Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Antitumor effects of combination toremifene and medroxyprogesterone acetate (MPA) in vitro and in vivo

The estrogen (ER) and progesterone (PgR) receptor levels in various gynecological tumors were measured. The same tumors were exposed in vitro to toremifene, MPA or their combination and the growth of the tumors was followed by measuring the adenosine triphosphate (ATP) within the cells by a simple bioluminescence assay. Altogether 34 clinical samples were studied. DMBA-induced mammary tumors bearing rats were treated in vivo with toremifene, MPA and their combination. About half of the ovarian cancers and 6 out of the 7 adenocarcinomas of uteri contained ER. The ovarian tumors were PgR rich in 25% and adenocarcinomas of uteri in 6 out of the 7 cases. When compared to control toremifene (concentration 1 mumol/l) was able to decrease the number of living cells to 50% or less in 9/34 samples, MPA (concentration 10 mumol/l) in 17/34 samples, and the combination in 25/34 samples. In five cases the antitumor effect of the combination was synergistic. In two cases signs of weak antagonism were seen. In vivo the antitumor effect of toremifene and MPA was clearly synergistic against DMBA-induced cancers. The effect was dose-dependent and at sufficiently high doses it was possible to eradicate the tumors and cure the animals.     

Review of the pharmacological properties of toremifene

New compounds were synthesized with the aim to develop new anti-estrogenic antitumor drugs. The biological properties of the molecules were screened by (1) estrogen receptor (ER) binding, (2) effect on MCF-7 cells, (3) uterotrophic effect and inhibition of estradiol induced uterotropic effect and (4) antitumor effect in DMBA induced rat mammary cancer. One of the molecules, Fc-1157a = toremifene, exhibited the following characteristics: competitive inhibition of [3H]estradiol binding to ER (IC50 = 0.3 mumol/l), inhibition of MCF-7 cell growth in a concentration-dependent manner and cell-killing effect at higher than 3 mumol/l concentrations. Minimal estrogenic dose of toremifene on rat uterus weight was about 40 times higher than that of tamoxifen. Toremifene had statistically significant effect against DMBA-induced rat mammary cancer. Further screening consisted of antitumor, pharmacokinetic and safety studies. Toremifene inhibited the growth of ER-negative, glucocorticoid sensitive, mouse uterine sarcoma in a dose-dependent manner. Pharmacokinetics and metabolism of toremifene resembled closely those of tamoxifen, but since the chlorine atom of the toremifene molecule was not metabolically cleaved tamoxifen and toremifene did not have chemically similar metabolites. Toremifene was well tolerated in animal toxicity studies. No hyperplastic or neoplastic nodules, which were seen in almost all high-dose (48 mg/kg for 24 weeks) tamoxifen-treated rats, were found in toremifene-treated rats (dose 48 mg/kg). In clinical phase I studies in healthy voluntary postmenopausal women, no side effects were reported, at doses less than or equal to 460 mg, neither after a single dose nor after five daily doses. At the dose of 680 mg two out of five persons experienced vertigo and headache. Toremifene, at the dose of 68 mg daily, had antiestrogenic effect on estradiol-induced human vaginal epithelial cells. Clinical phase II studies have confirmed that toremifene has a promising antitumor effect.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

Inhibition of fibroblast chemotaxis by recombinant human interferon gamma and interferon alpha

Interferons have recently been recognized as potent mediators in inflammatory processes, exerting profound effects on fibroblasts. The influence of interferons gamma and alpha on the chemotactic movement of fibroblasts toward various attractants was, therefore, investigated. Normal human adult and embryonal dermal fibroblasts, fibrosarcoma-derived fibroblasts and SV40-transformed fibroblasts were tested against conditioned medium from fibroblasts, the chemotactic peptide C-140 of fibronectin, platelet-derived growth factor, and leukotriene B4 as attractants in the presence or absence of the interferons. Interferons gamma and alpha inhibited chemotaxis in a dose-dependent manner and at concentrations at least as low as 10(-2) ng/ml. Inhibition was noticeable when the cells were exposed to interferon for as short a period as 60 minutes, and the effect was not readily reversible. Inhibition occurred when the cells came from sparse or dense cultures, but when platelet-derived growth factor was the attractant and the cells had been grown at low density there was no inhibition. It is concluded that this is a specific effect, not to be wholly explained by overall increase in membrane rigidity. Inhibition of fibroblast chemotaxis by interferons may be an important regulatory mechanism during wound healing or fibrosis and metastatic spread of tumor cells.     

Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.     

In vitro treatment of HEp-2 cells with human tumor necrosis factor-alpha and human interferons reduces invasiveness of Salmonella typhimurium

Enteroinvasive bacteria, like Salmonella typhimurium, can be internalized in in vitro cultured epithelioidal cells, like HEp-2 cells. This phenomenon is inhibited by pretreatment of cells with human tumor necrosis factor alpha (TNF-alpha) in a dose- and time-dependent manner. The effect was also reproduced in other cell types, including diploid embryo fibroblast cells. The TNF-alpha effect was neutralized by anti-TNF-alpha antibodies. No synergistic effect was produced by combinations of TNF-alpha with either interferon alpha or gamma. Unlike the effects of interferons, TNF-alpha inhibited the invasiveness of Shigella flexneri and the TNF-alpha effect was not inhibited by cycloheximide.     

The synergistic influence of human interferon-gamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with the enhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro

The influences of human interferons--natural gamma (2 X 10(7) NIH reference U/mg), recombinant gamma (approximately 5 X 10(6) U/mg), natural alpha (1.4 X 10(8) international reference U/mg), and natural beta (10(6) international reference U/mg)--were evaluated alone or in combination for their effects in vitro on colony formation by low density human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in normal incubator (approximately 20%) O2 tension or low (5%) O2 tension. Alone, these interferons demonstrated the same dose response inhibitory curves, as we reported previously, when cells were grown at 20% O2. Recombinant IFN-gamma gave the same dose response curve as natural IFN-gamma. Natural or recombinant interferon synergized with IFN-alpha to suppress colony formation at concentrations that were approximately 2 log units lower than that required by either interferon alone. Equal concentrations of these interferons were not needed for the synergistic effect and were still apparent when one was present at concentrations of 2 log units less than the other. IFN-gamma synergized to a lesser extent with IFN-beta, but IFN-alpha did not synergize with IFN-beta. Cells grown at 5% O2 were more sensitive to inhibition by 2 log units less IFN-gamma or IFN-alpha, and this effect was additive with the synergistic effects of IFN-gamma and IFN-alpha together. These results may have physiological, pathological, and/or clinical relevance.     

Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.     

Beta and gamma interferons act synergistically to produce an antiviral state in cells resistant to both interferons individually.

We showed previously that the mouse fibroblastoid cell line Ltk-aprt- is resistant to the antiviral effects of beta interferon. This lack of response reflects a partial sensitivity to the interferon that is accompanied by a failure to activate expression of several interferon-regulated genes, although certain other genes respond in a normal manner. We show here that Ltk-aprt- cells were also unable to establish an antiviral state and to activate expression of 2,5-oligo(A) synthetase when treated with gamma interferon. Strikingly, however, treatment with a combination of beta interferon and gamma interferon provided complete protection against viral replication. Although the cells were completely insensitive to up to 250 U of the interferons per ml added singly, essentially complete protection from viral cytopathic effects was achieved when as little as 10 U of each of the interferons per ml were combined. Expression of 2,5-oligo(A) synthetase was also sensitive to this synergistic effect. Activation of an antiviral state could also be achieved by sequential treatment, first with gamma interferon and then with beta interferon. Partial protection against viral replication could be achieved by pretreatment with gamma interferon for as little as 1 h before incubation with beta interferon and could be blocked by the addition of specific antibodies or by cycloheximide, indicating that gamma interferon induces the synthesis of a protein which can act synergistically with a signal produced by the beta-interferon receptor. We suggest that Ltk-aprt- cells suffer from defects in one or more components of the gene activation pathways for both type I and type II interferons. Nonetheless, gamma interferon is able to activate the expression of a gene encoding a protein required for signal transduction. This protein acts synergistically with a transient signal produced in response to beta interferon, thereby activating the expression of a further group of genes.     

Interferons in the management of neuroendocrine tumors and their possible mechanism of action.

Alpha interferons at doses of 3-9 MU subcutaneously, three to seven times/week, have been administered to 32 patients with malignant endocrine pancreatic tumors. The objective biochemical response rate was 63 percent with a median duration of 20.5 months. Significant reduction of tumor size was only noticed in 20 percent of the patients. Alpha interferon administered to 111 patients with malignant carcinoid tumors showed objective biochemical responses in 42 percent of the patients with a median duration of 32 months. Another 39 percent of the patients showed stabilization of disease without any further tumor growth. Subjective improvement was noticed in 70 percent of the patients. When survival data are analyzed in patients with malignant carcinoid tumors, the median survival from start of treatment was 80+ months in the group of patients treated with alpha interferon, which should be compared with only eight months in a historical group treated with chemotherapy (streptozotocin plus 5-fluorouracil). The adverse reactions to alpha-interferon treatment are dose-dependent and include, mainly, flu-like symptoms, fatigue, and low-grade weight loss. Autoimmune reactions are noted in about 20 percent of the patients. Patients treated with recombinant alpha interferons might develop neutralizing interferon antibodies (6-27 percent), which abrogate the anti-tumor response. The anti-tumor effect in neuroendocrine tumors includes anti-proliferation, apoptosis, differentiations, and cytotoxic/cytostatic effects. Furthermore, immunomodulation is obtained by increased expression of class I antigens on tumor cells. Four patients also developed antibodies directed against carcinoid tumor cells. Alpha interferons induce several nuclear enzymes such as 2'-5'-A synthetase, p-68 kinase, and Mx-A proteins, which are involved in a downregulation of expression of growth factors, oncogenes, and peptide hormones, leading to anti-proliferation and/or apoptosis. The response to alpha-interferon treatment might be predicted by analysis of the induction of 2'-5'-A synthetase in samples from neuroendocrine tumors. Stimulatory tests of hormone secretion, such as meal stimulation of pancreatic polypeptide secretion or secretin test, clearly demonstrate a normalization during alpha-interferon treatment, which might depend on reduced peptide production and/or secretion but also on eradication of malignant cell clones. In summary, alpha interferons have demonstrated significant anti-tumor effects in patients with malignant neuroendocrine gut and pancreatic tumors. The adverse reactions are dose-dependent and manageable. The anti-tumor effects of alpha interferons are pleiotropic and include several direct effects on tumor cells but also immunomodulation.     

Toremifene-atamestane; alone or in combination: predictions from the preclinical intratumoral aromatase model

Since most breast cancers occur in postmenopausal women and are hormone dependent, we developed a model system that mimics this situation. In this model, tumors of human estrogen receptor (ER) positive breast cancer cells stably transfected with aromatase (Ac-1) are grown in immune-compromised mice. Using this model we have explored a number of therapeutic strategies to maximize the antitumor efficacy of antiestrogens (AEs) and aromatase inhibitors (AIs). This intratumoral aromatase xenograft model has proved accurate in predicting the outcome of several clinical trials. In this current study we compared the effect of an AE toremifene and steroidal AI atamestane, alone or in combination, on growth of hormone-dependent human breast cancer. We have also compared toremifene plus atamestane combination with tamoxifen in this study. The growth of Ac-1 cells was inhibited by tamoxifen, toremifene and atamestane in vitro with IC(50) values of 1.8+/-1.3 microM, 1+/-0.3 microM and 60.4+/-17.2 microM, respectively. The combination of toremifene plus atamestane was found to be better than toremifene or atamestane alone in vitro. The effect of this combination was then studied in vivo using Ac-1 xenografts grown in ovariectomized female SCID mice. The mice were injected with toremifene (1000 microg/day), atamestane (1000 microg/day), tamoxifen (100 microg/day), or the combination of toremifene plus atamestane. In this study, our results indicate that the combination of toremifene plus atamestane was as effective as toremifene or tamoxifen alone but may not provide any additional benefit over toremifene alone or tamoxifen alone.     

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.