Tamoxifen-treated Cells Research Articles

The effect of high dehydroepiandrosterone sulfate levels on tamoxifen blockade and breast cancer progression

Background We investigated the stimulatory potential of dehydroepiandrosterone sulfate (DHEA-S) on tamoxifen-treated cells and assessed its effect on cancer progression in the adjuvant setting. Methods Mean serial serum levels of sex hormones from 44 patients receiving tamoxifen were correlated with follow-up status. T-47D (ER+/PR+) and HCC1937 (ER−/PR−) breast cancer cells were pretreated with 100 μM anastrozole, with or without tamoxifen, and stimulated with 22.8 μM DHEA-S. Rapid colorimetric assays allowed calculation of growth percent change. Results Clinically, development of metastatic disease correlated only with ≥90 μg/dL DHEA-S ( P = 0.005). In vitro, T-47D cells stimulated with DHEA-S after anastrozole showed 35% increased growth. Addition of 0.01 nM tamoxifen demonstrated −7% inhibition. Increasing the DHEA-S/tamoxifen ratio reversed suppression to +25%. Conclusions DHEA-S ≥90 μg/dL is a risk factor for recurrence in the adjuvant setting. In vitro, although tamoxifen inhibits cell growth at high doses it can be circumvented by DHEA-S. These results indicate that DHEA-S contributes to tamoxifen resistance and disease progression.

Changes in the expression and binding properties of the estrogen receptor in MCF-7 breast cancer cells during growth inhibition by tamoxifen and cisplatin.

Most mammary carcinomas contain estrogen receptors (ER), which are an important factor in diagnosis and prognosis, and in deciding on the type of therapy. ER-positive tumors are most commonly treated with the antiestrogen tamoxifen or with a combination of chemotherapeutic drugs. An important aspect for further treatment and anticipating possible side effects is the fate of the ER during the course of therapy. To study the effect of drug-induced growth inhibition on ER expression and binding properties. human breast cancer MCF-7 cells were treated with tamoxifen and cisplatin. and also estradiol (E2) for 5 days. Following this incubation, intact cells were incubated with [3H]E2 to determine the dissociation constant (KD) and maximal number of binding sites (Bmax) of the ER. The amount of ER protein per cell was quantified using anti-ER antibodies. For analysis of ER mRNA, total cellular RNA was subjected to Northern blotting. The 5-day treatment with E2 reduced Bmax and the amount of ER protein by about 70%, while the cellular level of ER mRNA was reduced by 40%. Treatment with E2 did not affect the subsequent growth inhibitory response to tamoxifen or cisplatin. In contrast, tamoxifen reduced the capacity for E2 binding; it caused about a 30-fold increase in the KD value. At the same time, Bmax and ER protein content were increased (about 3.5- and 2-fold, respectively), but the cellular level of ER mRNA was again reduced by 40%. The growth of tamoxifen-treated cells remained sensitive to subsequent treatment with estradiol, tamoxifen or cisplatin. Treatment of MCF-7 cells with cisplatin likewise reduced E2 binding due to a 20-fold increase in KD value. In this case, both Bmax and the amount of ER protein were decreased when calculated per milligram of protein, but were increased on a cellular basis due to an increase in cell size. The ER mRNA content was not altered in cisplatin-treated cells. Growth of these cells also remained sensitive to tamoxifen and cisplatin. In conclusion, drug-induced changes in ER expression and binding capacity do not necessarily indicate a loss of sensitivity of breast cancer cells to a subsequent chemotherapeutic treatment.

Protective action of 17β-estradiol and tamoxifen on glutamate toxicity in glial cells

Estrogens influence differentiation, growth and function of neurons, but less is known of their effects on glia. In our experiments reported here, the ovarian steroid, 17β-estradiol, and the “designer”, non-steroidal estrogen, tamoxifen, effectively protected C-6 glioma 2B clone cells from the cytotoxicity of the excitatory neurotransmitter, glutamate. Exposure of these cells to 10–20 mM glutamate induced 61–78% cell death. Pre-treatment of the cells with 0.01 mM estradiol or with 2 μM tamoxifen significantly reduced the glutamate-induced cell death, estradiol being the most effective in this regard. Estradiol- or tamoxifen-treated cells that had survived glutamate damage appeared more mature than controls. Thus, estrogens often used in therapy (estradiol as replacement after menopause and tamoxifen for treatment/prevention of breast cancer) may significantly protect glial cells against glutamate toxicity and stimulate cell differentiation.

Reduction of equilibrative nitrobenzylthioinosine-sensitive nucleoside transporter in tamoxifen-treated MCF-7 cells: an oestrogen-reversible phenomenon.

MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 microM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000+/-18000 to 136000+/-25000 sites per cell (mean+/-S.E.M.; n=5; P<0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37+/-0.05 to 0.45+/-0.08 nM (n=5; P>0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7+/-0.3 (n=8) to 8.4 +/- 2.2 microM/s (n=4; P < 0.05), whereas the Vmax of the ei component [from 4.7 +/- 0.5 (n=8) to 5.8 +/- 1.6 microM/s (n=4; P > 0.05)] and Km values for both components [es from 460 +/- 80 to 630 +/- 280 microM (n>/=4; P > 0.05) and ei from 355 +/- 115 to 440 +/- 220 microM (n>/=4; P > 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.

Tamoxifen enhances interferon-regulated gene expression in breast cancer cells.

The molecular basis for the enhanced growth inhibition of MCF-7 human breast cancer xenografts by a combination of human interferon-beta (IFN-beta) and tamoxifen was investigated. Treatment of MCF-7, MDA-MB-231, and BT-20 cells with the combination of IFN-beta and tamoxifen resulted in enhanced antiproliferative effects in vitro. Treatment with the combination of IFN-beta and tamoxifen enhanced the expression of several IFN-beta-inducible genes in human breast carcinoma cell lines relative to levels induced by IFN-beta alone. Tamoxifen alone did not induce transcription of IFN-stimulated genes (ISGs). Augmentation of ISG expression by the combination of IFN-beta and tamoxifen was noted in breast tumor cell lines irrespective of their functional estrogen receptor (ER) status or their dependence on estradiol for growth, suggesting that upregulation of ISGs was independent of ER status. Enhancement of IFN-stimulated gene expression by tamoxifen occurred at the transcriptional level. Expression of transfected reporter genes under the control of IFN-alpha/beta regulated promoters was also enhanced in IFN-beta and tamoxifen-treated cells. Similarly, transcriptional induction of chimeric reporter plasmids driven by an IFN-gamma inducible promoter (GAS; IFN-gamma activated site) was also enhanced by the combination of IFN-gamma and tamoxifen. In tamoxifen treated cells, IFN-beta and IFN-gamma readily activated transcription factors ISGF-3 and GAF, respectively. Therefore, augmentation of ISG expression by tamoxifen is an early event in the antitumoral activity of this drug combination.

Tamoxifen inhibits nitrobenzylthioinosine-sensitive equilibrative uridine transport in human MCF-7 breast cancer cells.

Tamoxifen inhibits the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to human MCF-7 breast cancer cells with an IC50 of 8 microM. Tamoxifen at 30 microM changed the apparent Kd for [3H]NBMPR binding from 0.63 +/- 0.12 to 4.75 +/- 0.58 nM, with little effect on the Bmax (311000 +/- 76000 and 263000 +/- 46000 sites per cell for untreated and tamoxifen-treated cells respectively). Corresponding to this decrease in binding of [3H]NBMPR in the presence of tamoxifen was an inhibition of NBMPR-sensitive equilibrative transport of 50 microM [3H]uridine (IC50 7-10 microM). In the presence of 15 microM tamoxifen, the apparent K(m) for [3H]uridine transport was increased from 390 +/- 30 to 1500 +/- 250 microM, with no change in Vmax (12.0 +/- 0.1 and 11.3 +/- 4.3 microM/s for untreated and tamoxifen-treated cells respectively). The inhibitory effect of tamoxifen on NBMPR-sensitive equilibrative uridine transport was specific, as similar results were also observed in HL-60 leukaemia and EL4 lymphoma cells. Furthermore a similar concentration of tamoxifen had no effect on the NBMPR-insensitive equilibrative transport of uridine in MCF-7, HL-60 and Morris 7777 hepatoma cells, and on the Na(+)-dependent transport of uridine in murine splenocytes. In this paper we demonstrate that tamoxifen by itself might have some antiproliferative effects through inhibition of DNA synthesis by blocking the nucleoside salvage pathway.

901–40 Gender-related Differences in Smooth Muscle Cell Proliferation in Response to Estradiol and Dihydrotestosterone

The cardioprotective effect of estradiol (E 2 ) is strongly supported by the reduced incidence of coronary artery disease (CAD) observed in premenopausal females versus age-matched males and in postmenopausal females on estrogen therapy. The favorable effect of estrogen on lipid profiles does not fully account for the observed cardiac benefits in females versus males. We have previously demonstrated that physiologic concentrations of E 2 inhibit female pig coronary vascular smooth muscle cells (VSMC) in vitro; this may partially account for estrogen's favorable cardiac effects. We determined the effect of sex steroids Idihydrotestosterone (DHT) and E 2 ) and tamoxifen Ian anti-estrogen) on proliferation of coronary VSMC obtained from male pigs. VSMC were cultured in phenol red-free Media 199 with 10% fetal calf serum. Cells obtained from mature adult male pigs were growth arrested in serum-free media for 24 hours, and treated with effectors in 2% charcoalstripped serum at concentrations ranging from 10 -11 to 10 -7 M for 24 hours. Cell proliferation was assessed by tritiated thymidine incorporation corrected for protein content. No significant effect on proliferation was observed in E 2 or tamoxifen-treated cells versus vehicle controls. Proliferation was significantly increased in cells treated with DHT at 10 -11 M. In conclusion, E 2 did not inhibit proliferation of coronary VSMC obtained from intact male animals, in contrast to the inhibition observed previously in VSMC obtained from intact female pigs. This gender-related differential response in vitro to E 2 may partially explain the reduction in CAD in estrogen-replete women compared to men. Tamoxifen had no effect on VSMC proliferation. The increased proliferation of VSMC in response to dihydrotestosterone may playa role in the pathogenesis of CAD in males.

Tamoxifen decreases the rate of proliferation of rat vascular smooth-muscle cells in culture by inducing production of transforming growth factor beta.

Tamoxifen selectively and reversibly decreased the rate of proliferation of adult rat aortic vascular smooth-muscle cells (VSMCs). Half-maximal inhibition of proliferation occurred at 2-5 microM tamoxifen for VSMCs and at > 50 microM for adventitial fibroblasts. The cell cycle time for all the VSMCs in the population was increased from 35 +/- 2 h to 54 +/- 4 h in the presence of 33 microM tamoxifen. Tamoxifen did not affect the time of entry into DNA synthesis, but delayed arrival at mitosis by > 24 h. It therefore extended the duration of the G2-to-M phase of the cell cycle. However, the rate of proliferation of VSMCs was not decreased by tamoxifen (at concentrations up to 50 microM) in the presence of neutralizing antibody to transforming growth factor beta (TGF-beta). The level of mRNA for TGF-beta 1 in VSMCs was strongly induced by 10 microM tamoxifen, and TGF-beta activity in conditioned medium from tamoxifen-treated cells was more than 50-fold higher than from control cells. Tamoxifen therefore extended the G2-to-M phase of the cell cycle in VSMCs by increasing TGF-beta activity in the culture.

Tamoxifen stimulates in vivo growth of drug-resistant estrogen receptor-negative breast cancer.

An estrogen receptor-negative, multidrug-resistant MDA-MB-A1 human breast cancer cell line was grown in culture with and without a noninhibitory concentration (0.5 microM) of tamoxifen for 122 days. Tamoxifen-treated and control cells were inoculated into opposite flanks of nine nude mice, where they produced measurable tumors in every case. Six of the animals were treated with tamoxifen at 500 micrograms/day for 22 days. Although no inhibitory nor stimulatory effect of tamoxifen was seen in vitro, tamoxifen had a clear tumor-growth-stimulating effect in mice. The most pronounced stimulatory effects were observed in the cells that had been cultured with tamoxifen. Within 3 weeks of the start of tamoxifen therapy, the cells grown in the presence of tamoxifen produced tumors with a mean size of 380 mm2, whereas the cells not pretreated with tamoxifen had tumors of 220 mm2. In contrast, in mice not receiving tamoxifen, the sizes of the tumors were 190 and 140 mm2, respectively. These preliminary results suggest that prolonged in vitro tamoxifen exposure induces cellular changes that result in tumors that are stimulated to grow faster in mice following tamoxifen treatment.

Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: 31P and 13C NMR studies.

Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with 31P and 13C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.

Novel antioestrogens

SynopsisRecently described 7α-alkylamide analogues of oestradiol, exemplified by ICI 164,384, have the pharmacological characteristics of pure antioestrogens. ICI 164,384 is completely free of oestrogenic activity in rodents and blocks the trophic actions of exogenous and endogenous oestradiol and of partial agonist antiooestrogens like tamoxifen. In addition, ICI 164,384 is peripherally selective, producing complete involution of the uterus without affecting LH secretion in intact female rats.In MCF-7 oestrogen-responsive, human breast cancer cells ICI 164,384 is a more potent and effective inhibitor of cell growth than tamoxifen. This difference in efficacy is reflected by a greater reduction in the proportion of cells continuing DNA synthesis in ICI 164,384-treated cultures than in tamoxifen-treated cells. This observation implies that a pure antioestrogen may provide more effective therapy of breast cancer but this will have to be demonstrated in clinical studies.

Isolation and characterization of a tamoxifen-resistant cell line derived from MCF-7 human breast cancer cells.

A tamoxifen-resistant variant (R27) was selected by cloning wild type MCF-7 cells. R27 has the same growth rate as MCF-7 under optimal conditions. But tamoxifen has no effect on the growth rate (growth constant of R27 under control condition, 0.071 +/- 0.007 day-1; tamoxifen-treated cells, 0.073 +/- 0.011 day-1) and minimal effects on thymidine incorporation in R27, whereas tamoxifen has a strongly suppressive effect on the growth rate and thymidine incorporation in MCF-7. The estradiol-mediated stimulation of growth in R27 is somewhat less than in MCF-7 (growth constants of R27 and MCF-7 are 0.112 +/- 0.015 and 0.125 +/- 0.009 day-1, respectively. R27 contains unoccupied receptors in both cytoplasmic and crude nuclear extract fractions with slightly higher numbers of cytosol receptors than wild type MCF-7. The same dissociation constant and the same molecular weight estimated by Sephadex chromatography and the same sucrose density behavior were observed in R27 and MCF-7. Extent of competition with estradiol and tamoxifen for cytoplasmic estrogen receptor is the same for R27 and MCF-7. Induction of progesterone receptor following treatment with estradiol is the same for R27 and MCF-7. Estrogen receptor is activated by salt and nucleotide in both MCF-7 and R27; however, the extent of activation is much higher in MCF-7 than R27.