Cell lines:
- It has been shown that VEGF is critical for the initial s.c. growth of T-47D breast carcinoma cells (BCC), whereas other angiogenic factors (basic fibroblast growth factor and transforming growth factor alpha) can compensate for the loss of VEGF after the tumors have reached a certain size (Yoshiji H. et al., 1997).
- Transfection of
TIMP-1 cDNA into MCF-7 BCC resulted in up-regulation of VEGF, with a linear relationship between
TIMP-1 and VEGF production in 9 cell clones examined. There was, however, no change in VEGF expression when the BCC were exposed to exogenous recombinant
TIMP-1. In a comparative study involving the MDA-MB-231, -415, -435, -468, -453, BT-483, BT-20, T-47D BCC lines and the non-neoplastic HBL-100 and Hs578Bst cell lines, there was significant correlation between
TIMP-1 and VEGF concentration in the serum-free culture supernatants (Yoshiji H. et al., 1998).
- VEGF expression was shown to vary widely between BCC lines. Their capacity to respond to hypoxia by an increase in VEGF level was also cell specific, relating mostly to the hypoxic sensing of the cell and the signal transduction mechanism. Such characteristics, if maintained
in vivo, have implications for the angiogenic potential of different tumor cells under normal and hypoxic conditions (Scott P.A. et al., 1998b).
- In T-47D BCC, progesterone was shown to induce a dose-dependent increase of 3-4-fold in media VEGF levels, with a maximum response occurring at a concentration of 10 nM. This effect was blocked by the antiprogestin RU 486. In addition to progesterone, a number of synthetic progestins used in oral contraceptives (e.g., norethindrone, norgestrel, and norethynodrel), hormone replacement therapy (medroxyprogesterone acetate), and high-dose progestin treatment of breast cancer (megestrol acetate) also increased VEGF in the media of cultured T-47D cells. This effect was hormone specific and was not produced by estrogens, androgens, or glucocorticoids. Collectively, these observations suggest that the increase in VEGF caused by progestins is mediated by progesterone receptors present in T-47D cells. The induction of VEGF by progestins was also cell type specific and did not occur in BCC lines MCF-7, ZR-75, or MDA-MB-231 (Hyder S.M. et al., 1998).
- Whether
bcl-2 could be involved in the modulation of the angiogenic phenotype of MCF-7/Adr (adriamycin-resistant) BCC was examined. Four
bcl-2-overexpressing clones, a control transfectant clone, and the MCF-7/Adr parental line were used for
in vitro and
in vivo experiments.
bcl-2 overexpression enhanced the synthesis of the hypoxia-stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in
bcl-2-overexpressing clones, and reverse transcription-polymerase chain reaction showed higher levels of the VEGF(121) and VEGF(165) mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of
bcl-2-transfected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from
bcl-2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in
in vivo xenografts was similar in control and
bcl-2 transfectants. The effect of
bcl-2 on angiogenesis was not mediated by p53 protein (Biroccio A. et al., 2000).
Tumors:
- VEGF is a regulator of tumor angiogenesis
in vivo. The molecule and its receptor could be targets for promising approaches to cancer therapy (Folkman J., 1995). However, breast cancer cell lines and primary human breast tumours express a wide range of vascular growth factors, including VEGF, but also placenta growth factor, pleiotrophin, TGF beta 1, acidic and basic FGF, and platelet-derived endothelial cell growth factor. Inhibiting angiogenesis by blocking vascular growth factors would be difficult with highly specific agents, but drugs with a broader spectrum of antagonism may be effective (Harris A.L. et al., 1996).
- In a series of 152 primary breast cancers, it was found that VEGF and platelet-derived endothelial cell growth factor (PD-ECGF) were frequently coexpressed in highly vascularized tumors with high microvessel counts (Toi M. et al., 1995b).
- In a series of 328 primary breast cancer patients, a close association was demonstrated between the increase in microvessel density (reflecting the angiogenetic process) and the expression of VEGF (p < 0.001) (Toi M. et al., 1995).
- VEGF mRNA expression was studied in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (4 specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (2 specimens), metastatic ductal carcinoma (3 specimens) and metastatic lobular carcinoma (1 specimen). VEGF mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VEGF mRNA was not expressed at high levels in infiltrating lobular carcinoma. The expression of two endothelial cell surface VEGF receptors, flt-1 and kdr was determined by in situ hybridization. VEGF receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue (Brown L.F. et al., 1995).
- In a series of 135 primary breast cancer tissues, intratumoral VEGF concentrations varied from 3.3 pg/mg protein to 2032 pg/mg protein (average 148 pg/mg protein). An immunocytochemical analysis using anti-VEGF antibody confirmed that VEGF was located mainly in the cytoplasm of the tumor cells. The VEGF concentrations were significantly higher in vascularly rich tumors than in vascularly poor tumors. No significant association was found between VEGF concentrations and the concentrations of two other endothelial growth factors, basic fibroblast growth factor and hepatocyte growth factor (Toi M. et al., 1996).
- Gene expression of VEGF; its receptor, flt-1; basic fibroblast growth factor; and transforming growth factors (TGFs) alpha and beta were examined in 18 paired cases of human breast carcinomas and the adjacent nonneoplastic tissues. In all of the paired cases, VEGF expression was markedly increased in the carcinomas. In contrast, an insignificant difference was observed in the expression of flt-1, basic fibroblast growth factor, TGF-alpha, and TGF-beta between the malignant breast tissue and the nonneoplastic counterpart. Immunostaining showed variable VEGF positivity of the malignant cells, whereas the nonneoplastic breast epithelial cells were negative (Yoshiji H. et al., 1996).
- Tumors from 247 (95%) of 260 node-negative breast cancer patients were found to contain detectable VEGF, ranging in concentration from 5.0 to 6523 pg/mg protein (median, 126.25 pg/mg protein). No statistically significant associations were found between VEGF and the other prognostic factors (e.g., age, menopausal status, histologic tumor type, tumor size, and hormone receptors) examined. Levels of VEGF were found to be prognostic for both relapse-free and overall survival in univariate and multivariate analyses (Gasparini G. et al., 1997).
- VEGF concentration and microvessel density were determined in 120 tissue specimens taken from breast fibromas (n = 23), normal epithelial breast tissue adjacent to fibromas (n = 8) and invasive breast cancer (n = 89). A significant correlation was found between VEGF concentration and microvessel count, but VEGF concentration did not significantly influence disease-free survival. Although VEGF protein was found at a significantly higher concentration in malignant than in non-malignant tissue, determination of intratumoral VEGF protein by an enzyme immunoassay was not prognostically relevant (Obermair A. et al., 1997).
- The mRNA and protein expression of the four alternatively spliced VEGF isoforms (121, 165, 189 and 206 amino acids) were examined in normal and malignant breast tissues. Three VEGF transcripts were detected in both (121>165>189), whereas only VEGF165 protein was detected. The tumours expressed more VEGF mRNA (P = 0.02) and protein (P < 0.0001). The expression of eIF-4E, a translation initiation factor, was examined. Increased eIF-4E mRNA levels were detected in the tumours (P < 0.0001) that correlated with VEGF mRNA (P = 0.0002), implying co-regulation of these genes. VEGF mRNA expression was elevated in tumours expressing the
epidermal growth factor receptor (P < 0.01), but there was no difference according to
oestrogen receptor status (P = 0.9), node status (P = 0.09) or between differing histologies (P = 0.4) (Scott P.A. et al., 1998).
- VEGF and its specific and functional receptor KDR were studied in a total of 13 mammary carcinomas, 3 fibroadenomas, 5 specimens with fibrocystic breast disease as well as normal (adjacent to malignant) breast tissue using immunohistochemistry and Western blot analysis. In all carcinomas examined, functional KDR protein was present independent of tumor type, tumor stage and histological grade as demonstrated by tyrosine phosphorylation analysis of KDR. When malignant tissues were compared with their neighboring non-neoplastic regions, activated KDR was found to be expressed to a much higher extent within the malignant tissue samples. In fibroadenomas, KDR was barely detectable, whereas in fibrocystic breast disease KDR expression was variable. Immunostaining of KDR was localized to endothelium and epithelium of mammary ducts in malignant and benign breast tissue, while VEGF immunoreactivity was primarily found in the endothelium and also in tumor cells and macrophages (Kranz A. et al., 1999).
- VEGF mRNA levels were determined by quantitative RT-PCR in the tumour and the adjacent non-neoplastic breast tissue of 19 breast cancer patients. In non-neoplastic breast specimens, the expression levels of VEGF isoforms 189, 165, and 121 were significantly higher in premenopausal compared to post-menopausal women and were inversely correlated with the patient's age. In contrast, in cancerous tissues menopausal status had no influence on VEGF expression levels (Greb R.R. et al., 1999).
- Expression of the angiogenic factor VEGF; the VEGF receptors flt-1 and
KDR;
thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin was investigated by mRNA
in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with
in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VEGF; strong endothelial cell expression of VEGF receptors; strong expression of
thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow (Brown L.F. et al., 1999).
- Both plasma (VEGFp) and serum (VEGFs) VEGF concentrations were examined in 201 blood samples from pre- and postmenopausal healthy controls and from patients with benign breast disease, localized breast cancer, breast cancer in remission, or metastatic breast cancer and related these to other clinicopathological markers. VEGFp but not VEGFs concentrations of patients with localized disease were significantly elevated compared with normal controls. Patients with metastatic disease had higher VEGFp and VEGFs levels than normal controls, and higher VEGFp, but not VEGFs, than patients with benign disease and patients with localized disease. However, the highest VEGFp and VEGFs concentrations were seen in patients in remission compared with normal controls. VEGFp concentrations in patients in remission were also higher than in patients with benign disease or patients with localized disease. Tamoxifen treatment was significantly associated with higher circulating and platelet-derived VEGF levels. Circulating VEGF did not correlate with any clinicopathological factor, including microvessel density (MVD) or VEGF expression. VEGF expression was significantly correlated with
estrogen receptor status and inversely correlated with tumor grade. MVD correlated with tumor size. Tamoxifen-induced increases in VEGF might be important in clinical prognosis or associated pathologies (Adams J. et al., 2000).
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