Correlation of the antiproliferative action of diphenylmethane-derivative antiestrogen binding site ligands with antagonism of histamine binding but not of protein kinase C-mediated phosphorylation

The nonestrogen receptor-mediated antiproliferative action of antiestrogen binding site (AEBS) ligands, including triphenylethylene antiestrogens and phenothiazines, has been linked to their ability to inhibit protein kinase C (PKC). Recent studies indicate that some diphenylmethane derivatives inhibit growth, are potent AEBS ligands, and antagonize histamine binding at an AEBS-related histamine site different from H1 and H2. Three novel diphenylmethane derivatives, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCI (DPPE), 4-decanoyl-DPPE (dec-DPPE), and 4-benzylphenyl decanoate (BPD) were studied in an attempt to determine whether PKC or histamine interactions best correlate with their antiproliferative effects. Platelet aggregation and the phosphorylation of a platelet Mr 47,000 protein (p47) induced by phorbol-12-myristate-13-acetate (PMA) represent two processes mediated by PKC. DPPE inhibits PMA-induced aggregation [50% inhibitory concentration (IC50) = 31.2 +/- 2.4 (SEM) x 10(-6) M] but does not significantly inhibit either PMA-induced phosphorylation of Mr 47,000 protein (IC50 greater than 500 x 10(-6) M), or binding of [3H]phorbol dibutyrate to platelets. dec-DPPE is a more potent inhibitor of PMA-induced platelet aggregation (IC50 = 18.8 +/- 0.7 x 10(-6) M), a weak inhibitor of Mr 47,000 phosphorylation (IC50 = 80-200 x 10(-6) M), but is without effect on [3H]phorbol dibutyrate binding. BPD, which lacks the alkylaminoethoxy side chain necessary for binding to the AEBS/DPPE site, is devoid of anti-PMA effects. These results are compared to the inhibition of [3H]histamine binding in rat cortex membranes (Ki value for DPPE = 0.83 +/- 0.62 x 10(-6) M; Ki value for dec-DPPE = 6.6 +/- 3.5 x 10(-6) M; BPD is inactive) and growth inhibition of MCF-7 cells (IC50 value for DPPE = 4.5 x 10(-6) M; IC50 value for dec-DPPE = 1.5 x 10(-5) M; BPD is ineffective at all concentrations tested). Thus, while dec-DPPE is a more potent inhibitor of PKC-mediated phosphorylation, DPPE is a more potent inhibitor of histamine binding and is correspondingly more antiproliferative than dec-DPPE. The results support a relationship between antagonism of histamine binding and growth inhibition but argue against an association between the antiproliferative effects of DPPE and dec-DPPE and inhibition of PKC. The findings for DPPE suggest that platelet response to PMA, antagonized by diphenylmethane-type AEBS-ligands, may be mediated, at least in part, by mechanisms other than activation of protein kinase C-dependent phosphorylation.     

Increased therapeutic index of antineoplastic drugs in combination with intracellular histamine antagonists

L-Histidinol, a protein synthesis inhibitor and structural analogue of L-histidine, has been demonstrated in chemotherapy-treated mice to be cytoprotective to normal stem cells but to enhance cytotoxicity to tumor cells. N,N-Diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine.HCl (DPPE) is an antagonist of recently described microsomal and nuclear intracellular histamine receptors implicated in the mediation of proliferation and modulation of prostaglandin synthesis. DPPE is cytotoxic to tumor cells in vitro and cytoprotective to the gut in vivo. Noting the similar pharmacologic profiles for histidinol and DPPE and the structural resemblance between histidinol and histamine, we tested 1) whether binding to intracellular histamine receptors may be important to the action of histidinol, 2) whether there exists a differential effect of DPPE and histidinol on proliferating normal and transformed or malignant cells, and 3) whether DPPE, like histidinol, protects host cells from the effects of chemotherapy while augmenting tumor cell kill in vivo. It was observed that histidinol does compete at intracellular histamine receptors in isolated microsomes and nuclei, but with significantly lower affinity than DPPE. Nevertheless, for each agent, potency at intracellular histamine receptors correlates with potency to inhibit DNA and protein synthesis, without cytotoxicity, in normal mitogen-stimulated murine lymphocytes and to kill transformed mouse lymphocytes or MCF-7 human breast cancer cells. As demonstrated previously for histidinol (1-2 g/kg), DPPE (4 mg/kg) protected murine bone marrow progenitors from doxorubicin or fluorouracil, while doses of 4-50 mg/kg significantly enhanced the antitumor activity of doxorubicin and daunorubicin in murine models of early cancer. One postulate to explain the effects of intracellular histamine receptor ligands is that intracellular histamine mediates DNA and protein synthesis, possibly through a downward modulation of growth-inhibitory prostaglandin levels. Antagonism of the intracellular action of histamine at intracellular histamine receptors by DPPE or histidinol may result in differential perturbations of growth/eicosanoid metabolism in normal and malignant cells, thus forming the basis of a new approach to chemotherapy.     

Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.     

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.     

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     

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.     

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.     

Flow cytometric determination of estrogen receptors in intact cells

Flow cytometry has been used to detect estrogen receptor (ER) in intact cells, using 1-(N)- fluoresceinyl estrone thiosemicarbazone ( 17FE ), a ligand shown to have sufficient affinity and specificity to identify high-affinity receptors. For each concentration of 17FE , two fluorescent distributions were obtained: (a) "total" fluorescence due to 17FE alone; and (b) diethylstilbestrol-inhibited fluorescence ("nonspecific"). The mean fluorescence intensity for both the "total" (MT) and "nonspecific" (MN) distributions was calculated at each ligand concentration by obtaining the total brightness of each sample, normalized to the number of cells analyzed. A specific binding curve was constructed over a broad range of 17FE concentrations (1.5 X 10(-9) to7 .5 X 10(-7) M) by plotting the mean specific fluorescence intensity (MS = MT-MN) at each 17FE concentration. Saturable binding was demonstrated for a known ER-positive cell line (MCF-7) at low ligand concentrations (5 to 10 X 10(-8) M), while no specific binding was obtained for a known ER-negative cell line (MDA-231). The Kd was estimated from the specific binding curve of MCF-7 cells as the concentration of 17FE at half-maximal binding. When corrected for the binding activity relative to estradiol, a Kd of 18 X 10(-10) M was obtained. The flow cytometer-generated distributions give a qualitative estimate of the proportions of ER-rich and ER-poor cells. Quantitation of ER heterogeneity will require a mathematical algorithm expressing the difference between the "total" and "nonspecific" distributions. These studies demonstrate that flow cytometry and an appropriate ligand can detect and partially describe ER heterogeneity in intact cells.     

All-trans-retinoic acid metabolites significantly inhibit the proliferation of MCF-7 human breast cancer cells in vitro.

All-trans-retinoic acid (ATRA) is well known to inhibit the proliferation of human breast cancer cells. Much less is known about the antiproliferative activity of the naturally occurring metabolites and isomers of ATRA. In the present study, we investigated the antiproliferative activity of ATRA, its physiological catabolites 4-oxo-ATRA and 5,6-epoxy-ATRA and isomers 9-cis-RA and 13-cis-RA in MCF-7 human breast cancer cells by bromodeoxyuridine incorporation. MCF-7 cells were grown in steroid- and retinoid-free medium supplemented with growth factors. Under these culture conditions, ATRA and its naturally occurring catabolites and isomers showed significant antiproliferative activity in MCF-7 cells in a concentration-dependent manner (10[-11] M to 10[-6] M). The antiproliferative activity of ATRA catabolites and isomers was equal to that of the parent compound ATRA at concentrations of 10(-8) M and 10(-7) M. Only at 10(-6) M were the catabolites and the stereoisomer 13-cis-RA less potent. The stereoisomer 9-cis-RA was as potent as ATRA at all concentrations tested (10[-11] M to 10[-6] M). In addition, we show that the catabolites and isomers were formed from ATRA to only a limited extent. Together, our findings suggest that in spite of their high antiproliferative activity the catabolites and isomers of ATRA cannot be responsible for the observed growth inhibition induced by ATRA.     

Binding of formyl peptides to Walker 256 carcinosarcoma cells and the chemotactic response of these cells

N-Formylmethionylleucylphenylalanine (fMLP) induces chemotaxis in leukocytes, the response being mediated by peptide binding to a receptor on the plasma membrane. In tumor cells, this peptide has been reported to induce cellular swelling and chemotaxis in vitro and to enhance the localization of circulating tumor cells in vivo. In the Boyden chamber, we evaluated the migratory responses of Walker carcinosarcoma 256 cells to varying concentrations of fMLP. Sigmoidal dose-response curves were obtained with the dose of chemotactic factor that elicits a half-maximal chemotactic response of 5.0 +/- 2.5 X 10(-8) M. Checkerboard analysis indicated that these responses were dependent upon a concentration gradient of fMLP with increases in migration of circa 2 to 2.5 times that of random movement. To examine the binding of fMLP, the tumor cells were incubated with 5 X 10(-9) M fML-[3H]P in Hanks' balanced salt solution. Specific binding (0.5 to 1% of total radioligand, to whole cells inhibited by 5 X 10(-6) M fMLP) approached equilibrium after 4 to 6 h at 4 degrees C and after 6 to 10 h at 22 degrees C. Autoradiographic studies demonstrated heterogeneous binding of the peptide by tumor cells and also showed its intracellular localization. In homogenates of Walker cells prepared in 0.1 M Tris HCl, pH 7.4, with 10 mM MgCl2 and bovine serum albumin (1 mg/ml), specific binding of approximately 0.5% of total fML-[3H]P reached equilibrium after 60 min at 4 degrees C. In whole cells and homogenates, binding was reversible by addition of unlabeled fMLP. In whole cells, displacement curves demonstrated a Kd of 1.9 +/- 0.1 X 10(-7) M, whereas in homogenates there was a background of low affinity (Kd greater than 10(-5) M) nonsaturable binding, but also a high-affinity component with Kd of 4.9 +/- 1.8 X 10(-8) M. Both chemotaxis and binding were inhibited by the oligopeptide, N-carbobenzoxy-L-phenylalanyl-L-methionine, which is a competitive inhibitor of formyl peptide-induced neutrophil chemotaxis. These data suggest that fMLP stimulates chemotaxis in tumor cells by a receptor-mediated pathway.     

Binding characteristics and biological activity of 17 alpha-[125I]iodovinyl-11 beta-methoxyestradiol, an estrogen receptor-binding radiopharmaceutical, in human breast cancer cells (MCF-7)

17 alpha-[125I]Iodovinyl-11 beta-methoxyestradiol ([125I]MIVE2), a gamma-emitting analogue of estradiol, previously shown to bind to rat uterine estradiol receptor, was studied to determine the binding characteristics and biological activity in human breast cancer cells. In vitro determination of receptor binding by dextran-coated charcoal assays indicates that [125I]-MIVE2 binds specifically and with a high affinity to cytosolic estrogen receptors in the human breast cancer cell line, MCF-7. [3H]Estradiol binds to the receptor with approximately four times the affinity of [125I]-MIVE2 (Kd = 2.55 X 10(-9) M for [125I]MIVE2; Kd = 6.4 X 10(-10) M for [3H]estradiol). Unlabeled MIVE2 produces estrogenic effects similar to those of estradiol such as progesterone receptor induction and increases in thymidine incorporation in MCF-7 cells in culture. Cytosolic progesterone receptor levels were elevated 2.8-fold over control levels by 6 X 10(-9) M MIVE2. Stimulation of thymidine incorporation (approximately 300% above control levels) was observed after exposure to 1 X 10(-9) M MIVE2. Preliminary data show receptor-mediated uptake by the uterus in biodistribution studies in athymic nude mice given injections of [125I] MIVE2 (32-34 microCi). At 4 h, uterus:blood ratios are 20.5 and target tissue:nontarget tissue ratios are 12.9. In light of the fact that this compound can be prepared with a high specific activity, [125I]MIVE2 may have potential as a radiotracer for imaging estrogen receptor-positive breast tumors or metastatic lesions in human breast cancer patients.     

Insulin receptor regulation in cultured human tumor cells

Insulin binding to monolayer cell cultures of human fibroblasts, human colon carcinoma (HCT-8, HT-29), human breast carcinoma (MCF-7, T-47D), and melanoma (MM-96) was measured using 125I-insulin. Binding was time and temperature dependent in all cell lines, and only one cell line (MM-96) degraded 125I-insulin. High-affinity insulin-binding sites (Kd = 1.4 X 10(-9) M to 0.4 X 10(-10) M) were detected in all cell lines, and insulin-binding capacity ranged from 0.6 to 14 fmol/10(6) cells. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabeled insulin, dissociating surface-bound insulin and measuring residual receptors by 125I-insulin uptake. Exposure of tumor cells to greater than 10(-6) M insulin for 2 hr at 37 degrees led to a decrease in the number of insulin binding sites in MM-96 and colon cell lines only, with maximum down-regulation ranging from 58% (MM-96) to 88% (HCT-8) receptor loss. The decrease in insulin binding was due to a decreased number of receptors per cell with no change in affinity. Monolayers exposed to 1.7 X 10(-5) M unlabeled insulin for 7 hr at 37 degrees invariably showed greater than 50% receptor loss. However, monolayers exposed to 1.7 X 10(-8) M unlabeled insulin for 7 hr at 37 degrees showed less marked (0 to 39%) down-regulation. In comparison, human fibroblasts showed 57% receptor loss after exposure to 3.5 X 10(-9) M unlabeled insulin for 7 hr. Thus, markedly supraphysiological concentrations of insulin are required to down-regulate insulin receptors in tumor cell lines compared with normal cells. This suggests a tumor-associated resistance to receptor down-regulation.     

Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells

Although antiestrogen therapy is effective in the treatment of hormone-responsive breast tumors, approximately 40% of the patients with estrogen receptor-positive tumors fail to respond to antiestrogens. To better understand the mechanisms by which antiestrogens inhibit the growth of hormone-dependent breast cancers, we have investigated the physicochemical properties and binding characteristics of the estrogen receptors with estradiol and antiestrogens and the occurrence of estrogen-noncompetible antiestrogen binding sites in two estrogen-sensitive but tamoxifen-growth-resistant estrogen receptor-positive MCF-7 cell variant clones, R3-98 and R27. In the variant cells, estradiol (10(-8) M) significantly stimulates cell proliferation as in the parent MCF-7 cells, but the antiestrogen tamoxifen (10(-6) M) has no significant effect on growth of the variant cells, whereas antiestrogen strongly inhibits proliferation of the parent MCF-7 cells. All three cell types contain high concentrations of estrogen receptor (150 to 250 fmol/mg protein), and competition binding analysis shows that the relative binding affinity of a series of compounds for estrogen receptor is similar among the three cell types with the affinity of trans-hydroxytamoxifen greater than estradiol greater than alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilben e greater than tamoxifen. Salt-extracted nuclear receptor complexes prepared from the three cell types showed similar sedimentation behavior on 0.4 M KCl-containing sucrose gradients with [3H]estradiol-labeled receptor complexes sedimenting at 4.2S, whereas receptors complexed with either of the antiestrogens trans-[3H]-hydroxytamoxifen or [3H]alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrosti lbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Sp gamma, to form complexes which sediment at 8.5S. The nuclear estrogen receptors from the parental MCF-7 and the two variant cells, when covalently labeled with [3H]-tamoxifen aziridine in intact cells and then salt extracted have identical molecular weights of approximately 62,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalently labeled nuclear and cytosol receptors in these 3 cell lines also show identical migration in 8 M urea polyacrylamide isoelectric focusing gels consistent with a predominant receptor species of isoelectric point approximately 5.7.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor cell surface-associated binding site for the M(r) 72,000 type IV collagenase.

We have studied the capacity of two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, to bind exogenous M(r) 72,000 type IV collagenase by both morphological and radioreceptor binding assays. By indirect immunofluorescence, staining with a specific anti-M(r) 72,000 type IV collagenase antibody was strongly induced when cells were preincubated with the purified enzyme. Scatchard plot analysis indicated the existence of a binding site for the M(r) 72,000 type IV collagenase with high affinity for both cell lines (Kd = 2 x 10(-9) M). These results are the first demonstration of the existence of a tumor cell membrane-associated putative receptor for a member of the matrix metalloproteinase family, as previously evidenced for the urokinase-type plasminogen activator.     

Antiestrogenic action of 3-hydroxytamoxifen in the human breast cancer cell line MCF-7

The antiestrogenic action of 3-hydroxytamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1-(3-hydroxyphenyl)-2 -phenylbut-1-ene] was characterized in vitro and compared with that of tamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. The relative binding affinities of 3-hydroxytamoxifen to estrogen receptor were 3.3% in cytosol of MCF-7 cells and 1.5% in human mammary carcinoma cytosol compared to values of 0.2 and 0.3% for tamoxifen (the affinity of 17 beta-estradiol considered to be 100%). The concentration of 3-hydroxytamoxifen necessary to suppress the 17 beta-estradiol-induced growth stimulation of MCF-7 cells was about tenfold lower than that for tamoxifen. The induction of progesterone receptor in MCF-7 cells by 17 beta-estradiol was inhibited by 3-hydroxytamoxifen. In the absence of 17 beta-estradiol, 3-hydroxytamoxifen gave rise to a moderate increase in the progesterone receptor levels, which demonstrates the partially estrogenic character of hydroxytamoxifen.     

Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells

We have examined the properties of the estrogen receptor and progesterone receptor in MDA-MB-134 human breast cells and have evaluated the effects of estrogen on cell proliferation and progesterone receptor levels in these cells as indices of hormonal sensitivity. These cells contain high levels of estrogen receptor (approximately 1.5 pmol/mg DNA) and low levels of progesterone receptor (0.15 pmol/mg DNA). More than 80% of the estrogen receptor is found in the nuclear fraction in the absence of estrogen, and the Kd of the receptor for estradiol is approximately 1.5 X 10(-10) M. Upon exposure to estradiol, the receptors become occupied, but there is no processing or apparent decrease in either nuclear or total cellular estrogen receptor content, as can be seen in MCF-7 human breast cancer cells. The nuclear estrogen receptor sediments as a 4.6 S species on high salt sucrose gradients, and it can be detected on sodium dodecyl sulfate-polyacrylamide gel immunoblot analysis as a species of molecular weight 65,000, identical to that of the MCF-7 estrogen receptor, using the monoclonal antibodies D75P3 gamma and H222Sp gamma prepared against the MCF-7 estrogen receptor. The estrogen receptor shows binding selectivity for estrogens and antiestrogens, and its affinity for ligands follows the order diethylstilbestrol (190%) greater than estradiol (100%) greater than estriol (13%) greater than tamoxifen (3%), as expected for estrogen receptor. Hence the receptor appears normal in many of its physicochemical properties and in terms of its binding affinity and specificity for estrogens and antiestrogens. Control cells contain low levels of progesterone receptor that display high affinity (Kd = 6 X 10(-9) M) for the synthetic progestin R5020, but exposure to estradiol (10(-11)-10(-7)M) fails to increase cellular progesterone receptor levels. In contrast, estradiol markedly stimulates the rate of cell proliferation, while tamoxifen suppresses the growth of control and of estradiol treated cells. Hence, our data show that these cells, which contain substantial levels of estrogen receptor, respond to estrogen with enhanced cell proliferation but fail to have their progesterone receptor level modulated by estradiol. These cells represent an interesting and unusual situation in which estrogenic regulation of proliferation and the stimulation of progesterone receptor are dissociated. These cells should prove useful in further evaluation of estrogenic regulation of cell proliferation and specific protein synthesis in human breast cancer.     

The antiproliferative properties of tamoxifen and phenothiazines may be mediated by a unique histamine receptor (?H3) distinct from the calmodulin-binding site

Summary N,N-diethyl-2-[(4-phenylmethyl)-phenoxyl]-ethanamine HCl (DPPE), a novel histamine antagonist (?H₃), which selectively binds with high affinity to the antiestrogen-binding site (AEBS/?H₃), inhibits the activity of calmodulin-dependent myosin light chain kinase (MLCK) only at concentrations >1 mM, as opposed to tamoxifen (TAM), which has an IC₅₀=4 M in the same assay. This suggests that the antiestrogen-binding site is distinct from the site on calmodulin which binds TAM and phenothiazines. However, at an in vitro concentration of 1×10^-6 M, the antiproliferative effects of DPPE and several phenothiazines, which also compete for binding to AEBS/?H₃, are about equal; this suggests that affinity for AEBS/?H₃ rather than that for the calmodulin-binding site may correlate with clinically relevant antigrowth effects of these compounds.This work was supported by grants from the National Cancer Institute of Canada, the Medical Research Council of Canada, and by a generous gift from the estate of Alice Christle.     

Effects of antiestrogens and medroxyprogesterone acetate on the clonogenic growth of tamoxifen-sensitive and resistant human breast cancer cells

Effects of tamoxifen (TAM), nafoxidine (NFA), 4-hydroxytamoxifen (4-OH-TAM), 3-hydroxytamoxifen (3-OH-TAM), and medroxyprogesterone acetate (MPA) on the clonogenic growth of a hormone-responsive human breast cancer cell line (MCF-7) and its tamoxifen-resistant variant (R-27) were studied. TAM, (10(-6)M) showed an inhibitory effect on the colony formation in a plastic dish of MCF-7 cells only in medium containing DDC-treated FCS (E2(-) medium). With the presence of E2 (10(-8)M) in the medium (E2(+) medium), TAM did not show any effect on cell growth. Irrespective of the presence or absence of E2 in the medium, there was no inhibitory effect of TAM on the clonogenic growth of R-27 cells. The ID50 values, expressed as the suppression of plating efficiencies, obtained by adding NFA, 4-OH-TAM, 3-OH-TAM, and MPA to the MCF-7 cells were shown to be 2 X 10(-7)M, less than 10(-8)M, 1 X 10(-7)M, and 4 X 10(-7)M, respectively in the E2 (-) medium, and 2 X 10(-6)M, 2 X 10(-7)M, 2 X 10(-6)M, and 4 X 10(-8)M respectively in the E2 (+) medium. For the R-27 cells, ID50 values obtained by adding NAF, 4-OH-TAM, 3-OH-TAM, and MPA were 7 X 10(-7)M, 5 X 10(-8)M, 4 X 10(-7)M, and 6 X 10(-8)M, respectively in the E2(-) medium, and 2 X 10(-6)M, 2 X 10(-6)M, greater than 5 X 10(-6)M, and 1 X 10(-8)M, respectively in the E2(+) medium. These results suggest that the antiestrogens used produce their suppressive effects on cell growth depending on the E2 concentration in the medium in these estrogen-responsive cell lines, and that the monohydroxytamoxifens are more potent than TAM in suppressing cell growth. The effects of MPA are shown to be different from the antiestrogen used in that MPA inhibited the growth of both TAM-sensitive and-resistant cells, independent of the presence or absence of E2 in the medium. The R-27 cell line, a variant of the MCF-7 cell line, appears to be a good model for studying antiestrogen resistance and for evaluating the effectiveness of agents against endocrine-resistant breast cancer cells.     

Transient Receptor Potential Vanilloid 1 Expression and Functionality in MCF-7 Cells: A Preliminary Investigation

Purpose

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel belonging to the transient receptor potential family, and it is expressed in different neoplastic tissues. Its activation is associated with regulation of cancer growth and progression. The aim of this research was to study the expression and pharmacological characteristics of TRPV1 in cells derived from human breast cancer MCF-7 cells.

Methods

TRPV1 presence was assessed by binding studies and Western blotting. Receptor binding characteristics were evaluated through competition assays, while 3-(4,5-dimethylthiazol-2-yl)-2,5,-dipheyltetrazolium bromide reduction assays were performed to confirm an early hypothesis regarding the modulation of cancer cell proliferation. The functionality of TRPV1 was evaluated by measuring Ca²⁺ uptake in the presence of increasing concentrations of TRPV1 agonists and antagonists.

Results

Binding studies identified a single class of TRPV1 (B_max 1,492±192 fmol/mg protein), and Western blot showed a signal at 100 kDa corresponding to the molecular weight of human TRPV1. Among the different tested agonists and antagonists, anandamide (Ki: 2.8×10^-11 M) and 5-iodoresiniferatoxin (5-I-RTX) (Ki: 5.6×10^-11 M) showed the highest degrees of affinity for TRPV1, respectively. All tested TRPV1 agonists and antagonists caused a significant (p<0.05) decrease in cell growth rate in MCF-7 cells. For agonists and antagonists, the efficacy of tested compounds displayed the following rank order: resiniferatoxin>anandamide>capsaicin and 5-I-RTX=capsazepine, respectively.

Conclusion

These data indicate that both TRPV1 agonists and antagonists induce significant inhibition of MCF-7 cell growth. Even though the mechanisms involved in the antiproliferative effects of TRPV1 agonists and antagonists should be further investigated, it has been suggested that agonists cause desensitization of the receptor, leading to alteration in Ca²⁺-influx regulation. By contrast, antagonists cause a functional block of the receptor with consequent fatal dysregulation of cell homeostasis.     

The equilibrium and kinetic drug binding properties of the mouse P-gp1a and P-gp1b P-glycoproteins are similar.

The gene encoding the multidrug resistance P-glycoprotein (P-gp) is duplicated in rodent species and the functional basis for this remains unresolved. Despite a high sequence similarity, the mouse P-gp1a and P-gp1b isoforms show distinct patterns of tissue distribution which suggest a specific role of the P-gp1b isoform in steroid transport. In the present study possible biochemical differences between the isoforms were directly investigated at the level of drug interaction. There was no detectable difference in the affinity or binding capacity of the two isoforms towards [3H]vinblastine at equilibrium. Similarly, the rate at which [3H]vinblastine associates with P-gp was indistinguishable between the two isoforms. Some modest differences were observed in the relative abilities of the multidrug-resistant (MDR) reversing agents CP100-356, nicardipine and verapamil to displace equilibrium [3H]vinblastine binding to P-gp1a and P-gp1b. The steroid hormone progesterone displayed a low affinity (Ki = 1.2 +/- 0.2 microM for P-gp1a and 3.5 +/- 0.5 microM for P-gp1b), suggesting an unlikely role as a physiological substrate. Thus the mouse isoforms do not appear to exhibit functional differences at the level of initial substrate interaction with protein.