Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids

The interaction between the estrogen receptor and a variety of flavonoids was studied in the presence or absence of estradiol using a stably-transfected human breast cancer cell line (MVLN). On the other hand, flavonoids were evaluated for their effects on proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions and/or substituents essential for estrogenic or antiestrogenic activities. In contrast, we did not find the same relationship for cell proliferation. Among all flavonoids used, only 7-methoxyflavanone and 7,8-dihydroxyflavone at high concentrations (50 μM) possess antiestrogenic and antiproliferative activities. These results suggest that two hydroxyls (in positions 7 and 8) or 7-methoxy substituents are essential for the antiestrogenic activity of flavonoids. However, it seems that flavonoids at high concentrations exert their antiproliferative activity through other estrogen receptor-independent mechanisms.     

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     

Two new estrogen-supersensitive variants of the MCF-7 human breast cancer cell line

Two new estrogen-sensitive variants of MCF-7 human breast cancer cells, CG-4 and CG-5, are described in this report. These cells were derived from a casual contamination by MCF-7 cells of primary cultures from a human adenocarcinoma of the breast and a pleural effusion of a patient with advanced breast cancer, respectively. Careful characterization of these variants revealed chromosomal properties highly similar to and alloenzyme phenotypes identical to those of MCF-7 cells which were simultaneously cultured in the laboratory. MCF-7, CG-4 and CG-5 cells were tested for estrogen responsiveness under identical growth conditions, that is in the presence of culture medium supplemented with 5% charcoal-treated serum. While the number of MCF-7 cells increases by about 40% over the controls in the presence of physiological concentrations of estradiol, the number of CG-4 cells doubles and the number of CG-5 cells triples. All these cell lines have approximately the same number of estrogen, androgen, glucocorticoid, and progesterone receptor sites/cell. Since several difficulties arise in demonstrating the estrogen responsive growth stimulation of currently available human breast cancer cells, these two new variants, characterized by a high and reproducible estrogen responsiveness, afford a new model for studying the mechanisms by which estrogen regulates cell proliferation. The problems related to the careful characterization of every established cell line are discussed.     

Effects of type I and II interferons on cultured human breast cells: interaction with estrogen receptors and with tamoxifen

The combined effects of tamoxifen, a competitive inhibitor of estrogen, and type I or II interferons on the proliferation of several human breast cancer cell lines in vitro were examined. Additive antiproliferative effects were observed with interferons and tamoxifen, in two estrogen receptor positive cell lines, MCF-7 and T-47D. In MCF-7 cells neither beta ser interferon, gamma interferon, nor alpha 6 interferon were able to significantly alter estrogen receptor levels. Antigrowth activities of beta ser interferon and gamma interferon in an estrogen receptor negative cell line, HS578T, were equivalent to those in estrogen receptor-positive cell lines. Consistent with the antiproliferative effects of interferons, high affinity beta ser interferon receptors and interferon inducible 2'5'-oligoadenylate synthetase were present in both MCF-7 and HS578T cell lines. Thus steroid hormone receptor status did not influence the antiproliferative effects of interferons on breast carcinoma cells in vitro.     

Estrogen control of lactate dehydrogenase isoenzyme-5 in human breast cancer

Determinations of estradiol receptor (ER), progesterone receptor (PR), total lactate dehydrogenase activity (LDH) and electrophoretic separation of LDH isoenzymes were performed in cytosols from 118 samples of primary infiltrating ductal mammary carcinoma. ER + PR+ tumors demonstrated a significant increase in the proportion of the LDH muscle-type isoenzyme (LDH5) as compared to ER-PR- samples (p less than 0.002). Tumors lacking one of the receptors presented intermediate LDH5 percentages. As total LDH activity bore no relation to either the presence or absence of receptors, the increased proportion represents an absolute elevation of LDH5, suggesting that LDH5 may be a promising hormone-dependence marker. As an in vitro model to study whether LDH5 was induced by estradiol via ER, we have used two human breast cancer cell lines, MCF-7 and T47D. In both cell lines LDH5 was the sole isoenzyme. Total ER and PR have been determined by a whole-cell method. In MCF-7 cells (with high ER levels), after incubation with 10(-10) M estradiol, maximal induction of LDH had already been achieved. In relation to T47D (low ER levels) estradiol did not evoke an induction of LDH5 at any concentration examined. In MCF-7 cells, the level of LDH5 induction paralleled processing of ER. The processing effect was rapid, beginning within 5 min of estradiol addition, and was completed within 1 hr. With 10(-6) M tamoxifen, LDH5 induction was suppressed and this effect was reversed by estradiol. Such antiestrogenic effects of tamoxifen on LDH5 have not been observed in T47D cells. Agonistic effects of low doses of tamoxifen on LDH5 were not observed. Our studies suggest that estrogen stimulation of LDH5 involves ER.     

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.     

Antiproliferative activities of interferon-alpha

The antiproliferative activities of human lymphoblastoid interferon-alpha (MOR-22) against 22 cultured human cell lines were examined. Daudi cells and ACHN cells were highly sensitive to interferon compared with other human cells. The cell lines derived from renal and brain tumors showed concentration-dependent growth inhibition as determined from a 7-day growth curve but the concentration dependency was different in various cell lines. It was shown that longer exposure to interferon was necessary to obtain antiproliferative activity, and cell growth inhibition was completely reversible by washing the cultures. It was suggested that interferon had a time-dependent antiproliferative action.     

Mild hyperthermia modulates biological activities of interferons.

A significant enhancement of antiviral activity of human IFN-alpha, -beta and -gamma and murine IFN-gamma is observed when cells are treated with mild hyperthermia (39 degrees C) during antiviral assays. Treatment of primary human fibroblast cells with mild hyperthermia for 4 and 24 hours prior to interferon antiviral assays (pre-assay hyperthermia) further enhances interferon antiviral activity. An enhancement of interferon induced enzyme, 2,5-oligoadenylate synthetase, is also observed in cells treated with interferon and mild hyperthermia. This increase in enzyme activity is, in part, responsible for the observed increase in interferon antiviral activity with hyperthermia. Besides antiviral activity, mild hyperthermia also increases interferon antiproliferative activity on different tumour cells beyond its effect at normal physiological temperatures. On the other hand, mild hyperthermia decreases human interferon production in both human and murine cells when challenged with a viral or non-viral inducer. Also, mild hyperthermia suppresses interferon-mediated enhancement of natural killer (NK) cell activity in human and murine cells. The findings demonstrate that, although mild hyperthermia has suppressive effects upon interferon production and NK cell activity, it significantly increases both antiviral and antiproliferative activities of all three human interferons. These observations have direct bearing upon clinical utilization of exogenously administered interferons to late stage cancer patients who for the most part have a weaker immune system. In these patients, the antiviral and antiproliferative efficacies of administered interferon can be enhanced by combining interferon and hyperthermia.     

Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line)

The effects of trans-4-hydroxytamoxifen (OHTam) on proliferation of cells of the Ishikawa human endometrial adenocarcinoma line were studied under serum-free, phenol red-free conditions and compared to those of estradiol. The addition of OHTam (1 microM) to basal medium (BM), consisting of equal parts of Dulbecco's modified Eagle's medium and Ham's F-12 with additional glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, resulted in significant increases in cell numbers relative to controls. These effects were even greater than those obtained with estradiol (10 nM-1 microM) or 1% charcoal-treated fetal bovine serum (ctFBS). Addition of 1% ctFBS to BM containing 1 microM OHTam further increased cell numbers whereas addition of estradiol (10 nM) did not do so. The stimulation of growth was positively correlated with OHTam concentrations in the range of 10 nM to 1 microM. Dissociation of estradiol and OHTam proliferative effects was observed in a variant of Ishikawa cells in which estradiol did not increase proliferation while OHTam had a strong stimulatory effect. The growth-promoting effects of OHTam were also observed in BM containing 5% or 15% ctFBS. In contrast, in parallel experiments in which BM was replaced by minimal essential medium (Eagle's) with Earle's salts, OHTam (1 microM) did not stimulate proliferation under these conditions and acted as an antiestrogen, inhibiting the proliferative effects of estradiol. These results illustrate marked effects of medium composition on proliferation and antiestrogenic actions of OHTam. Alkaline phosphatase activity was strongly stimulated by estradiol (10 nM) but only very weakly affected by OHTam (1 microM); at these concentrations, OHTam inhibited the effect of estradiol, both in serum-free BM and in minimal essential medium plus 15% ctFBS, demonstrating dissociation in its actions on proliferation and on enzymatic activity. These findings suggest that OHTam may stimulate the proliferation of particular clones of endometrial cancer cells in human tumors. They also suggest that OHTam can exert effects not mediated by the estrogen receptor system, or form OHTam-estrogen receptor agonistic complexes unlike those resulting from estradiol-estrogen receptor interactions. Clearly, Ishikawa cells provide a useful model to investigate mechanisms of action of antiestrogens.     

Histamine and growth: interaction of antiestrogen binding site ligands with a novel histamine site that may be associated with calcium channels

N,N-Diethyl-2-[(4-phenylmethyl)-phenoxy]ethanamine hydrochloride (DPPE) is a novel paradiphenylmethane derivative with antiproliferative and antiestrogenic properties. Like tamoxifen (TAM), DPPE binds to the microsomal antiestrogen binding site with high affinity (Kd approximately 50 nM), but, conversely, not to estrogen receptor or calmodulin. We now demonstrate that DPPE competes for [3H]histamine binding in rat cerebral cortex with an affinity (Ki = 4.5 +/- 2.6 X 10(-6) M) significantly greater than that of the H1 antagonist pyrilamine (Ki = 7.2 +/- 2.2 X 10(-5) M), despite the previous demonstration that pyrilamine is up to 1000 times more potent than DPPE in antagonizing histamine-induced contraction in canine tracheal smooth muscle. DPPE demonstrates antiproliferative activity against MCF-7 cells at concentrations between 1 X 10(-7) and 1 X 10(-5) M; the IC50 value of DPPE for growth inhibition at 7 days in this assay is 5 X 10(-6) M, a value equivalent to its Ki value for histamine binding. DPPE also competes for [3H]verapamil binding in membranes from whole rat brain with an affinity equal to that for verapamil (Kd = 4.0 +/- 1.8 X 10(-7) M); however, verapamil competes for [3H]DPPE binding in brain membranes and rat liver microsomes with an affinity markedly lower (Ki approximately 1 X 10(-4) M) than that of DPPE, suggesting allosteric interactions between the verapamil and DPPE sites. Unlike DPPE, verapamil is not antiproliferative in vitro against MCF-7 cells at concentrations up 1 X 10(-5) M, but, like DPPE, is cytotoxic at concentrations of 1 X 10(-4) M. In immature oophorectomized rats, verapamil or DPPE alone is antiuterotropic; however, verapamil shows no antagonism of exogenous estradiol on uterine growth, as opposed to DPPE which is a partial antagonist. Thus, the antiproliferative and antiestrogenic properties of DPPE either are not associated with calcium channel antagonism, or result from a qualitatively different effect on channels than verapamil. The in vitro antiproliferative effect of DPPE (7.5 X 10(-6) M) on MCF-7 cells at 72 h is significantly reversed by 10 mM L-histidine (70.2 +/- 12.6% reversal) and L-methionine (92.4 +/- 11.1% reversal), but not by L-ornithine, L-arginine, L-phenylalanine, or exogenous histamine. At lower concentrations of TAM (0.75 X 10(-6) M), where growth inhibition is estrogen-reversible, L-ornithine, but not L-histidine or L-methionine, causes significant reversal of growth inhibition (66.8 +/- 13.3%; p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Shape-dependent regulation of proliferation in normal and malignant human cells and its alteration by interferon

The relationship between cell morphology, proliferation, and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes by plating onto plastic surfaces coated with poly(2-hydroxyethyl methacrylate). Poly(2-hydroxyethyl methacrylate) can precisely vary the shape of cells without toxicity. Cell proliferation was quantitated by cell counts and labeling indices were determined by autoradiography. The normal JHU-1 foreskin fibroblasts and IMR-90 lung fibroblasts exhibited contact-inhibited growth with a saturation density of 2.9 X 10(5) and 2.0 X 10(5) cells/cm2, respectively. These cells also exhibited stringent dependency on cell shape with a mitotic index of less than 3% at poly(2-hydroxyethyl methacrylate) concentrations at which the cells were rounded versus a labeling index of 75-90% when the cells were flat. The malignant bladder carcinoma line RT-4 exhibited partial contact-inhibited growth. Its dependency on cell shape was less stringent than that of normal cells with a mitotic index of 37-40% when rounded and 79% when flat. The malignant fibrosarcoma line, HT1080, was not contact inhibited and was entirely shape independent with a mitotic index of 70-90% regardless of cell shape. Treatment of HT1080 cells with low concentration of human fibroblast interferon (less than 40 units/ml) restored shape-dependent proliferation while having little effect on normal cells. Subantiproliferative doses of interferon were also shown to restore contact-inhibited proliferation control to malignant cells previously lacking it. The concordant restoration of contact inhibition and shape-dependent proliferation in malignant cells by interferon suggest that these two types of proliferation controls represent a manifestation of common regulatory mechanisms. However, since these effects occurred at interferon concentrations below that required to produce an antiproliferative effect, these actions of interferon may be distinct from the antiproliferative action.     

Combined recombinant human interferon alpha 2 and cytotoxic agents studied in a clonogenic assay

A human tumor clonogenic assay has been used to test the antiproliferative effect of recombinant human leukocyte interferon alpha 2 alone and in combination with each of 8 cytotoxic agents. Cell lines derived from 6 human tumors and primary tumor cells from 13 patients have been used in these clonogenic assay studies. Results show that interferon as a single agent causes insignificant reduction in tumor cell colony survival if the short-term 1-hr cell exposure method is used; only high concentrations of interferon used in continuous cell exposure in the clonogenic assay can demonstrate a reduction in colony survival to below 50% of control values. Combinations of interferon with either doxorubicin or cisplatin frequently show additive and occasionally synergistic antiproliferative effects on tumor cell colony formation. Variations in drug concentrations and sequencing of drugs have been tested, showing that optimal antiproliferative effects of combined interferon and doxorubicin are realized when maximal concentrations of interferon and prolonged cell exposure time of both interferon and doxorubicin are employed. Combinations of interferon and doxorubicin tested in the clonogenic assay demonstrate cytotoxicity superior to that of either agent tested alone.     

A potent specific pure antiestrogen with clinical potential.

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells. Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.     

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.     

Inhibition of human HT29 colon carcinoma growth in vitro and in vivo by swainsonine and human interferon-alpha 2

Swainsonine, a plant alkaloid and potent inhibitor of Asn-linked oligosaccharide processing, has previously been shown to inhibit organ colonization by metastatic murine tumor cells and to inhibit the growth of transformed fibroblasts in soft agar. In this report, we show that swainsonine has antiproliferative activity against human tumor cells growing in tissue culture and as tumor xenografts in nude mice. The antiproliferative activity of swainsonine was additive with that of human interferon-alpha 2 (HuIFN-alpha 2) in cultures of HT29 colon carcinoma, SN12 renal carcinoma, and A375 melanoma cells. In vivo, the growth rate of HT29m human colon carcinoma tumors in athymic nude mice was reduced by supplementing their drinking water with swainsonine (49%) or by administering HuIFN-alpha 2 systemically (53%); combining these treatments reduced tumor growth by 78%. Combining swainsonine and HuIFN-alpha 2 treatments enhanced the activity of the interferon-inducible enzyme 2',5'-oligoadenylate [2',5'-oligo(A)] synthetase in HT29m tumors compared to that observed in tumors from mice treated with interferon alone. In vitro, swainsonine enhanced interferon-dependent induction of 2',5'-oligo(A) synthetase activity in low-density cultures of HT29m cells. However, swainsonine alone did not stimulate 2',5'-oligo(A) synthetase activity in vivo or in vitro, indicating that the antiproliferative effect of swainsonine is independent of interferon production. The results suggest that in addition to the previously reported antimetastatic activity of swainsonine, the plant alkaloid has antiproliferative activity that is independent from, but additive with, that of interferon in vivo and in vitro.     

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.     

Interferon effects upon the adenocarcinoma 38 and HL-60 cell lines: antiproliferative responses and synergistic interactions with halogenated pyrimidine antimetabolites

In order to gain insight into the mechanisms affecting combination treatment of tumor cells with interferon and halogenated pyrimidine antimetabolites, in vitro studies of murine colon adenocarcinoma 38 and HL-60 cell lines were undertaken. Interferons exhibited modest antiproliferative effects against these lines with DNA synthesis inhibited greater than RNA greater than protein synthesis, as studied by 3H-precursor incorporation. The adenocarcinoma cell line was considerably more sensitive to recombinant gamma-than to purified alpha/beta-interferon, while HL-60 was slightly more sensitive, in short term studies, to antiproliferative effects of recombinant alpha- than to gamma-interferon. Interferon treatment was further associated with suppression of a hyperdiploid component of the adenocarcinoma cell line, as detected by flow cytometry. Combination treatment of the adenocarcinoma cell line with interferon and halogenated pyrimidines, under 4-day continuous exposure conditions, revealed significant synergy for growth inhibition with gamma- much greater than alpha/beta-interferon and with 5-fluorodeoxyuridine greater than 5-fluorouracil much greater than 5-fluorouridine. Thus, synergy was much greater with the more antiproliferative interferon and with the antimetabolite derivative most likely to lead to thymidylate synthetase inhibition rather than RNA incorporation. Sequential 2-day + 2-day treatment revealed greater synergy when interferon preceded 5-fluorouracil or 5-fluorodeoxyuridine rather than the reverse protocol. The synergy of gamma-interferon and 5-fluorodeoxyuridine could be blocked by thymidine, which bypasses inhibition of thymidylate synthetase. HL-60 also exhibited thymidine antagonized synergistic growth-inhibitory effects of interferon and 5-fluorouracil. Analysis of this interaction by [3H]thymidine incorporation, which can reflect thymidylate synthase inhibition, revealed exaggerated responses of interferon-treated cells to either 5-fluorouracil or 5-fluorodeoxyuridine. These results indicate that interferon-halogenated pyrimidine antimetabolite synergistic interactions may be common to several cell types. Evidence is further presented for a mechanism of synergy entailing enhanced thymidylate synthetase inhibition by the antimetabolite of interferon-treated cells.     

Combined effect of interferons alpha, beta and gamma on tumor growth in vitro

The antiproliferative effect of human recombinant interferon alpha, beta and gamma was studied in vitro in combination. Growth of human gastric cancer cell lines and a pancreatic cancer cell line was inhibited markedly by combination of interferons beta and gamma or a combination of all three interferon types. Combination of low-dose interferons (50-100 units/ml) was more effective than a high dose of single interferon. Moreover, human pancreatic cancer cells were completely eliminated after incubation with 5-fluorouracil (0.001 microgram/ml) or methotrexate (0.1 microgram/ml) combined with interferons beta and gamma (50 units/ml each). These results suggested that combination of interferons beta and gamma could be the most effective interferon therapy for cancer.     

Effects of recombinant human interferon-alpha, beta and gamma on the antiproliferative activity of cytarabine in K562 human myeloid leukemia clonogenic cells.

The effects of recombinant human interferon-alpha, beta and gamma (IFN) on the antiproliferative activity of cytarabine (ara-C) in K562 human myeloid leukemia clonogenic cells were studied in an agar capillary microassay. The addition of IFN-alpha did not affect the antiproliferative activity of ara-C in K562 cultures treated with low concentrations of ara-C: 10-50 nM, whereas in those treated with high concentrations of ara-C: 100-500 nM, IFN-alpha significantly reduced the antiproliferative activity of ara-C. The addition of 3 x 10(4) U/ml IFN-alpha to ara-C-treated K562 cultures increased the IC50 of ara-C on day 5 from 102 to 214 nM, i.e. ara-C+IFN-alpha was about twofold less potent than ara-C alone. Low concentrations of IFN-beta and IFN-gamma did not affect the antiproliferative activity of ara-C on K562 colonies, but high concentrations of these two interferons reduced the antiproliferative activity of ara-C. The addition of 4 x 10(3) U/ml IFN-beta or 10(4) U/ml IFN-gamma increased the IC50 of ara-C on day 5 to 304 nM or to 316 nM, respectively, i.e. ara-C+IFN-beta or IFN-gamma was about threefold less potent than ara-C alone. The significant reduction of the desired antiproliferative activity of ara-C by the three interferons was reproduced in liquid suspension cultures of K562 cells on day 4 in the following order: IFN-gamma greater than IFN-beta greater than IFN-alpha. The present negative interactions of the three interferons with ara-C particularly at high concentrations, may caution against the clinical use of the combination of ara-C and interferon in the treatment of myeloid leukemia patients.