Measurement of beta-galactosidase tissue levels in a tumor cell xenograft model.

The nude mouse xenograft model is commonly used to examine the growth and development of human cancer cells in vivo. Tumor cells transfected with the Lac-Z reporter gene for beta-galactosidase (beta-gal) enzyme activity can be used to quantify tumor and metastatic development in this model. The present study was designed to develop methodology to accurately measure beta-gal in tumor and tissue samples from a nude mouse model. In this study, we developed tissue extraction procedures and compared the sensitivity and accuracy of o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red beta-D-galactopyranoside (CPRG); two beta-gal substrates. Our results demonstrated that the CPRG substrate is more sensitive and accurate in the measurement of beta-gal activity than the ONPG substrate. In addition, matrices and blood in tissue samples are less likely to interfere with the CPRG assay. We concluded that the CPRG substrate-based assay represents a reliable technique for the determination of beta-gal activity in transfected cancer cells present in tumor and tissue specimens from the nude mouse xenograft model.

Influence of antiestrogens on NIH-3T3-fibroblast-induced motility of breast cancer cells.

The motile behavior of tumor cells is regulated in part by growth factors, cytokines, and other endogenous factors. In some instances, stromal tissue surrounding the tumor cells produces these growth factors which interact with tumor cells and thus may play an important role in tumor proliferation and progression. We and others have shown that conditioned media from NIH 3T3 fibroblasts (3T3-CM) increases the invasiveness of breast cancer cells. The present study characterized the influence of 3T3-CM on breast cancer cell motility and examined the hypothesis that antiestrogens inhibit this 3T3-CM-induced effect. In this study we observed that 3T3-CM added to MCF-7 cells produced an immediate cell-scattering effect as determined by time-lapse videomicroscopy, scanning electron microscopy, and F-actin labeling. The results of this study indicate that keratinocyte growth factor in 3T3-CM is largely responsible for the 3T3-CM-induced scattering motility of MCF-7 cells. These results emphasize the importance of stromal-tumor cell (epithelial-mesenchymal) interactions in the motility of breast cancer cells. Further, our results demonstrate that antiestrogens (tamoxifen, ICI-182,780 and Analog II) inhibit 3T3-CM-induced motility of MCF-7 breast cancer cells. Antiestrogen treatment reduced membrane movements and the motile morphology of MCF-7 cells induced by 3T3-CM. These results suggest that antiestrogens inhibit breast cancer cell motility and that antiestrogen treatment may be used to reduce the metastatic spread of breast cancer.

Keratinocyte growth factor-induced motility of breast cancer cells.

Endogenous growth factors and cytokines are known to have a major influence on the progression, motility and invasiveness of tumor cells. We have reported previously that conditioned media from mouse fibroblasts increases the motility of breast cancer cells. Further, we determined that keratinocyte growth factor (KGF) was an active factor from mouse fibroblasts responsible for most of the motility response in breast cancer cells. The present study examined the effect of Human KGF on the motility of estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines in culture using time-lapse videomicroscopy to quantify cell motility. In the present study we observed that recombinant human KGF enhanced several parameters of cellular motility in ER-positive cells but not in ER-negative cell lines. Further, we observed that the level of KGF receptor (KGFR) expression in ER-positive cells was much greater than in the ER-negative cell lines. The motility response to KGF was found to be both dose-and time-dependent. Of the three ER-positive breast cancer cell lines tested. MCF-7 cells were the most responsive to KGF stimulation. Finally, MCF-7 cells grown in estrogen-depleted media did not respond to KGF. These results suggest that KGF from stromal tissue surrounding a primary tumor mass can enhance tumor cell motility and may be an early signal in the progression of breast cancer cells to a more motile and metastatic phenotype. Thus, KGF, KGFR and/or the KGF signaling pathway may be important therapeutic targets for the treatment or prevention of breast cancer metastasis.

Influence of antiestrogens on the invasiveness and laminin attachment of breast cancer cells.

Metastatic spread of breast cancer is responsible for most of the morbidity and mortality associated with this disease. Thus, it is important to identify agents with antimetastatic activity. Because invasiveness and tumor cell attachment are fundamental steps in the metastatic cascade, the major objective of the present study was to evaluate the antimetastatic potential of three antiestrogens, each with different chemical structure and mechanism of action, on breast cancer cell invasiveness and laminin attachment. The antiestrogens examined were tamoxifen, a mixed antagonist/agonist; Analog II, a cyclopropyl antiestrogen with pure antagonist activity; and ICI-182,780, a steroidal antiestrogen with pure antagonist activity. Our results indicate that MDA-MB-231 human breast cancer cells are much more invasive and have a higher affinity for laminin than do MCF-7 human breast cancer cells. All three antiestrogens, at a concentration of 10(-6) M, produced a reduction in MDA-MB-231 cell invasiveness, which was comparable in magnitude to their inhibition of MDA-MB-231 attachment to laminin. Evaluation of MDA-MB-231 cell morphology using scanning electron microscopy revealed the involvement of cellular pseudopodia and microvilli in the process of invasion. The results of this study suggest that antiestrogen-induced inhibition of breast cancer cell invasiveness could be due in part to a decrease in the attachment of tumor cells to laminin in the basement membrane.

The influence of antiestrogens on the release of plasminogen activator (uPA) by MDA-MB-231 and MCF-7 breast cancer cells.

Plasminogen activators are known to be involved in the metastatic spread of breast cancer. In the present study we examined the effects of antiestrogens [Analog II (1,1-dichloro-cis-2,3-diphenyl cyclopropane) (AII), ICI-182,780 (ICI) and tamoxifen (TAM)], on the in vitro release of uPA from estrogen receptor (ER)-positive MCF-7 (MCF) and ER-negative MDA-MB-231 (MDA) human breast cancer cell lines. Using a solid-phase radioassay, uPA activity was found to be higher in the culture medium from MDA cells compared to MCF cells. Aminocaproic acid, a specific plasmin inhibitor, produced a 50-60% reduction in the degradation of labeled substrate, in the solid phase assay, using culture medium from both cell lines, thus indicating that most of the proteolysis observed was due to uPA-mediated plasmin generation from plasminogen. In the absence of plasminogen, the enzyme activity was not detected in either the quantitative assay or by zymography. In MDA cells, uPA release was not altered by any of the antiestrogens used alone or in the presence of estradiol. In contrast, in MCF cells, ICI alone produced maximal inhibition (40%) of enzyme release, while estradiol alone produced a 120% increase in enzyme activity. When co-administered with estradiol, in MCF cultures, each antiestrogen reduced enzyme activity to control levels. Substrate gel zymography revealed that the urokinase-type PA is the predominant form of PA released by both cell lines. Comparison of the activity of all three antiestrogens used in this study indicates that ICI is the most potent inhibitor of enzyme activity in ER-positive MCF cells.

Influence of antiestrogens on the migration of breast cancer cells using an in vitro wound model.

The metastasis of malignant tumor cells to other organs in the body is the major cause of cancer-related patient mortality. Therefore, the inhibition of tumor cell motility is critical in the prevention or control of tumor malignancy. In the present study, the antimetastatic potential of antiestrogens [tamoxifen (TAM); ICI-182,780 (ICI); and Analog II (AII)] on highly invasive, estrogen receptor (ER)-negative MDA-MB-231 (MDA) and non-invasive, ER-positive MCF-7 (MCF) human breast cancer cell lines was investigated using an in vitro wound model. Wounds were created in confluent cell cultures and repopulation of the wound space was evaluated by counting the number of cells that migrated into the wound area and by measuring the maximum distance traveled. In addition, the number of cells that were passively seeded into the wounded area was determined. ICI and AII reduced the number of MCF cells that migrated into the wounded area and reduced the number of viable passively seeded MDA cells. Unlike ICI and AII, TAM appeared to enhance MCF and MDA cell movement. This study indicates that the in vitro wound technique is applicable to the study of breast cancer cell movement in response to antiestrogens and other antimetastatic agents. It also demonstrates that antiestrogens differ in their influence on breast cancer cell migration.

Differential influence of antiestrogens on the in vitro release of gelatinases (type IV collagenases) by invasive and non-invasive breast cancer cells.

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and cancer metastasis. Accordingly, a higher level of these enzymes has been associated with the invasive phenotype. In the present study the effect of the antiestrogens, Analog II (AII), ICI-182,780 (ICI), and tamoxifen (TAM), on the in vitro release of MMPs, particularly gelatinases A and B by the MDA-MB-231 (MDA) and MCF-7 (MCF) human breast cancer cell lines was investigated using a solid-phase radioassay and substrate gel zymography. Quantitatively, the enzyme activity was found to be higher in the incubation medium from estrogen receptor (ER)-negative and more metastatic MDA cells compared to ER-positive and less metastatic MCF cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1) reduced the enzyme activity in media from both MDA (56.36%) and MCF (71.03%) cells. Differential antiestrogen effects on the two cell lines were observed following 4 days of treatment of cells at a concentration of 10(-6)M. The enzyme activity from MDA cells was not influenced by treatment with any of the antiestrogens, whereas, in MCF cells, ICI produced the greatest enzyme inhibition (47.93%), followed by AII (36.51%) and TAM (24.05%). Concurrent treatment of MCF cells with 17-beta-estradiol (10(-9)M) partially reversed the AII- and TAM-induced but did not alter ICI-induced inhibition of enzyme activity. Substrate gel zymography revealed that among the MMPs, the MDA cells released predominantly progelatinase A (72 kDa) along with minor bands of activated forms, 62 kDa and 59 kDa, whereas progelatinase B (92 kDa) was detected predominantly in the medium from MCF cells. Comparison of the overall antiestrogen effect indicates that ICI is the most potent inhibitor of enzyme activity in ER-positive MCF cells and that antiestrogen treatment may limit the metastatic potential of ER-positive breast cancer.

Antitumor activity of a novel antiestrogen (Analog II) on human breast cancer cells.

Analog II (1,1-dichloro-cis-2,3-diarylcyclopropane), previously shown to be a pure antiestrogen in mice, was examined for potential antitumor activity on human breast cancer cells in culture. In this study, Analog II produced a dose-related antiproliferative effect on the growth of estrogen receptor (ER)-positive MCF-7 human breast cancer cells over a concentration range of 10(-11) to 10(-5) M. Analog II increased the fraction of MCF-7 cells in the G2/M phase of the cell cycle. Further, this compound inhibited the growth of ER-negative MDA-MB-231 human breast cancer cells over a concentration range of 10(-9) to 10(-6) M. Using scanning electron microscopy to evaluate drug-induced changes in cellular morphology, it was observed that Analog II decreased the length and density of microvilli on both MCF-7 and MDA-MB-231 cells. The effects of Analog II on MCF-7 and MDA-MB-231 cell proliferation and morphology were not reversed in the presence of estradiol. In addition, the induction of estrogen-dependent genes in MCF-7 cells was not reversed by Analog II. It was observed that non-specific cytotoxicity may be responsible for part of the Analog II-induced inhibition on MCF-7 and MDA-MB-231 cell proliferation. However, the antitumor activity of this compound was found to be specific to human breast cancer cells since it did not alter the proliferation or viability of non-breast A-549 human lung cancer cells. In conclusion, these results indicate that Analog II is a potent antitumor agent, has a unique antitumor mechanism in breast cancer cells and may be effective in the treatment of breast cancer.

The influence of antiestrogens on pS2 and cathepsin D mRNA induction in MCF-7 breast cancer cells.

Tamoxifen, although widely used in the treatment of estrogen-dependent tumors, is a partial estrogen agonist producing undesirable effects in breast cancer patients. ICI 182,780 a steroidal antiestrogen displays pure antagonist activity which is due to its ability to prevent dimerization of the estrogen receptor (ER). Our previous studies have shown that 1,1-dichloro-cis-2,3-diaryl cyclopropane (Analog II), a diarylcyclopropyl compound is devoid of estrogenic activity, has a weak binding affinity for the estrogen receptor in the mouse uterine tissue and inhibits the growth of breast cancer cells in culture. These findings suggest that Analog II may not inhibit tumor cell growth at the cellular level by an ER-mediated mechanism of action. Since these three antiestrogens appear to have different mechanisms of antiestrogenic activity, the purpose of this study was to compare the influence of the three antiestrogens on estradiol-induced expression of pS2 and cathepsin D (cath-D). These genes are known to be primarily under the influence of estrogen in ER positive MCF-7 human breast cancer cells. The results of this study demonstrate different mechanisms of regulation of the cath-D and pS2 genes by antiestrogens in MCF-7 cells. This study indicates that ICI 182,780 is a pure antagonist at the levels of gene regulation and cell proliferation. The relative order of inhibitory action was found to be ICI 182,780 > tamoxifen > Analog II.

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

Cyclopropyl compound 7b [(Z)-1,1-dichloro-2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxyphenyl)-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.

Influence of cyclopropyl antiestrogens on the cell cycle kinetics of MCF-7 human breast cancer cells.

Five cyclopropyl compounds, previously shown to exhibit pure antiestrogenic activity in the mouse uterotropic assay and antiproliferative activity of MCF-7 human breast cancer cells in culture, were examined for their influence on the cell cycle kinetics of MCF-7 cells. The DNA-histogram of a single cell suspension was obtained on Coulter Epics V after fixing the cells in 70 % ethyl alcohol and staining in propidium iodide. Tamoxifen increased the percentage of cells in G1-phase with a concomitant decrease in percentage of cells in S-phase, in an estradiol reversible manner. Cyclopropyl compound 7a increased the percentage of cells in G1-phase, in an estradiol-irreversible manner. Further, compounds 5a, 5c, 7a and 7b decreased the percentage of cells in S-phase and increased percentage of cells in the G2M-phase, in an estradiol-irreversible manner. Of the five cyclopropyl compounds tested, only 4d had no influence on the cytokinetic parameters, even though this compound was found to exhibit antiproliferative activity on MCF-7 cells equal to that of tamoxifen. In conclusion, all of the cyclopropyl compounds, except 4d, altered cell cycle parameters of MCF-7 cells in a manner different than that of tamoxifen. Thus, the results of this study indicate that, although these cyclopropyl compounds are antiestrogenic, they produce antiproliferative activity by a distinct mechanism of action in estrogen receptor positive breast cancer cells.

The mutagenic potential of antiestrogens at the HPRT locus in V79 cells.

Tamoxifen, a nonsteroidal antiestrogen is widely used in the treatment of breast cancer. Although clearly of clinical value, it produces adverse side effects associated with its estrogen agonist activity. This has led to the development of antiestrogens with less estrogen agonist activity. Analog II (1,1-dichloro-cis-2,3-diaryl cyclopropane) is a cyclopropyl compound which produces pure antiestrogenic activity in mice and inhibits the proliferation of breast cancer cells. Since the genotoxicity of Analog II has not been examined, the aim of the present study was to investigate the mutagenic potential of Analog II. The mutagenic effects of Analog II were studied at the hypoxanthine phosphoribosyl transferase (hprt) locus in Chinese hamster lung fibroblasts (V79 cell line) and compared to tamoxifen and estradiol. In this study Analog II was not mutagenic at the hprt locus in V79 cells and appeared to have less mutagenic potential than either estradiol or tamoxifen. However, an examination of the genotoxicity of metabolic products of these compounds will be necessary before definite conclusions can be drawn concerning their genotoxicity in vivo.

A comparison of the antitumor activity of two triarylcyclopropyl antiestrogens (compounds 4d and 5c) on human breast cancer cells in culture.

Compound 4d ((E)- and (Z)-1,1-Dichloro-2-[4-(benzyloxy)-phenyl]2,3-bis(4-methoxyphenyl) cyclopropane) and compound 5c ((Z)-1,1-Dichloro-2-[4-(benzyloxy)-phenyl]- 2-(4-methoxyphenyl)-3-phenylcyclopropane) are two members of a novel series of triarylcyclopropyl compounds which have been shown to be pure antiestrogens. In the present study, the antiproliferative activity of 4d and 5c 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 4d inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-13) to 10(-5) M while compound 5c inhibited MCF-7 cell growth in a dose-related manner over a concentration range of 10(-9) to 10(-5) M. Further, neither compound altered the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol reversed the antiproliferative activity of 4d but not 5c on MCF-7 cells. Both compounds bound specifically to ER in MCF-7 cells; however, the relative binding activity of 4d was five times greater than estradiol and 5000 times greater than 5c. The influence of 4d and 5c on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy. Both compounds, at a concentration of 10(-6) M, reduced the density of microvilli on MCF-7 cells, which was reversed by co-administration of estradiol (10 (-8) M). These compounds did not alter the cell surface morphology of ER-negative MDA-MB-231 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. The antiproliferative activity of 7a (10(-7) M) on MCF-7 cells was reversed by co-administration of estradiol (10(-8) M). 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(-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). 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture.

The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.

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.