tretinoin and afimoxifene

The sodium iodide symporter (NIS) mediates active iodide uptake in lactating breast tissue, and when its levels are enhanced by all-trans retinoic acid (atRA), NIS has been proposed as a target for the imaging and radiotherapy of breast cancer. Importantly, the estrogen receptor α (ERα) is an important regulator of atRA induced NIS gene expression in breast cancer cells. In this study, we investigated the effect of an ER agonist (17β-estradiol, E(2)) or antagonist [trans-hydroxytamoxifen (TOT) or raloxifene (RAL)] treatment on the regulation of NIS gene expression and iodide uptake in an ERα-positive breast cancer (MCF-7) model.. NIS functional activity was measured in vitro by (125)I uptake assay after incubation with E(2) (from 10(-15) to 10(-5) M), TOT (from 5×10(-8) to 5×10(-6) M), or RAL (from 5×10(-8) to 5×10(-6) M) in the presence or absence of atRA (10(-7) M). Under the same conditions, NIS mRNA expression was examined by reverse transcriptase polymerase chain reaction. Athymic mice with MCF-7 xenograft tumors were treated with atRA alone or atRA together with E(2) to evaluate the change of (125)I uptake in tumor tissues in vivo.. In the iodide uptake study in cells, E(2), TOT, or RAL treatment alone did not stimulate (125)I uptake. However, when iodide uptake was stimulated by atRA, cotreatment with E(2), TOT or RAL decreased (125)I uptake in a concentration-dependent manner. The hormone effects on NIS mRNA expression levels in MCF-7 cells were similar. The results of the in vivo biodistribution study showed that (125)I uptake was reduced 50% in tumor tissues of mice treated with atRA/E(2) as compared to tumors treated only with atRA.. Our results suggest that combination treatment of atRA and ER ligands could limit the functional activity of the NIS gene induced by atRA, thereby compromising its use as a target for diagnosis or radiotherapy in breast cancer.

Topics: Animals; Biological Transport; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Interactions; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Ligands; Mice; Mice, Inbred BALB C; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; Symporters; Tamoxifen; Tretinoin; Xenograft Model Antitumor Assays

The anti-estrogen 4-hydroxytamoxifen (TAM) and vitamin A-related compounds, the retinoids, in combination act synergistically to inhibit growth of breast cancer cells in vitro and in vivo. To clarify the mechanism of this synergism, the effect of TAM and all trans-retinoic acid (AT) on proliferation of MCF-7 breast cancer cells was studied in vitro. TAM and AT acted synergistically to cause a time-dependent and dose-dependent inhibition of MCF-7 cell growth. In a temporally related manner, TAM+AT acted synergistically to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis. TAM and AT each blocked cell cycle progression throughout 7 days of treatment but without any synergistic or additive effect on this process, indicating a selective synergism for apoptosis. The negative growth factor-transforming growth factor beta (TGFbeta) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM+AT on apoptosis. TAM+AT acted synergistically to induce a fivefold increase in TGFbeta1 secretion over 72 h. TGFbeta1 alone had no apoptotic effects on these cells; however, TGFbeta1 in combination with AT acted synergistically to inhibit growth, to downregulate Bcl-2 mRNA and Bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner comparable with that noted for TAM+AT. The synergism of both TAM+AT and TGFbeta1+AT for apoptosis was suppressed by estradiol. Co-incubation of TAM+AT with anti-TGFbeta antibody did not block down-regulation of Bcl-2 protein expression or stimulation of apoptosis. The synergistic effects of TAM+AT on apoptosis therefore occur independently of TGFbeta, although TGFbeta may interact with AT in a novel manner to provide another important anti-proliferative mechanism for breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Estrogen Receptor Modulators; Female; Humans; Stimulation, Chemical; Tamoxifen; Time Factors; Transforming Growth Factor beta; Tretinoin

To gain insight into the molecular regulation of the human vitamin D3 receptor (hVDR), we have cloned and sequenced the 5' flanking region of exon 1c and examined promoter activity of this region in breast cancer cells. Sequence analysis of the first 1300 bp upstream of exon 1c reveals several characteristics of a class II promoter, including GC-rich regions and the presence of a TATA box at -29 bp. Putative transcription factor binding sites identified in this potential hVDR promoter include AP-2, Sp-1, and glucocorticoid response elements. No consensus vitamin D3 (VDRE) or estrogen (ERE) responsive elements were identified in the promoter sequence. Primer extension analysis performed with a primer specific for exon 1c confirms that transcription initiated in the 5' flanking region of exon 1c occurs in MCF-7 cells. Transient transfection of MCF-7 cells with this putative promoter region cloned into the pRLnull luciferase reporter vector generates significant reporter gene activity that is enhanced by treatment with forskolin, retinoic acid, and 17beta-estradiol. The enhancement of exon 1c promoter activity by 17beta-estradiol is blocked by the selective estrogen response modifier (SERM) tamoxifen and is not observed in estrogen receptor-negative breast cancer cells. In summary, we have cloned and characterized a TATA containing promoter upstream of exon 1c of the hVDR and provide evidence that this region represents a hormonally regulated hVDR promoter.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Cholecalciferol; Colforsin; Estradiol; Estrogen Antagonists; Estrogens; Exons; Gene Expression Regulation; Hormones; Humans; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Calcitriol; Response Elements; Tamoxifen; TATA Box; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.

Topics: Animals; Apoptosis; CD11 Antigens; Cell Aggregation; Cell Differentiation; Cell Size; DNA Replication; DNA, Complementary; Enzyme Activation; Estradiol; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Integrins; Interleukin-3; Leukemia, Erythroblastic, Acute; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Inbred DBA; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myeloid Progenitor Cells; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein Subunits; Proto-Oncogene Proteins c-raf; Receptors, Cytokine; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured

Although retinoic acid has been shown to inhibit proliferation in human breast cancer cells, the mechanisms by which these effects are mediated are not known. Since several steroid hormones and their synthetic antagonists also inhibit proliferation of human breast cancer cells, we investigated the interactions between retinoic acid, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and antioestrogens in the control of human breast cancer cell proliferation in vitro. When T-47D cells, the most sensitive of six human breast cancer cell lines to the growth inhibitory effects of retinoic acid, were treated with retinoic acid and 1,25-(OH)2D3, a synergistic inhibitory effect on cell growth was observed. Retinoic acid also enhanced the growth inhibitory effect of various antioestrogens (4-hydroxytamoxifen, 4-hydroxyclomiphene or LY117018). However, retinoic acid did not affect oestradiol-induced growth stimulation. Measurement of the cellular receptors for 1,25-(OH)2D3 and oestrogen revealed no significant change in receptor levels following treatment with concentrations of retinoic acid which modulated growth. These results indicate that retinoic acid not only has direct growth inhibitory effects on breast cancer cell proliferation but also augments the effects of some other known regulators of breast cancer cell replication including 1,25-(OH)2D3 and antioestrogens. Synergism appears to involve interactions with steroid hormone action distinct from changes in steroid hormone receptor levels.

Topics: Breast Neoplasms; Calcitriol; Cell Division; Cell Line; Clomiphene; Drug Synergism; Estradiol; Estrogen Antagonists; Female; Humans; Kinetics; Pyrrolidines; Receptors, Calcitriol; Receptors, Estradiol; Receptors, Steroid; Tamoxifen; Thiophenes; Tretinoin

The ability of all-trans-retinoic acid, 13-cis-retinoic acid, the free acid of etretinate (RO 10-1670), the 'arotinoid' RO 13-6298 and its free acid RO 13-7410 to affect the growth of T47D human breast cancer cells in vitro was investigated. The growth of T47D cells was inhibited by all of the retinoids tested, with the arotinoids being up to 100 times more effective than all-trans-retinoic acid. The presence of cellular retinoic acid binding protein (cRABP) was indicated by the cellular uptake of [3H]all-trans-retinoic acid. Maximum binding was 460 fmol/micrograms DNA. All of the retinoids with a polar terminal free carboxyl group readily competed for the binding sites, but none of the retinoids competed for the estrogen or progesterone receptor. Co-treatment of the T47D cells with 0.1 microM all-trans-retinoic acid and either tamoxifen (1 microM) or hydroxytamoxifen (10 nM or 0.1 microM) produced an additive effect on growth inhibition. No such additive effect was observed when T47D cells were co-treated with arotinoids and antiestrogens. The results showed that the T47D cells can serve as a useful model in vitro to test the effects of the synthetic retinoids and antiestrogens on steroid receptor-positive human breast cancer.

Topics: Binding, Competitive; Breast Neoplasms; Carrier Proteins; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Estrogen Antagonists; Female; Humans; Receptors, Retinoic Acid; Retinoids; Tamoxifen; Time Factors; Tretinoin

Using established breast cancer cell lines in a cell culture model we studied the growth effect of retinoic acid (RA) alone or in combination with the antiestrogen 4-hydroxytamoxifen (OHT). Cytoplasmic 3HRA binding sites were determined by sucrose density gradient centrifugation analysis. Of the three cell lines Hs578T, BT 20, and 734 B only the last showed a significant amount of specific RA binding (10(5) sites/cell). This cell line showed a dose dependent decrease in proliferation after a long-term incubation with RA whereas the 3H-thymidine uptake was highly significantly increased after incubation with 10(-6)M of RA for 20 hr. Growth inhibition was not further increased by the addition of OHT (10(-6) M), but the increase in thymidine incorporation due to RA was neutralized by OHT. Hs578T and BT 20 cells were not affected by any of the treatments. The different action of RA on proliferation and thymidine incorporation suggests a cell cycle specific mechanism.

Topics: Breast Neoplasms; Carrier Proteins; Cell Division; Cell Line; Cytosol; Estrogen Antagonists; Female; Humans; Neoplasm Proteins; Receptors, Retinoic Acid; Tamoxifen; Thymidine; Tretinoin