Angiotensin II Type 1 Receptor (AT-1R) Expression Correlates with VEGF-A and VEGF-D Expression in Invasive Ductal Breast Cancer

Recent studies point to the involvement of angiotensin II (Ang II) receptor type 1 (AT-1R) on processes of metastasing, stimulation of invasiveness and angiogenesis in tumours. In this study, the correlation between intensity of AT-1R expression and expression of lymph- and angiogenesis markers in invasive ductal breast cancers (IDC) was examined. Immunohistochemical studies (IHC) were performed on archival material of 102 IDC cases. Only 28 (27.5%) cases manifested low AT-1R expression while 74 (72.5%) cases demonstrated a moderate or pronounced AT-1R expression. Expression intensity of AT-1R was found to correlate with expressions of VEGF-A (r = 0.26; p = 0.008) and VEGF-D (r = 0.24; p = 0.015). Out of the examined markers of angiogenesis and lymphangiogenesis only the pronounced expression of VEGF-C was found to correlate with patient poor clinical outcome (p = 0.009). The positive correlation between AT-1R and VEGF-A and VEGF-D could point to stimulatory action of Ang II on their expression what might result in augmented lymph- and angiogenesis in IDC

Galactosylceramide affects tumorigenic and metastatic properties of breast cancer cells as an anti-apoptotic molecule.

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ

Response to doxorubicin of MDA/LUC-shUGT8 cells with silenced expression of UGT8 gene and control MDA/LUC cells.

<p>(A) Western blot analysis of anti-caspase-3 mAbs binding to cellular proteins of control MDA/LUC and MDA/LUC-shUGT8 cells with decreased expression of UGT8, grown at 70% confluence in the presence of increasing amounts of doxorubicin (0.005–0.5 µM) for 48 h. Cell lysates, equivalent to 40 µg protein, were separated by SDS-PAGE under reducing conditions on a 13% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control. (B) Survival of control MDALUC cells and MDA/LUC-shUGT8 cultured in the presence of increasing concentrations of doxorubicin (0.001–1.0 µM) for 48 h. Cell viability was determined using MTT assay as described in the “Materials and Methods”. Data represent the mean ± SD of six replicates from two independent experiments. (^*p = 0.0472, two-way ANOVA test).</p

Immunohistochemical analysis of tumor xenografts of MDA/LUC-shUGT8 cells with silenced expression of UGT8 gene and control MDA/LUC cells.

<p>(A) Immunohistochemical staining with monoclonal antibody against Ki67 antigen of tumor sections after subcutaneous implantation of control MDA/LUC cells (I) and sh-transduced MDA/LUC-shUGT8 cells with decreased expression of UGT8 (II) into nu/nu mice. The numbers of Ki67-positive cells in MDA/LUC (I) and sh-transduced MDA/LUC-shUGT8 tumors were compared by Mann-Whitney U-test (^***p<0.0001). (B) TUNEL technique after subcutaneous implantation of MDA-M/LUC cells (I) and MDA/LUC-shUGT8 cells (II) breast cancer cells into nu/nu mice. Original magnification: x100. The numbers of apoptotic cells in MDA/LUC (I) and sh-transduced MDA/LUC-shUGT8 tumors were compared by Mann-Whitney U-test (^*p = 0.0432).</p

Characteristic of MDA-MB-231/LUC-shUGT8 cells with silenced UGT8 gene expression (A).

<p>Expression of UGT8 and GCS mRNAs in control MDA-MB-231 cells transduced with vector alone (MDA/LUC) and MDA-MB-231 cells tranduced with pLVTHM/LUC-shUGT8 construct (MDA/LUC-shUGT8). UGT8 and GCS levels were normalized against β-actin. (B) Western blot analysis of anti-UGT8 rabbit polyclonal antibodies binding to cellular proteins of control MDA/LUC and sh-transduced MDA/LUC-shUGT8 cells. Cell lysates, equivalent to 40 µg protein, were separated by SDS-PAGE under reducing conditions on a 10% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control. (C) Immunostaining of neutral glycolipids from control MDA/LUC and sh-transduced MDA/LUC-shUGT8 breast cancer cell lines, separated by HP-TLC, with anti-GalCer rabbit polyclonal antibodies. For the analyzed cell lines, an aliquot of total neutral glycolipids corresponding to 1×10⁷ cells were applied to the HP-TLC plate. (D) proliferation of control MDA/LUC and sh-transduced MDA/LUC-shUGT8. Cell proliferation was determined using SRB assay as described in the “Materials and Methods”. The values are shown as the mean ± SD of eight independent replicates.</p

Expression of Metallothionein and Ki-67 Antigen in GISTs of Different Grade of Malignancy*

Abstract Background. Metallothioneins (MTs) are low molecular weight proteins (6-7 kDa), which have been shown to regulate zinc ion homeostasis. MTs exert anti-apoptotic and pro-proliferative effect on cancer cells. Overexpression of MT-I and MT-II isoforms has been noted in many malignant tumors, but the role of their expression in gastrointestinal stromal tumors (GISTs) remains unclear. Objectives. The aim of the study was to examine the relationship between expression of MT-I/II and K-67 proliferation antigen in a subset of GISTs presenting differential grade of malignancy. Material and Methods. The study was conducted using immunohistochemical methods on archival paraffin sections of 34 cases of GISTs. Of those, 17 tumors were classified as benign (GISTB) and 17 tumors as malignant (GISTM). Results. The GISTM cases demonstrated higher MT-I/II expression as compared to the GISTB cases, but not significantly higher (p = 0.08). The GISTM tumors showed significantly higher expression of Ki-67 antigen than the GISTB cases (p = 0.01). MT-I/II and Ki-67 expression positively correlated in GISTBs (r = 0.48, p = 0.0463), but not in GISTMs. Conclusion. The results of this study may point to a potential role of MT-I/II in the proliferation of GIST cells and disease progression (Adv Clin Exp Med 2013, 22, 4, 513-518)