Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.     

Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules

Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.     

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.     

Stromal influences on breast cancer cell growth.

Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue (2p less than 0.05, n = 8). The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid-receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells.     

Carboxymethyl benzylamide dextran and tamoxifen combination inhibits tumor growth and angiogenesis.

We showed previously that a carboxymethyl dextran benzylamide (CMDB7) blocks angiogenesis of MDA-MB-435 carcinoma and its lung metastases in nude mice. In this study, we examined the combination effects of CMDB7 and tamoxifen (TAM) on cell proliferation, tumor growth, and angiogenesis on the MCF-7RAS cells. We showed that CMDB7 and TAM acted in a synergistic manner to inhibit the growth of MCF-7RAS cells, blocking them in G(0)/G(1) phase of the cell cycle. For 7 weeks, the CMDB7- (300 mg/kg/week) and TAM- (20 mg/kg/week) treated groups showed tumor growth inhibition of about 66% and 76%, respectively. Combined treatments with CMDB7 and TAM block the tumor development by 94% and induce a complete regression of 4 of 8 mice. Histological analysis showed markedly less neovascularization (88%) in the tumors treated with a combination of CMDB7 and TAM. This antiangiogenic activity was further demonstrated by direct inhibition of endothelial cell proliferation. Overall, this study points to the potential use of a combination of CMDB7 and TAM to inhibit tumor angiogenesis that can prevent tumor progression.     

Modulation of the insulin-like growth factor-I system by N-(4-hydroxyphenyl)-retinamide in human breast cancer cell lines.

The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.     

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Effects of liarozole, a new antitumoral compound, on retinoic acid-induced inhibition of cell growth and on retinoic acid metabolism in MCF-7 human breast cancer cells.

Liarozole is a new imidazole derivative with antitumoral properties. Effects of the compound alone and in combination with all-trans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentration dependently inhibited by all-trans-retinoic acid (drug concentration resulting in 50% growth inhibition, 2 x 10(-8) M), while liarozole at 10(-5) M inhibited cell growth by only 35%. When MCF-7 cells were incubated with a combination of all-trans-retinoic acid and liarozole, the antiproliferative effect of all-trans-retinoic acid was clearly enhanced. This enhancement was dependent on the liarozole concentration and was more than 10-fold. A combination of 10(-8) M all-trans-retinoic acid and 10(-6) M liarozole resulted in a greater antiproliferative effect than that obtained with 10(-7) M all-trans-retinoic acid alone. When MCF-7 cells were incubated for 4 h with [3H]all-trans-retinoic acid, the radioactivity in the supernatant consisted of unaltered retinoid. However, when cells had been pretreated with 10(-6) M all-trans-retinoic acid overnight, they were able to substantially metabolize [3H]all-trans-retinoic acid during a subsequent 4-h incubation. High-performance liquid chromatography analysis of the supernatants revealed that the reaction products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-trans-retinoic acid in MCF-7 cells with 10(-5) M liarozole reducing the amount of polar metabolites by 87%. It is concluded that the enhancement by liarozole of the antiproliferative effects of retinoic acid on MCF-7 human breast cancer cells is probably due to inhibition of retinoic acid metabolism. Further research into these effects in MCF-7 cells as well as in other cancer cell lines will provide more information concerning the exact mechanism of action of liarozole and the use of inhibitors of retinoid metabolism in cancer treatment.     

Role of androgens on MCF-7 breast cancer cell growth and on the inhibitory effect of letrozole

Previous work has shown that androgens inhibit breast cancer cells and tumor growth. On the other hand, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Here, we report that androgens, such as the aromatizable androstenedione and the non-aromatizable 5alpha-dihydrotestosterone, inhibit MCF-7 cell proliferation. This effect is observed only in the absence or at a low concentration of estrogens and is evident in cells with low aromatase activity. Growth of a new aromatase stably transfected MCF-7 cell line (Ac1) was stimulated by conversion of androstenedione into estrogens and was sensitive to aromatase inhibitors. We show that blockade of the androgen receptor (AR) in these cells by the antiandrogen casodex or by the anti-AR small interfering RNA inhibited the antiproliferative effect of dihydrotestosterone and letrozole (aromatase inhibitor). We also show that suppression of the estrogen-induced antiapoptotic protein Bcl-2 may be involved in the antiproliferative effects of androgens and letrozole. These effects can be reversed by casodex. In conclusion, the results suggest that aromatase inhibitors may exert their antiproliferative effect not only by reducing the intracellular production of estrogens but also by unmasking the inhibitory effect of androgens acting via the AR.     

Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.

The 3T3-L1 cell line is a preadipocyte cell line derived from the Swiss 3T3 mouse fibroblast cell line. We have compared the effect of 3T3-L1 conditioned medium (3T3-L1 CM) and Swiss 3T3 conditioned medium (3T3 CM) on the growth of normal mouse mammary cells (NMMG) and the human MCF-7 breast carcinoma cell line. 3T3 CM increased the growth of both NMMG and MCF-7 cells by 19 +/- 2% (SD) and 24 +/- 3%, respectively, and increased thymidine incorporation by 74 +/- 4% and 104 +/- 8%, respectively. Conditioned medium from 3T3-L1 cells stimulated the growth of NMMG cells by 64 +/- 2%; in contrast, 3T3-L1 CM inhibited the growth of MCF-7 cells by 36 +/- 1%. In parallel with these growth studies, thymidine incorporation increased by 20 +/- 4% in NMMG cells and decreased by 72 +/- 5% in the MCF-7 cells. Moreover, a similar effect was also noted in NCI H630 colon cancer cells, where 3T3-L1 CM produced a 58 +/- 4% decrease in growth and a 82 +/- 6% decrease in thymidine incorporation. Heating the 3T3-L1 CM at 100 degrees C for 30 min destroyed all inhibitory activity. Several known inhibitory growth factors (fibroblast growth factor, 20 ng/ml; interleukin 6, 1000 units/ml; tumor necrosis factor alpha, 15 ng/ml; transforming growth factor beta, 1 ng/ml) were tested for activity in the MCF-7 cells. Tumor necrosis factor alpha and transforming growth factor beta produced a 97% and 67% inhibition of thymidine uptake, respectively, whereas interleukin 6 and fibroblast growth factor had no effect. Neither transforming growth factor beta nor tumor necrosis factor alpha activity was detectable in 3T3-L1 CM using an enzyme-linked immunosorbent assay. High-performance liquid chromatography fractionation of the 3T3-L1 CM revealed that the inhibitory activity eluted at a molecular weight of 67,000; moreover, silver staining of these eluates on a denaturing polyacrylamide gel revealed that M(r) 69,000 peptide was the predominant protein band in the inhibitory fractions. Thus 3T3-L1 CM stimulates the growth of normal breast epithelial cells and inhibits the growth of MCF-7 breast cancer cells. This inhibitory activity appears to be due to a protein secreted by 3T3-L1 preadipocytes.     

Anti-proliferative and antitumoral activities of a functionalized dextran (CMDBJ) on the 1205 L-U human tumor melanoma cells

Carboxy methyl dextran benzylamide jorge (CMDBJ) is a derivatized dextran prepared from native dextran after random carboxymethylation of hydroxyl groups on D-glucose units (CM) and consecutive conversion of some carboxylate groups to benzylamide structures (B). This polymer exhibits an inhibitory action upon the proliferation of 1205 L-U human melanoma cells. At low concentrations, this compound exerts a cytostatic effect whereas, at higher concentrations, a cytotoxicity appears within 24 hours of treatment. The 1205 L-U cell line forms subcutaneous angiogenic tumors in athymic mice and, after several weeks, spontaneously forms micrometastasis in the lungs. We demonstrated that the CMDBJ treatment of animals not only reduces the rapid growth of primary tumors but also induces tumor regression and tumor necrosis. Moreover, CMDBJ treatment blocks the appearance of lung metastasis. Pleiotrophin (PTN), heparin-binding angiogenic growth factor, is secreted by 1205 L-U cells and breast tumor MDA-MB 231 cells. CMDBJ, as an inhibitor of heparin-binding growth factor activities, suppresses the mitogenic activity of conditioned media from 1205 L-U and MDA-MB 231 on endothelial HUVEC cells. We conclude that CMDBJ can inhibit the in vitro cell proliferation of 1205 L-U cells and 1205 L-U tumor development in athymic mice and that PTN secreted by these cells could be involved in this inhibition.     

The antiandrogen flutamide inhibits growth of mcf-7 human breast-cancer cell-line

The antiandrogen flutamide (FLU) has been reported to exert antiproliferative action on both male and postmenopausal breast cancer and to inhibit growth of chemically induced rat breast cancer. We studied the effects of various concentrations of FLU on the growth of the ER+, AR+ and PR+ MCF-7 and the ER-, AR- and PR- MDA-MB-231 human breast cancer cell lines. The growth of MCF-7 cells in both steroid free and estradiol supplemented media was inhibited by FLU. MDA-MB-231 cell growth was not affected by FLU. Our data show a direct inhibitory action of FLU on human breast cancer cells and suggest a different susceptibility to the antiproliferative action of FLU, which seems to be related to the steroid receptor status.     

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture

 Cyclopropyl compound 7b [(Z)-1,1-dichloro2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxy-phenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development. Received: 6 January 1995/Accepted: 9 October 1995     

Human breast carcinoma desmoplasia is PDGF initiated

The desmoplastic response to human breast carcinoma is a host myofibroblast-mediated collagenous response exhibiting synergistic effects on tumor progression. Although many paracrine interactions between breast carcinoma cells and myofibroblasts have been characterized, the event(s) which initiate desmoplasia have remained undefined. Our studies utilized c-rasH transfected MCF-7 cells which overexpress ras p2l and which are weakly tumorigenic in ovariectomized nude mice. The xenografts are desmoplastic and comprised of 30% myofibroblasts and 60 mg/g of interstitial collagen. In situ hybridization studies of these xenografts reveal a stromal gene expression pattern (stromelysin-3, IGF-II and TIMP-1) identical to that observed in human tumor desmoplasia. 17-beta estradiol increases c-rasH MCF-7 growth but abolishes desmoplasia. c-rasH MCF-7 in vitro constitutively produce myofibroblast mitogenic activity which competes with PDGF in a receptor binding assay. This myofibroblast mitogenic activity is unaltered by 17-beta estradiol/tamoxifen pretreatment in vitro. Transfection of c-rasH MCF-7 with a PDGF-A dominant negative mutant, 1308, produced by site-directed mutagenesis (serine-->cysteine129) reduces both homo- and heterodimer secretion of PDGF by as much as 90% but does not interfere with the secretion of other growth factors. Clones with low PDGF, though tumorigenic, are non-desmoplastic. Our results suggest that breast carcinoma-secreted PDGF is the major initiator of tumor desmoplasia.     

Inhibition of estrogen-dependent breast cell responses with phenylacetate

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.     

The influence of a novel cyclopropyl antiestrogen (compound 7a) on human breast cancer cells in culture

Compound 7a ([Z]-1,1,-dichloro-2,3-diphenyl-2-(4-(2-dimethylamino)ethoxy)phenyl) cyclopropane, dihydrogen citrate salt) is a novel cyclopropyl antiestrogen which was shown to be an estrogen antagonist without estrogen agonist activity. The antiproliferative activity of 7a was examined on estrogen receptor (ER)positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells. Compound 7a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10^–9 to 10^–5M, but did not alter the growth of MDA-MB-231 or A-549 cells. The antiproliferative activity of 7a (10^–7M) on MCF-7 cells was reversed by co-administration of estradiol (10^–8M). An ER-dependent mechanism of action is also supported by the specific ER binding of 7a in MCF-7 cells observed in this study. A study of cell surface morphology using scanning electron microscopy (SEM) revealed that compound 7a at 10^–6M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10^–8M). Compound 7a did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7a inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study support the antiestrogenic action of 7a previously observedin vivo and suggest that 7a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Role of polyamines in the growth of hormone-responsive and -resistant human breast cancer cells in nude mice.

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells. DFMO, while inhibiting E2-supported MCF-7 breast cancer growth, did not inhibit E2-stimulated progesterone receptor synthesis. Cellular levels of polyamines were highest in MCF-7 cells and lowest in the BT-20 cell line. Tumor content of spermidine was similarly suppressed by DFMO treatment in the 3 cell lines, while the spermine level was unaffected. Cellular putrescine levels were suppressed in MCF-7 and BT-20 cells. Administration of DFMO prior to implantation of fragments of MCF-7 or MDA-MB-231 tumors in nude mice significantly inhibited tumor development to a similar extent. The action of DFMO seemed to be predominantly tumoristatic since new tumors develop in some mice upon discontinuation of the drug. We conclude that the hormone-independent breast cancer cell lines tested do not exhibit increased polyamine biosynthesis or resistance to antipolyamine therapy when grown in vivo in nude mice.     

Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture

Stromal and epithelial components surrounding neoplastic cells are believed to be important in tumor regulation. We have studied the effects of stromal and epithelial cells on the proliferation of a variety of breast-cancer epithelial cell lines. Co-culture experiments were performed in which the 2 cell types were separated by a microporous membrane. Under these conditions, fibroblasts from normal breast tissues inhibited the proliferation of MCF-7 cells, but not that of immortalized normal S2T2 cells. In contrast, fibroblasts from cancerous breast tissues did not influence the proliferation of the 2 cell lines tested. Conditioned media (CM) of breast fibroblasts derived from normal tissues were not able to affect MCF-7 cell growth, suggesting complex paracrine interactions between both cell types. Normal breast epithelial cells (NBEC) have also been tested for their ability to regulate the proliferation of breast-cancer epithelial cell lines. Co-culture experiments demonstrated that NBEC inhibited a variety of breast-cancer cell lines. CM from NBEC induced similar results and the inhibitory effect appeared to be specific for epithelial cells from tumorous breast. Moreover, CM from NBEC and normal fibroblasts were shown to contain more TGF_β1 and amphiregulin than those of MCF-7 cells. We conclude that both the tissue origin and the target tumor cell's phenotype will determine the extent of proliferative response. More important, the tumor-cell growth inhibition induced by fibroblasts and epithelial cells of normal breast tissue may constitute a tumor-growth-regulatory mechanism. Int. J. Cancer, 71:42–48, 1997. © 1997 Wiley-Liss, Inc.     

Novel therapeutic approach: organic arsenical melarsoprol) alone or with all-trans-retinoic acid markedly inhibit growth of human breast and prostate cancer cells in vitro and in vivo

The organic arsenical known as melarsoprol (Mel-B) is used to treat African trypanosomiasis. Recently, another arsenical, As2O3 was shown to be effective in treatment of acute promyelocytic leukaemia. We have investigated the anti-tumour activities of Mel-B either with or without all-trans-retinoic acid (ATRA) using the MCF-7 human breast cancer cells, as well as the PC-3 and DU 145 human prostate cancer cells both in vitro and in vivo. The antiproliferative effects of Mel-B and/or ATRA against breast and prostate cancer were tested in vitro using clonogenic assays and in vivo in triple immunodeficient mice. Furthermore, the mechanism of action of these compounds was studied by examining the cell cycle, levels of bcl-2, apoptosis and antiproliferative potency using a pulse-exposure assay. Clonogenic assays showed that the cancer cell lines were sensitive to the inhibitory effect of Mel-B (effective dose that inhibited 50% clonal growth [ED50]: 7 x 10(-9) M for MCF-7, 2 x 10(-7) M for PC-3, 3 x 10(-7) M for DU145 cells. Remarkably, the combination of Mel-B and ATRA had an enhanced antiproliferative activity against all three cancer cell lines. Furthermore, the combination of Mel-B and ATRA induced a high level of apoptosis in all three cell lines. Treatment of PC-3 and MCF-7 tumours growing in triple immunodeficient mice with Mel-B and ATRA either alone or in combination markedly retarded tumour size and weight of the tumours without major side-effects. In conclusion, our results suggest that either Mel-B alone or with ATRA may be a useful, novel therapy for breast and prostate cancers.