Enhancement of tumorigenic, metastatic and in vitro invasive capacity of rat mammary tumor cells by transforming growth factor-beta.

We investigated the effects of transforming growth factor-beta (TGF-beta) on biological behavior of a weakly malignant rat mammary carcinoma ER-1 cell line. TGF-beta enhanced the tumorigenic and metastatic capacity of ER-1 cells and their in vitro invasiveness to rat mesothelial and endothelial cell. Further cell biological analysis indicated that the increased invasive and metastatic capacity of ER-1 cells by TGF-beta was due to the increase in cell motility and adhesion to the mesothelial and endothelial cell monolayers. Thus, it is suggested that TGF-beta acts on ER-1 cells as a progression-enhancing factor which stimulates their adhesive and motile activities.     

Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide

Vascular endothelial growth factor receptor-1 (VEGFR-1) exists in two isoforms: a membrane-bound isoform (mVEGFR-1) and a soluble one (sVEGFR-1). mVEGFR-1 is involved in endothelial cell migration and survival supported by VEGF-A and placenta growth factor (PlGF), whereas the biologic function of sVEGFR-1 has not been fully elucidated. We previously reported that sVEGFR-1 induces endothelial cell motility and promotes endothelial cell adhesion. In this study, we tested a set of VEGFR-1-derived peptides for their ability to interfere with endothelial cell migration. Peptide B3 was found to specifically inhibit cell migration induced by sVEGFR-1 and by mVEGFR-1-specific ligands. Moreover, peptide B3 markedly hampered angiogenesis in vitro and in vivo and was found to interfere with VEGFR-1 homodimerisation. Altogether, these data demonstrate that peptide B3 might be a useful tool for the specific inhibition of VEGFR-1 function and might represent a basis for the development of new anti-angiogenic compounds.     

Transforming growth factor-beta1 increases survival of human melanoma through stroma remodeling.

Transforming growth factor (TGF)-beta is growth inhibitory for normal epithelial cells and melanocytes but can stimulate mesenchymal cells. Resistance to its inhibitory effects is characteristic of human melanoma, the growth of which may instead be promoted by TGF-beta, because its production is increased with melanoma progression. Whether TGF-beta has an autocrine function for melanoma cells or is important for paracrine stimulation of the tumor stroma is not known. In this study, TGF-beta1 was expressed in melanoma cells via adenoviral gene transfer, and tumor growth was analyzed in vitro, in human skin grafts, and in mixtures with fibroblasts that were injected s.c. into immunodeficient mice. The TGF-beta1 produced by the melanoma cells activated the fibroblasts to produce matrix within and around the tumor mass, whereas control tumors showed less stroma and more cell death. High expression of collagen, fibronectin, tenascin, and alpha2 integrin was detected in the TGF-beta1-expressing tumors by immunohistochemistry. Number and size of lung metastases were significantly increased. cDNA expression array analysis of TGF-beta1-transduced fibroblasts embedded in type I collagen and of TGF-beta1-transduced melanoma cells demonstrated induction of types XV, XVIII, and VI collagens, tenascin, plasminogen activator inhibitor-I, vascular endothelial growth factor, cysteine-rich fibroblast growth factor receptor-1, and platelet-derived growth factor receptor-beta, which could be linked to promotion of growth and survival in melanoma. These data suggest that remodeling of the neighboring stroma, which provides a supporting scaffolding and a positive feedback stimulation of tumor growth, is an important function of TGF-beta1 in melanoma.     

The expression of transforming growth factor-beta1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma.

BACKGROUND: Transforming growth factors beta (TGFs beta) are involved in a variety of important cellular functions, including cell growth and differentiation, adhesion, migration, extracellular matrix formation, and immune function. Moreover, it has been reported that TGFs beta are correlated with angiogenesis. However, the role of TGF-beta as an angiogenic factor in gastric carcinoma is still unclear. METHODS: TGF-beta1 expression was determined in 101 patients with gastric carcinoma by immunohistochemical procedures, and this expression was compared in the current study with both the expression of vascular endothelial growth factor (VEGF), which is thought to be the most potent angiogenic factor, and microvessel density, to evaluate the effect of TGF-beta1 on the angiogenesis of gastric carcinoma tissues. RESULTS: TGF-beta1 expression was detected in 23 tumors (22.8%). TGF-beta1 expression was more frequent in differentiated than in undifferentiated gastric carcinoma. Furthermore, TGF-beta1 expression was significantly correlated with the depth of invasion and the stage of disease. There was a close correlation between TGF-beta1 expression and VEGF expression. There was no correlation between TGF-beta1 expression and microvessel density, whereas VEGF expression was significantly correlated with microvessel density. With regard to prognosis, the 5-year survival rate was 55.9% for patients with TGF-beta1 positive tumors and 67.0% in patients with TGF-beta1 negative tumors. Accordingly, the prognosis for patients with TGF-beta1 negative tumors was significantly better than that for patients with TGF-beta1 positive tumors. Multivariate analysis indicated that lymph node metastasis, tumor size, and TGF-beta1 expression were independent prognostic factors. CONCLUSIONS: These results suggest that TGF-beta1 might be associated with tumor progression by indirectly stimulating angiogenesis through the up-regulation of VEGF expression in gastric carcinoma.     

The effects of exogenous growth factors on matrix metalloproteinase secretion by human brain tumour cells

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-beta1,2) and vascular endothelial growth factor (VEGF)].on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-beta1 and TGF-beta2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.     

Synergy between anti-endoglin (CD105) monoclonal antibodies and TGF-beta in suppression of growth of human endothelial cells

Endoglin (CD105) is a proliferation-associated cell membrane antigen of endothelial cells and strongly expressed in the angiogenic vasculature of solid tumors. Endoglin is essential for angiogenesis/vascular development and an ancillary transforming growth factor beta (TGF-beta) receptor. Certain anti-endoglin monoclonal antibodies (mAbs), termed SN6 series mAbs, inhibited angiogenesis, tumor growth and metastasis in mice. We investigated the mechanisms by which anti-endoglin mAbs suppress growth of proliferating endothelial cells. We found that 4 SN6 series mAbs suppressed growth of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner in the absence of any effector cells or complement. Significant differences in the growth suppression between the 4 anti-endoglin mAbs defining different epitopes were observed. These differences were not determined by antigen-binding avidities of the mAbs. Combination of TGF-beta1 and each of the 4 anti-endoglin mAbs exerted synergistic growth suppression of HUVECs. Binding of anti-endoglin mAbs to endoglin-expressing cells did not block the subsequent binding of TGF-beta1. Conversely, preincubation of HUVECs with TGF-beta1 did not change cell surface expression of endoglin. The present results suggest that direct suppression of the endothelial cell growth by SN6 series mAbs is one of the underlying mechanisms by which anti-endoglin mAbs exert antiangiogenic and tumor-suppressive activity in vivo. The results further suggest that TGF-beta1 plays an important role in the in vivo antiangiogenic efficacy of anti-endoglin mAbs by synergistically enhancing the activity of these mAbs. Further studies of the present novel findings may provide valuable information about the functional roles of endoglin and anti-endoglin mAbs in the TGF-beta-mediated cell regulation.     

Expression of transforming growth factor beta 1 by human endometrial carcinoma cell lines: inverse correlation with effects on growth rate and morphology

We examined the effects of transforming growth factor beta 1 (TGF-beta 1) on various aspects of the cell biology of human endometrial carcinoma (HEC) cell lines in vitro, as well as the expression of TGF-beta 1 mRNA by these cell lines. Cell lines from eight HEC tumors, representing a variety of histological subtypes, were studied in order to test the generality of conclusions regarding the effects of TGF-beta 1 on this particular tumor cell type. The growth of five HEC cell lines was inhibited by TGF-beta 1 (10 ng/ml), while growth of three cell lines was not inhibited. The effects on growth correlated with morphological alterations induced by TGF-beta 1; the cell lines with inhibited growth displayed a larger, flatter, more contact-inhibited phenotype, while the cell lines whose growth ws not inhibited showed few discernible morphological alterations in response to TGF-beta 1. Northern analysis of TGF-beta 1 mRNA levels revealed that the three HEC cell lines unresponsive to TGF-beta 1 treatment expressed relatively large amounts of TGF-beta 1. Correspondingly, the five HEC cell lines which responded to TGF-beta 1 with growth and morphological changes expressed much lower levels of TGF-beta 1 mRNA. These results suggest that the sensitivity of human HEC cell lines to TGF-beta 1 is variable and that this sensitivity is inversely correlated with the level of expression of TGF-beta 1.     

Effects of androgen, fibroblast growth factors or other various growth factors on growth of Shionogi carcinoma cells in a protein-free medium

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. We have recently established an androgen-dependent cloned cell line (SC-3) from a SC115 tumor. We found in the present study that the growth of SC-3 cells can be stimulated by 10(-8) M testosterone (up to 100-fold) even in a protein-free medium beginning from plating [Ham's F-12: Eagle's minimum essential medium (1:1, v/v)]. In the protein-free culture, the proliferation of SC-3 cells was also found to be stimulated by acidic or basic fibroblast growth factor (FGF) alone (up to 50-fold) among various growth factors examined such as FGFs, insulin, insulin-like growth factor (IGF)-I, IGF-II, nerve growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor (TGF)-alpha and TGF-beta. The testosterone (10(-8) M)- or FGF (10 ng/ml)-induced growth of SC-3 cells was abolished only by TGF-beta (greater than or equal to 1 ng/ml) among various growth factors examined. We show for the first time in this study an androgen-dependent growth of cancer cells (SC-3) in a protein-free medium. In the protein-free medium, growth of SC-3 cells is also stimulated by FGFs, and the androgen- or FGF-induced growth is inhibited only by TGF-beta.     

Differential effects of transforming growth factor beta 1 on the growth of poorly and highly metastatic murine melanoma cells

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth of paired murine melanoma cell clones that differ with respect to their experimental metastatic potential. Neither poorly (clone 16) nor highly (clone M2) metastatic cells were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium in the absence of serum. However, both clones were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium containing 10% calf serum. Colony formation in the presence of 10% calf serum was enhanced in a dose-dependent manner by TGF-beta 1 (half-maximal dose, 0.1 ng/ml) and was 5- to 10-fold greater than colony formation in the presence of 10% calf serum alone. Under anchorage-dependent (monolayer) conditions, neither clone grew in the absence of serum or in medium containing less than 1% calf serum. The monolayer growth of poorly metastatic cells (clone 16) was enhanced in a dose-dependent manner by TGF-beta 1 in medium supplemented with calf serum. Growth was 3.5-fold and 2.3-fold greater than untreated controls after 5 days in submitogenic (0.5%) and mitogenic (10%) concentrations of calf serum, respectively. In contrast, TGF-beta 1 had no effect on the monolayer growth of highly metastatic cells (clone M2) either in submitogenic (0.5%) or mitogenic (10%) concentrations of serum. TGF-beta 1 did not directly stimulate DNA synthesis by either poorly or highly metastatic cells when measured 24 h after TGF-beta 1 treatment. The ability of TGF-beta 1 to stimulate the anchorage-independent growth of metastatic melanoma cells suggests that this potent growth factor may play a role in the growth of these cells in vivo. In addition, the differential sensitivity of poorly and highly metastatic cells to TGF-beta 1 may be relevant to their metastatic potential in vivo. While the mechanism(s) by which TGF-beta 1 stimulates the growth of these cells remains unknown, these differentially metastatic clones of the K-1735 murine melanoma should provide a useful model in which to study the effects of transforming growth factor beta on the metastatic phenotype.     

Roles of transforming growth factor beta in inhibition of androgen-induced growth of Shionogi carcinoma cells in serum-free medium

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. In a serum-free culture system we have established [Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin], 10(-8) M testosterone markedly stimulates the growth of SC-3 cells (a cloned cell line from a SC115 tumor) via androgen receptor. The testosterone-induced growth of SC-3 cells, which has been shown to be mediated through autocrine fibroblast growth factor (FGF)-like peptide, was almost completely abolished by 1 ng/ml of transforming growth factor beta (TGF-beta). In the present study, mechanisms of the inhibitory effect of TGF-beta on the testosterone-induced growth of SC-3 cells were examined in the serum-free medium. Although the testosterone-induced growth was almost completely inhibited by TGF-beta, basic FGF- or FGF-like peptide (secreted from SC-3 cells by testosterone)-induced growth was only partially inhibited (45%) by TGF-beta. This difference can be explained by the fact that TGF-beta decreased the amount of testosterone-induced FGF-like peptide secreted from SC-3 cells to 18% of control. The TGF-beta-induced inhibition was found to be reversible. Furthermore, no significant effects of the TGF-beta treatment on number or affinity of both androgen and FGF receptors were demonstrated. The present findings show that TGF-beta markedly inhibits testosterone-induced secretion of FGF-like peptide from SC-3 cells and also inhibits growth-stimulatory effects of the secreted factor on SC-3 cells, probably via postreceptor mechanisms.     

Growth stimulation, altered regulation of epidermal growth factor receptors, and autocrine transformation of spontaneously transformed normal rat kidney cells by transforming growth factor beta

The tumorigenic NRK-PT14 cell line requires exogenous epidermal growth factor (EGF), but has lost the requirement for transforming growth factor beta (TGF-beta) for anchorage-independent growth, compared to normal rat kidney (NRK) cells. Development of an optimized serum-free medium for the growth of these cells revealed that NRK-PT14 cells also exhibit a qualitatively altered sensitivity to exogenous type 1 TGF-beta, compared to NRK cells. EGF-induced serum-free monolayer growth of NRK-PT14 cells was stimulated 2-fold by TGF-beta under conditions where growth of NRK cells was inhibited by 67%. TGF-beta only stimulated the growth of NRK-PT14 cells when EGF was present and when EGF was added before TGF-beta. In addition, the stimulation of EGF-induced NRK-PT14 cell growth by TGF-beta was associated with a specific, reversible loss of the high-affinity subpopulation of EGF receptors from the surface of these cells. Treatment of NRK cells with TGF-beta resulted in an increase in this EGF receptor population. Finally, EGF-induced anchorage-independent growth of NRK-PT14 cells was shown to be dependent on secreted TGF-beta, demonstrating an autocrine role for TGF-beta in the transformed phenotype of these cells. Autocrine transformation of NRK-PT14 cells by TGF-beta may result directly from the acquisition of an altered (positive) sensitivity to this growth factor.     

Induction of anchorage-independent growth by epidermal growth factor and altered sensitivity to type beta transforming growth factor in partially transformed rat kidney cells

A partially transformed cell line (NRK-PT14) was isolated from normal rat kidney (NRK) cells. Like NRK cells, NRK-PT14 cells required epidermal growth factor for anchorage-independent growth, but lost the additional requirement for exogenous type beta transforming growth factor (TGF-beta). Compared to NRK cells, NRK-PT14 cells did not secrete elevated levels of TGF-beta, but exhibited an altered response to this growth factor. Monolayer growth of NRK cells in a serum-free medium was inhibited by TGF-beta, whereas growth of NRK-PT14 cells was stimulated by TGF-beta. In addition, TGF-beta stimulated epidermal growth factor binding to high affinity sites in NRK cells, but decreased epidermal growth factor binding to NRK-PT14 cells during growth of the cells in serum-free medium. These qualitative changes in the response to TGF-beta may be representative of an intermediate stage in the spontaneous transformation of NRK cells.     

Secretion and dual regulation between epidermal growth factor and transforming growth factor-beta1 in MDA-MB-231 cell line in 42-hour-long cultures

MDA-MB-231 is a breast cancer cell line which possesses large quantities of epidermal growth factor (EGF) receptors and specific high-affinity transforming growth factor-beta1 (TGF-beta1) receptors. We have established that these cells secrete constitutively measurable levels of EGF and TGF-beta1 in conditioned medium. The constitutive secretion of EGF decreased over time in culture (42 h), while the constitutive secretion of TGF-beta1 remained constant. TGF-beta1 secretion in EGF-treated cells was lower than in controls (P < 0.0001), but EGF concentrations were not modified after TGF-beta1 supplement. We postulate that in MDA-MB-231 cell line there is a dual regulation between both growth factors.     

The suppression of fibroblast growth factor 2/fibroblast growth factor 4-dependent tumour angiogenesis and growth by the anti-growth factor activity of dextran derivative (CMDB7).

Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) blocks basic fibroblast growth factor (FGF-2)-dependent cell proliferation of a human breast epithelial line (HBL100), suggesting its potential role as a potent antiangiogenic substance. The derived cell line (HH9), which was transformed with the hst/FGF4 gene, has been shown to be highly proliferative in vitro and to induce angiogenic tumours in nude mice. We show here that CMDB7 inhibits the mitogenic activities of the conditioned media from HBL 100 and HH9 cells in a dose-dependent manner. When HH9 cells were injected s.c. into nude mice, CMDB7 treatment (300 mg kg(-1) week(-1)) suppressed the tumour take and the tumour growth by about 50% and 80% respectively. Immunohistochemical analysis showed a highly significant decrease, by more than threefold, in the endothelial density of viable tumour regions, together with a significant increase in the necrosis area. This antiangiogenic activity of CMDB7 was further demonstrated by direct inhibition of calf pulmonary artery (CPAE) and human umbilical vein (HUVEC) endothelial cell proliferation and migration in vitro. In addition, we showed that CMDB7 inhibits specifically the mitogenic effects of the growth factors that bind to heparin such as FGF-2, FGF-4, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-beta1), but not those of epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). These results demonstrate that CMDB7 inhibits FGF-2/FGF-4-dependent tumour growth and angiogenesis, most likely by disrupting the autocrine and paracrine effects of growth factors released from the tumour cells.     

The concentration of serum transforming growth factor beta-1 (TGF-beta1) is decreased in cervical carcinoma patients

Transforming growth factor beta-1 (TGF-beta1) is a multifunctional growth factor and known to inhibit the proliferation of epithelial cell. The relationship between serum TGF-beta1 level and the presence of cervical cancer was investigated in this study. The patients with cervical squamous cell carcinoma (SCC) had significantly lower level of serum TGF-beta1 (39.14 +/- 9.03 ng/mL; mean +/- SD) than those with myoma (49.17 +/- 9.38 ng/mL) and normal subjects (49.13 +/- 8.81 ng/mL), (p < 0.007 and p < 0.001, respectively). TGF-beta1 level was also lower in the patients with cervical intraepithelial neoplasia (CIN3) (42.07 +/- 9.38 ng/mL) than in normal controls (49.13 +/- 8.81 ng/mL) (p < 0.04). It suggested that diminution of the production of TGF-beta1 has close association with the neoplastic transformation of cervical epithelium.