The antiproliferative effects of Uncaria tomentosa extracts and fractions on the growth of breast cancer cell line.

Uncaria tomentosa, also known as "Uña de gato", is a Rubiaceae species widely used in South-American folk medicine for the treatment of cancer, arthritis, gastritis and epidemic diseases. Extracts of the plant have been shown to possess cytostatic and anti-inflammatory activity as well as mutagenic and antimutagenic properties. However, to date no studies have been carried out to verify the direct antitumor activity of the extracts. The present study investigates the effects of some extracts and their chromatographic fractions from the bark of U. tomentosa on the growth of a human breast cancer cell line (MCF7). Our data indicated that, in addition to the antimutagenic activity, U. tomentosa extracts and fractions exert a direct antiproliferative activity on MCF7. The bioassay-directed fractionation from barks and leaves resulted in the isolation of two active fractions, which displayed an IC50 of 10 mg/ml and 20 mg/ml, respectively and an antiproliferative effect, with about 90% of inhibition at a concentration of 100 mg/ml.

Genistein blocks breast cancer cells in the G(2)M phase of the cell cycle.

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 microM genistein showed a dose-dependent accumulation in the G(2)M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G(2)M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G(2)M arrest was coupled with a parallel depletion of the G(0)/G(1) phase. To understand the mechanism of action underlying the block in G(2)M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G(2)-->M transition. As expected, p34(cdc-2) expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34(cdc-2) catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 microM genistein, cyclin B(1) (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G(2)M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34(cdc-2)/cyclin B(1) complex only in all of the studied cell lines.

Time-dependent relevance of steroid receptors in breast cancer.

PURPOSE: To analyze the time-dependent prognostic role of the investigated variables, considered, when appropriate, on a continuous scale, for the purpose of evaluating and describing the interrelationships between clinically relevant patient and tumor characteristics (age, size and histology, and estrogen receptor [ER] and progesterone receptor content) and the risk of new disease manifestation. PATIENTS AND METHODS: We applied a flexible statistical model to a case series of 1,793 patients with axillary lymph node-negative breast cancer with a minimal potential follow-up of 10 years. To avoid a potential confounding effect of adjuvant treatment, only patients given local-regional therapy until relapse were considered. RESULTS: ER content and tumor size (adjusted for all the other covariates) showed a time-dependent relationship with the risk of new disease manifestations. In particular, ER content failed to show a prognostic effect within the first years of follow-up; thereafter, a positive association with risk of relapse was observed. For tumor size, within the first years of follow-up, the risk of relapse was directly related to size for only tumors up to 2.5 cm in diameter; thereafter, the impact on prognosis progressively decreased. CONCLUSION: The availability of a long follow-up on a large breast cancer series, as well as the use of innovative statistical approaches, allowed us to explore the functional relation between steroid receptors and clinical outcome and to generate a hypothesis on the involvement of ER in favoring long-term metastasis development.

Relationship between steroid receptors (as continuous variables) and response to adjuvant treatments in postmenopausal women with node-positive breast cancer.

In current clinical practice for breast cancer patients, estrogen (ER) and progesterone receptor (PgR) concentrations, quantified by the dextran-coated charcoal assay, are categorized by an arbitrary cutoff into a negative or positive status. However, although the results obtained with this approach are easy to interpret, such a representation could oversimplify the relationship between ER and PgR content and patient outcome and imply an assumption of monotonicity, which is generally expected but rarely proven. We evaluated the relationship between ER and PgR content (considered on a continuous scale) and clinical outcome, using a flexible statistical model, in a group of postmenopausal patients with N-positive operable tumors who were submitted to surgery and different adjuvant treatments (tamoxifen or CMF). Univariate analysis indicated that in the tamoxifen-treated group, ER level, number of metastatic nodes (pN) and age, but not PgR, were significant indicators of clinical outcome (p = 0.032, p = 0.021 and p = 0.029, respectively). Multivariate analysis indicated that in this group of patients there was no interaction between variables, and in the final model for disease-free survival (DFS) only ER and pN were retained with an overall predictive ability of the regression model of 0.723, as evaluated by Harrell's c. However, pN markedly contributed to the predictive ability of the model with respect to ER, since a marked decrease in Harrell's c statistic (c = 0.582) was observed when pN was removed from the model. In the CMF-treated group, only pN affected clinical outcome. When the estimated DFS curves obtained from the final Cox regression models were plotted according to four values of ER (in the tamoxifen-treated group) or three values of pN (in the CMF-treated group) we observed that in the tamoxifen-treated group patients with an ER concentration equal to 0 fmol/mg cytosol protein had the worst prognosis, whereas a marked improvement of the expected DFS was observed for patients with a low but detectable ER level (generally classified as ER-negative because falling below the conventional cutoff value of 10 fmol/mg cytosol protein). Our results seem to suggest that the use of steroid receptor concentrations on a continuous scale, instead of dichotomous "status", is to be preferred in the choice of adequate therapeutic strategies.

The two phyto-oestrogens genistein and quercetin exert different effects on oestrogen receptor function.

We compared the oestrogenic and anti-oestrogenic properties of the two well-known phyto-oestrogens, genistein and quercetin, on the oestrogen-sensitive breast cancer cell line MCF-7. Genistein exerted a biphasic effect on growth of MCF-7 cells, stimulating at low and inhibiting at high concentrations, whereas quercetin was only growth inhibitory. At doses which did not inhibit cell growth, respectively 5 and 1 microM, genistein and quercetin counteracted oestrogen- and transforming growth factor-alpha-promoted cell growth stimulation. Furthermore, genistein promoted transcription of the oestrogen-regulated genes pS2 and cathepsin-D, whereas quercetin interfered with the oestrogen-induced expression of the proteins. In in vitro binding experiments, genistein competed with oestradiol for binding to the oestrogen receptor (ER), but quercetin did not. Quercetin and genistein down-regulated cytoplasmic ER levels and promoted a tighter nuclear association of the ER, but only genistein was able to up-regulate progesterone receptor protein levels. In gel mobility assays, ER preincubation with oestradiol or with the two phyto-oestrogens led to the appearance of the same retarded band, excluding differences between the various complexes in binding to the consensus sequence. The data allowed us to conclude that quercetin acts like a pure anti-oestrogen, whereas genistein displays mixed agonist/antagonist properties, and to formulate a hypothesis on the possible mechanism of action of such phyto-oestrogens.

Is steroid receptor profile in contralateral breast cancer a marker of independence of the corresponding primary tumour?

We compared oestrogen receptor (ER) and progesterone receptor (PgR) profiles between primary and corresponding contralateral breast cancer (CBC) to investigate whether CBC should be considered relapse of a primary or as a feature of the multicentric origin of breast cancer. We adjusted for patient age, menopausal status, histology and adjuvant therapy. In spite of the general application of a cut-off value to dichotomise ER and PgR, we considered them as continuous variables. Moreover, we considered as synchronous cancers only simultaneously occurring lesions. For 399 patients, ER and PgR receptor levels in primary and CBC did not differ significantly, but were significantly correlated within the same patient. The correlation was higher for synchronous than for metachronous lesions when considering ER, but not PgR. The correlation between ER and PgR levels in the same tumour (primary or CBC) appeared stronger than the correlation of either receptor type (ER or PgR) between primary and CBC. Age, histology and adjuvant treatment affected ER concentration, whereas age, menopausal status and histology affected PgR concentration. The analysis indicated that primary and CBC tend to be characterised by a similar steroid receptor profile. The finding may support the hypothesis of CBC as a second primary arising in a common predisposing milieu, rather than a primary-dependent contralateral lesion. In this light, the clinical management of patients with a bilateral breast cancer should be similar to that of a unilateral breast cancer.

Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro.

Genistein significantly inhibited cell growth (IC50 around 10 microM) of MCF-7, MDAMB-231 and HBL-100 cell lines, but not of skin-derived fibroblasts and counteracted the growth-stimulatory effects exerted by estradiol and growth factors. It abolished the paracrine stimulation observed in MCF-7 cells in co-culture with MDAMB-231 or fibroblasts. Genistein-treated cells accumulated in the S and G2/M phases of the cell cycle and underwent apoptosis. Genistein decreased tyrosine phosphorylation induced upon treatment with transforming growth factor-alpha. Finally, genistein bound the estrogen receptor (ER) (relative affinity constant Kd = 4 nM), induced pS2 and cathepsin-D transcription and increased nuclear ER levels.

Effect of steroid receptors, pS2 and cathepsin D on the outcome of elderly breast cancer patients: an exploratory investigation.

In 83 elderly breast cancer patients, oestrogen and progesterone receptors (ER, PgR), pS2 and cathepsin D (CathD) were evaluated for their possible prognostic role on disease-free survival (DFS). The biomarkers were determined on the same cytosol by using immunoradiometric assays, and the variables were considered on a continuous scale. Univariate analysis indicated a linear relationship between logarithmic hazard ratio (log(HR)) and the log(ER) and log(PgR) concentration, but a non-linear relationship between log(HR) and CathD. As regards pS2, there was no evidence of a relationship with log(HR). In multivariate analysis, log(ER) content did not have a significant prognostic role, whereas log(PgR) retained a significant prognostic role. As regards the predictive ability, log(PgR) was the best discriminator of outcome followed by CathD, whereas the contribution of log(ER) was negligible. In multivariate analysis, 2 models were considered: one with log(ER), pS2, CathD and the interaction between pS2 and CathD, and another with log(PgR), pS2, CathD and the interaction between pS2 and CathD. In the first model, log(ER) content did not have a significant prognostic role, whereas in the second model log(PgR) retained a significant prognostic role. Our findings indicate that the quantitative determination of pS2 and CathD, in addition to steroid receptors, on the same cytosolic fraction could be a complementary tool to describe all breast cancer patients rather than just the elderly and that the use of a continuous scale, instead of a simple dichotomous "status", may improve the biological information supplied by the variables.

Estrogen-receptor status of patients who underwent mastectomy for breast cancer with a disease-free interval of not less than 8 years.

The authors present a series of 1,226 patients who underwent total or partial mastectomy for breast cancer, who for a period of no less than 8 years from surgery did not have a relapse or relapsed only after 8 years. The patients were evaluated for estrogen-receptor (ER) content of the primary tumor. In the group of 237 patients who relapsed, only 8.8% (21 of 237) were ER negative; in the group of 989 patients without a relapse, 24.1% (239 of 989) were ER negative. The difference was significant (p < 0.001). Therefore, the absence of hormone receptors (ER negative) indicates a favorable prognosis after a period of 8 years. For comparison, ER content in the group of patients who relapsed before 8 years was evaluated; the results showed a different prognosis from the ER-negative patients in this group. Because the behavior of ER-negative tumors in the cancer-affected breast is similar to that of non-hormone-dependent carcinomas arising in other parts of the body (which generally are considered cured after a long disease-free interval from surgery), we conclude that this repudiates the theory that confers to breast cancer the character of a systemic disease.

Expression of the kidney-associated differentiation glycoprotein gp160 and resistance to the antitumor effects of interferon alpha in renal cell carcinomas.

BACKGROUND: Alpha interferon (IFN-alpha) is commonly used to treat patients with advanced renal cell carcinoma (RCC). We previously reported that resistance of RCCs to IFN-alpha in vitro correlated with the expression of a cell-surface glycoprotein of 160,00 kD molecular weight (gp160) which we subsequently identified as aminopeptidase A. MATERIALS AND METHODS: To directly test the role of gp160/APA in IFN-resistance, we stably introduced the gp160/APA cDNA into IFN-sensitive SK-RC-49 cells resulting in the expression of an enzymatically active gp160/APA protein. In addition, to determine if gp160/APA expression could function as a marker of IFN-resistance in vivo, we assessed gp160/APA protein levels in autologous normal kidney and primary renal cancer specimens from 29 patients half of which were randomized to receive adjuvant IFN-alpha therapy following nephrectomy. RESULTS: Four clones which possessed varying amounts of gp160/APA specific enzyme activity were assayed for sensitivity to the antiproliferative effects of IFN-alpha. All four clones exhibited sensitivity to IFN-alpha similar to that observed with parental SK-RC-49 cells. The analysis of tumor tissue detected no significant difference between the mean level of gp160/APA in tissue from control and IFN-alpha treated patients (1.33 A.U. versus 0.9981 A.U., p = 0.23); however, the mean gp160/APA level was significantly less in tumor tissue (mean = 1.15 A.U.) compared to normal tissue (mean = 2.15 A.U.; p < 0.00001). Within the IFN-alpha treated group, tumor gp160/APA levels did not correlate with the development of metastases or survival (p = 0.469). CONCLUSIONS: These data indicate that gp160/APA does not directly convey IFN-resistance to RCC cells and suggest that expression of gp160/APA in primary RCCs does not predict the benefit of IFN-alpha therapy.

Effect of liposome-encapsulated alpha- or beta-interferon on breast cancer cell lines.

Experimental evidence and clinical studies have indicated that interferons (IFN) inhibit proliferation in a wide panel of neoplasms, including breast cancer. However, the antitumor activity of IFN requires the continuous presence of high concentrations of the drug and is associated with side effects. To explore the potential of liposomes as an IFN delivery system, we compared the effect of free or liposome-encapsulated alpha-IFN and beta-IFN on the growth of two breast cancer cell lines (MCF7 and MDA-MB231). Cells were cultured in the presence of IFN at different concentrations (500, 1000, 2000 IU/ml) or in the presence of multilamellar liposomes (phosphatidylcholine-phosphatidylserine at a molar ratio of 7:3) containing saline buffer, alpha-IFN or beta-IFN. Additional control groups consisted of cells cultured with alpha-IFN or beta-IFN plus empty liposomes. Empty liposomes were not cytotoxic and did not interfere with IFN activity. In both cell lines liposomes encapsulating alpha-IFN (at the highest lipid:drug ratio) inhibited cell growth in a manner similar to that of free alpha-IFN, whereas liposomes encapsulating beta-IFN showed slightly, lower inhibition than free beta-IFN, this was more evident in MCF7 cells. The present results indicate that liposomes encapsulating alpha-IFN or beta-IFN were effective on the growth of both breast cancer cell lines, which are characterized by a different estrogen responsiveness, and that they might be a useful carrier system for the delivery of high doses of IFN.

P53 accumulation in primary breast cancer: a comparison between immunohistochemistry and a novel luminometric immunoassay.

The accumulation of p53 protein was evaluated by a novel luminometric immunoassay (LIA) in cytosol samples from a series of 245 primary breast cancers. The cytosolic p53 content was not related to nodal status or hormone receptor status, but it was significantly and directly associated with tumor size and cell proliferation. A matched comparison between immunohistochemistry (IHC) and LIA results of individual tumors showed a significant association, albeit with a correlation coefficient of only 0.41. The agreement of results from the two assays was higher in node-positive, estrogen-receptor-negative and rapidly proliferating tumors than in the complementary subgroups. Overall, there was a significant trend in favor of an increase in p53 levels as determined by LIA with the increase in p53-positive cells shown by IHC. However, taking IHC detection as a reference, the sensitivity of the LIA was better for negative (86%) than for positive (61%) values. Based on these findings, a comparative assessment of the clinical relevance of LIA versus IHC results has to be recommended.

Simultaneous but not sequential treatment with sodium butyrate improves the antiproliferative effect of alpha- or beta-interferon on a breast cancer cell line.

Clinical evidence indicates that alpha- and beta-interferon (alpha-IFN, beta-IFN) are only partially effective in human breast cancer. To improve their effectiveness, it has been proposed that differentiation inducers, such as sodium butyrate (NaB), be used to increase the IFN sensitivity of tumors. Therefore, we assessed concomitant or sequential combinations of low/intermediate concentrations of alpha-IFN or beta-IFN (10, 50 and 100 IU/ml) with a low concentration (0.1 mM) of NaB on a human breast cancer cell line (MDA-MB231), which exhibited a moderate sensitivity to IFN. Moreover, to verify the capability of NaB to potentiate IFN effectiveness by increasing IFN receptor (IFN-R) concentration, we investigated the effect of NaB on the synthesis of IFN-R. The concomitant presence of NaB and alpha-IFN or beta-IFN significantly improved the antiproliferative effect of the corresponding IFN alone. Conversely, the sequential treatment NaB-IFN did not enhance the inhibitory activity of the cytokine, although NaB was able to induce the expression of IFN-R. More likely, NaB induced the expression of some component of the IFN system, such as Stat1, Stat2 or p48, whose higher availability in the cytoplasmic compartment promotes formation of the multimeric transcription factor ISGF3, which induces the transcription of IFN-stimulated genes.

Effect of sodium butyrate on human breast cancer cell lines.

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 mM. The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G2M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.

Combined effect of tamoxifen or interferon-beta and 4-hydroxyphenylretinamide on the growth of breast cancer cell lines.

To improve the effectiveness of 4-hydroxyphenylretinamide (4-HPR), an analogue of retinoic acid used in chemoprevention and treatment of breast cancer, we investigated the effect of concomitant administration of 4-HPR (0.1, 1 microM) and tamoxifen (TAM, 0.1, 1 microM), or 4-HPR and interferon-beta (IFN-beta, 10, 100, 500 IU/ml) on the growth of four cell lines (MCF7, T47D, MDA-MB231 and BT20) characterized by a different steroid receptor profile. A high concentration of 4-HPR caused a significant inhibitory effect not only on the estrogen receptor-positive cell lines (MCF7 and T47D), but also on one (BT20) of the two estrogen receptor-negative cell lines. IFN-beta displayed a dose-dependent inhibitory effect in all cell lines, but it was most evident in MCF7 cells. In all cell lines, the combination of 4-HPR (0.1 microM) and TAM (1 microM) or IFN-beta (500 IU/ml) generally caused additive or synergistic effects. In particular, the finding that in estrogen receptor-negative MDA-MB231 cells 4-HPR (which at 1 microM was singly ineffective) in combination with TAM at 1 microM or any concentration of IFN-beta produced a synergistic effect suggests that the compound could act through a pathway independent of specific receptors for retinoids. Our results indicate that intrinsic characteristics of cells can influence responsiveness to 4-HPR, TAM and IFN-beta, singly or in association, ever within cell lines with similar steroid receptor profiles. Thus, more attention should be payed to the biological characteristics of the single tumor in order to help choose the best combination of drugs to be applied.

Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line.

In vivo and in vitro studies have shown an antitumor activity of Lactobacilli in colon cancer, and some epidemiologic studies have indicated a reduced risk of breast cancer in women who consume fermented milk products. We studied the direct effect of milk fermented by five bacteria strains (Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus paracasei) on the growth of the MCF7 breast cancer cell line. Our results showed a growth inhibition induced by all fermented milks, even though B. infantis and L. acidophilus were the most effective (85% inhibition after 9 days). The antiproliferative effect was not related to the presence of bacteria in fermented milk, and neither whole milk (crude or ultrahigh temperature sterlizied) nor its main fractions (lactalbumin or beta-lactoglobulin fraction) affected cell growth. Our findings suggest the presence of an ex novo soluble compound produced by lactic acid bacteria during milk fermentation or the microbial transformation of some milk components in a biologically active form. Although the mechanism of the antitumor activity is not clear, the present study suggests the potentiality offered by fermented milk as producers of compounds with antiproliferative activity useful in the prevention and therapy of solid tumors like breast cancer.

int-2 oncogene amplification and prognosis in node-negative breast carcinoma.

The role of int-2 oncogene amplification on the prognosis of breast cancer patients was investigated in 128 patients with node-negative primary breast cancers given first-line local-regional treatments until relapse and with a median follow-up of 65 months. Tumours had been previously characterised for oestrogen (ER) and progesterone receptor (PgR) status and proliferative activity (3H-thymidine labelling index). Amplification of the int-2 oncogene occurred in 18% of cases and was significantly related to the presence of hormone receptors and to menopausal status or age, but not to proliferative status. Patients with tumours exhibiting int-2 amplification had a lower probability of disease-free survival than patients with non-amplified tumours and frequently developed local-regional recurrence. Disease-free survival analysis, adjusted for the prognostic contribution provided by tumour size, steroid receptors and proliferative rate, indicated that the association between int-2 amplification and risk of relapse was maintained and remained constant even in the presence of the other co-variates. Interestingly, int-2 amplification was a further prognostic discriminant within subsets of patients with a putatively good (i.e., tumour size <20 mm, ER+ and PgR+) or poor prognosis (i.e., high labelling index). Our exploratory study suggests that within node-negative patients, int-2 amplification could be a valuable and independent prognosticator, useful to identify patients at high risk of local-regional recurrence.

Modulation of cathepsin-D and pS2 protein levels in human breast cancer cell lines.

Cathepsin-D and pS2 are two estrogen-regulated proteins in human breast cancer cell lines. They have been considered possible prognostic factors in breast cancer, but results have been contradictory. To better understand the regulation of these proteins, we investigated the role of estradiol (E2), serum, and growth factors in hormone-dependent (MCF-7, ZR75.1) and hormone-independent (MDAMB-231, BT20) breast cancer cell lines. E2 treatment in serum-free conditions increased intracellular and secreted levels of pS2 in ZR75.1 and in MCF-7, secreted levels only of cathepsin-D in MCF-7, and both levels of cathepsin-D in ZR75.1. Insulin-like growth factor I (IGF-I) and progesterone receptors were also stimulated by E2, whereas the estrogen receptor was down-regulated. Following treatment with epidermal growth factor (EGF), secreted pS2 levels doubled only in MCF-7 cells. IGF-I did not modify cathepsin-D or pS2 levels in either cell line, but caused an increase in its own receptor. Cathepsin-D and pS2 doubled in MCF-7 cells grown in medium supplemented with denaturated serum, but estrogen regulation of these proteins was still maintained. Cathepsin-D was expressed in MDAMB-231 and BT20, but its levels were modified by neither E2 nor growth factor treatment. Conversely, neither cell line expressed detectable levels of pS2 before or after treatment. In conclusion, our results show that in different types of breast cancer cells, some estrogen-regulated proteins (e.g. pS2) are also regulated by growth factors-such as EGF and other unknown serum factors. This may account for the contradictory results obtained regarding the prognostic relevance of cathepsin-D and pS2.

Effect of progestin treatment on estradiol-and growth factor-stimulated breast cancer cell lines.

BACKGROUND: Reports about the effects of progestins on cell proliferation are contradictory. We investigated the effect of progesterone, medroxyprogesterone acetate, megestrol acetate, ORG 2058 and the antiprogestin RU 486 on two hormone-dependent cell lines, T47D and MCF-7 (characterized by a different content of PgR). The aim of the study was to understand the eventual ability of progestins to interfere with cell proliferation stimulated by estradiol and various growth factors (TGF-a, IGF-I, IGF-II). MATERIAL AND METHODS: MCF-7 and T47D cells were maintained in DMEM/F12 medium supplemented with 2% FCS while experiments were carried out in the same culture medium using DCC-stripped FCS. RESULTS: In the absence of estradiol, all tested progestins generally tended to stimulate cell growth in the T47D cell line, but in the MCF-7 cell line only the highest concentrations (10(-6) M and 10(-7) M) were found to be stimulatory. In contrast, in the presence of 10(-8) M estradiol, progestins tended to inhibit cell growth stimulation in MCF-7 and T47D cell lines. The antiprogestin RU 486 exerted a stimulatory effect similar to that promoted by estradiol itself in MCF-7 cells. Instead, in T47D cells, RU 486 did not modify cell growth in the absence of estradiol, but tended to counteract the estradiol-promoted cell proliferation. In MCF-7 cells, medroxyprogesterone acetate and megestrol acetate were also able to effectively counteract the cell growth induced by TGF-alpha. However, none of these progestins was able to abolish cell proliferation promoted by IGF-I or IGF-II. CONCLUSION: We therefore concluded that failure to respond to progestin treatment may be due to the very heterogeneous nature of human breast tumors and to the inability of these molecules to interfere with the IGF-R pathway.

The effect of ICI 164384 and beta-interferon on the growth and steroid receptor profile of breast cancer cell lines.

We investigated the effect of a concomitant treatment of ICI 164384 and B-interferon (beta-IFN) on the growth of estrogen-receptor-positive (ER+) and estrogen-receptor-negative (ER-) breast cancer cell lines and on their steroid receptor profiles. ICI 164384 reduced cell proliferation not only in ER+ but also in ER- cell lines and completely suppressed the stimulation induced by estradiol (E2) in hormone-sensitive cell lines, MCF7 and T47D. When associated with beta-IFN, ICI 164384 increased the inhibitory effect exerted by the low concentration of beta-IFN. Moreover, ICI 164384, singly or in association with beta-IFN, did not affect ER and PgR concentration in ER- cell lines, whereas in ER+ cell lines we observed an almost total disappearance of ER and PgR. In conclusion, our study seems to indicate that, although beta-IFN is able to control the proliferation of hormone-sensitive and hormone-independent subclones, it does not further improve the antiproliferative activity of ICI 164384. In contrast, the presence of ICI 164384, which does not induce the selection of resistant subclones under the same experimental conditions in which TAM does, may improve the efficacy of low concentration of beta-IFN and prevent the development of a secondary TAM-induced resistance.