about 65% lower than control).</p><p><b>CONCLUSIONS</b>HSG exibits multiple anticancer functions in breast cancer cells including inhibition of proliferation and in vitro invasion, G(0)/G(1) arrest and promotion of apoptosis. Besides, inhibition of Ras-dependent signaling pathway may be involved in these processes.</p>

Expression of E-cadherin, beta-catenin, cathepsin D, gelatinases and their inhibitors in invasive ductal breast carcinomas / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>E-cadherin, beta-catenin, cathepsin D, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 are all invasion-related proteins. The expression patterns of these proteins in invasive ductal breast carcinomas, and their associations with known clinicopathological parameters, tumor recurrence and expressions of estrogen receptor (ER), progesterone receptor (PR), PS2 and c-erbB2 were not well studied in Chinese patients.</p><p><b>METHODS</b>In a set of 94 invasive ductal breast carcinomas, protein expressions of these molecular markers were investigated by immunohistochemistry, and their associations with known clinicopathological parameters, tumor recurrence and expressions of ER, PR, PS2 and c-erbB2 were also examined. In addition, the interrelationship between the expressions of these proteins were studied.</p><p><b>RESULTS</b>Preserved membrane E-cadherin expression was associated with late tumor stage and tumor recurrence, whereas the reduced junctional beta-catenin associated with positive lymph node status and c-erbB2 overexpression. Positive staining of cathepsin D in tumor stromal cells displayed a significant association with late tumor stage. High expression of MMP-2 in cancer cells was associated with large tumor size and PR positive expression. TIMP-2 expression was positively associated with tumor recurrence. In addition, inter-relationship between the expressions of these biomarkers was also assessed. Cathepsin D staining in cancer cells was inversely correlated with its staining in stromal cells, and also inversely correlated with MMP-2 staining in tumor stromal cells. MMP-2 expression in stromal cells displayed an inverse correlation with TIMP-2 expression. MMP-9 expression displayed parallel associations with TIMP-1 and TIMP-2 expression.</p><p><b>CONCLUSION</b>Evaluation of E-cadherin, beta-catenin, cathepsin D, MMP-2 and TIMP-2 expression may be of some help in more accurately predicting the prognosis of invasive ductal breast carcinomas.</p>

Effects of beta1-integrin, fibronectin and laminin on invasive behavior of human gliomas / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of beta1-integrin, fibronectin (FN) and laminin (LN) on the invasive behavior of human gliomas.</p><p><b>METHODS</b>Functional impacts of beta1-integrin, fibronectin and laminin on cell adhesion, migration and metastasis of U251 malignant glioblastoma cells were investigated by in vitro adhesion, migration and invasion assays. The amount and distributions of cellular microfilaments and pseudopodia were studied by fluorescent cytochemistry, confocal laser scanning microscope and scanning electron microscope. Lastly, beta1-integrin, fibronectin and laminin were investigated for their roles in cellular microfilament skeleton.</p><p><b>RESULTS</b>(1) Fibronectin did not affect cell adhesion of U251MG cells, but anti-beta1 integrin antibodies inhibited cell adhesion (P < 0.01); Laminin stimulated cell adhesion of U251MG cells (P < 0.01) but anti-beta1 integrin antibodies had little effect on the laminin-mediated cell adhesion. (2) The migration of U251MG cells on dishes coated with FN was inhibited by anti-beta1 integrin antibodies (P < 0.05). (3) F-actins formed strong and dense stress fibers in U251MG cells on dishes coated with FN and LN. Anti-beta1 integrin antibodies disrupted the microfilament network and F-actin aggregation. (4) FN and LN increased the number of pseudopodia on cell surface, whereas anti-beta1 integrin antibodies reversed this function. (5) FN and anti-beta1 integrin antibodies had little effects on the invasive ability of U251MG cells in vitro. The invasion was increased by LN, but inhibited by anti-beta1 integrin antibodies.</p><p><b>CONCLUSIONS</b>(1) The interaction between beta1-integrin, FN may stimulate U251MG cell migration via changing the structures of microfilament skeleton and the number of pseudopodia. (2) beta1-integrin may play a role in the LN-mediated in vitro invasion of U251MG cells.</p>

Mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells.</p><p><b>METHODS</b>Glioblastoma U87MG cells, which over-express epidermal growth factor receptor (EGFR), were transfected with antisense-EGFR constructs. Several clones with stable expression of lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR on cell differentiation was studied using morphological evaluation and western blotting analysis of glial fibrillary acidic protein (GFAP) expression. The effect of antisense-EGFR on cell cycle was studied by flow cytometry and immunohistochemical analysis of p53, Rb, p16 and CDK4 expressions. The effect of antisense-EGFR on telomerase activity was studied by telomeric repeat amplification protocol (TRAP) assay.</p><p><b>RESULTS</b>U87MG cells that were transfected with antisense-EGFR constructs had smaller cell bodies and longer processes, and expressed higher level of GFAP compared with that of the control cells. Flow cytometric analysis showed that the proportion of cells in G(0)/G(1) phases of the cell cycle in the antisense EGFR cDNA transfected clones increased significantly when compared with control cells, whereas the proportion of cells in S phase decreased markedly. In addition, immunohistochemical analysis showed that the expression of wild-type p53 was significantly increased in the antisense-EGFR cDNA transfected clones, whereas the expressions of Rb, p16 and CDK4 were not altered. TRAP assay revealed that telomerase activity in the antisense-EGFR clones was significantly decreased.</p><p><b>CONCLUSIONS</b>Antisense-EGFR transfection inhibits U87MG cell growth by inducing cell differentiation and p53 expression, G(1) cell cycle arrest and inhibition of telomerase activity.</p>