A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Cura?ao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.     

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.     

Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells

We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha. We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A). In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner. The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium. TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2. Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa). Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa. Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active. TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2. These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro. The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.     

Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide

Bax, one of the bcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax; and two unique clones, MCF-7/bax-1 and MCF-7/ bax-2, that expressed different levels of bax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level of bax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenous bax-alpha overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.     

Affinity for the insulin-like growth factor-II (IGF-II) receptor inhibits autocrine IGF-II activity in MCF-7 breast cancer cells

We have investigated the autocrine regulation of insulin-like growth factor-II (IGF-II) signaling by the insulin-like growth factor-I receptor (IGF-IR) and the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) in MCF-7 breast cancer cells, employing retroviruses encoding both IGF-I, IGF-II, and IGF-I and II mutants with reductions in affinity for either the IGF-IR or the IGF-IIR. These studies revealed reciprocal roles for IGF-IR and IGF-IIR affinity in the regulation of autocrine IGF-II activity. IGF-IR affinity was required for serum-free proliferation but also for efficient IGF-II secretion. In contrast, cellular proliferation, receptor tyrosine kinase-dependent signaling, and extracellular IGF-II protein accumulation were all reduced in the presence of IGF-IIR affinity. Inhibition of IGF-II signaling appeared to be the sole consequence of IGF-IIR affinity, as no cellular responses attributable to selective IGF-IIR binding by a reduced IGF-IR affinity IGF-II mutant could be detected. By operating as an IGF-II antagonist, the IGF-IIR has tumor suppressor-like properties, a suggestion consistent with reports of loss of heterozygosity at the IGF-IIR locus in a variety of human malignancies.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.     

MGBG analogues as potent inhibitors of S-adenosylmethionine decarboxylase of Onchocerca volvulus

The county organization, including health care, is reorganized in the province of Scania in southern Sweden. As part of the restructuring of health care, a program for digitalization of the departments of diagnostic imaging, as well as for teleradiology, has been set up. Standards for network, radiology information systems, and workstations have been settled, and teleradiology links both for on-call consultations and for on-line consultations day-time have been implemented, mainly running at 10 Mb/s. Further digitalization and implementation of teleradiology is planned for the nearest years. Parallel to this, a video conference system including several disciplines, hospitals and health care levels in the whole of southern Sweden has been implemented. The links are now also used for education, both in the province and internationally.     

5-En-androstene-3 beta,17 beta-diol inhibits the growth of MCF-7 breast cancer cells when oestrogen receptors are blocked by oestradiol

Adrenal androgens show a dual and apparently opposite effect on the growth of oestrogen-responsive breast cancer: they stimulate growth on their own, but counteract the growth-stimulatory effect of oestrogens. Focusing on the inhibitory action we have studied the effects of 5-en-androstene-3 beta,17 beta-diol (ADIOL) on the growth of oestrogen-responsive MCF-7 breast cancer cells in the presence of oestrogens (oestradiol and diethylstilboestrol), antiestrogens (tamoxifen) and antiandrogens (hydroxyflutamide). The inhibition of oestrogen-stimulated growth, attained with nanomolar concentrations of ADIOL, was not modified by increasing concentrations of diethylstilboestrol up to 100 nM. This inhibition was counteracted by antiandrogens, which were unable to block the ADIOL stimulatory effect in steroid-free medium. On the other hand, in the presence of tamoxifen ADIOL showed an additive antiproliferative activity also in steroid-free medium, rather than the usual stimulatory effect. These results suggest that ADIOL stimulates breast cancer cell growth via oestrogen receptors, but inhibits oestrogen-stimulated growth via androgen receptors.     

Fibroblast growth factor-2 has opposite effects on human breast cancer MCF-7 cell growth depending on the activation level of the mitogen-activated protein kinase pathway

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway.     

Regulation of transforming growth factor-beta type II receptor expression in human breast cancer MCF-7 cells by vitamin D3 and its analogues

In view of the tumor suppressor role of the transforming growth factor-beta (TGFbeta) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFbeta growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFbeta, was not affected by these two compounds. TGFbeta-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFbeta activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells.     

Interaction of vault particles with estrogen receptor in the MCF-7 breast cancer cell

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.     

Lipotrope deficiency inhibits cell growth and induces programmed cell death in human breast cancer cell line MCF-7

BACKGROUND: We examined the effect of growth hormone (GH) administration on the psychological capacity and sense of well-being in 25 patients with adult-onset GH-deficiency (GHD). METHODS: Very low dosages [0.5-1.0 UIday(-1) s.c. at bed-time] of recombinant human (rh)-GH (n = 13; aged 50+/-15 years, mean+/-SD) or placebo (n = 12, 53+/-14 years) were given at random for a 6-month period. Quality of life was assessed by using the Italian version of the self-rating Kellner Symptom Questionnaire (KSQ) and the Hamilton Depression Scale (HDS). RESULTS: No difference in insulin-like growth factor I (IGF-I) levels was noted between groups on entry to the study. A significant increase in IGF-I [month 0 56.2+/-10.4 microg L(-1) vs. month 6 125.7+/-16.7 microg L(-1); P < 0.001] levels was noted only in the rh-GH-treated group. There was no difference in overall scores on the KSQ between the rh-GH-treated and control groups on entry. A slight, non-significant, decrease in overall scores was noted in both groups of subjects. Subsection analysis of items from the KSQ did not show significant differences in either group during the 6-month period. A significant decrease (month 0 28+/-1 vs. month 6 25+/-1; P = 0.02) in the HDS score was noted in rh-GH-treated but not in placebo-treated patients. There was a significant correlation (rs, -0.56, P = 0.05) between increase in IGF-I levels and decrease in HDS scores in rh-GH treated patients. CONCLUSION: The data demonstrate that low rh-GH dosages significantly improve psychological profiles as rated by HDS evaluation in adult-onset patients with GHD. On the other hand, a 6-month period of treatment does not produce any significant differences in quality of life as measured by KSQ between treated patients and placebo controls.     

Tamoxifen restores the E-cadherin function in human breast cancer MCF-7/6 cells and suppresses their invasive phenotype

Tamoxifen is an antiestrogen used in adjuvant therapy of breast carcinoma and could potentially prevent the development of mammary cancer. While it is widely clinically used, its exact mechanisms of action are not yet fully elucidated. MCF-7/6 cells are estrogen receptor-positive invasive human breast cancer cells with a functionally inactive cell surface E-cadherin. In this study, we report that tamoxifen, and to a lesser extent its metabolites 4-OH-tamoxifen and N-desmethyltamoxifen, restore the function of E-cadherin in MCF-7/6 cells. In an aggregation assay, 10(-6) M tamoxifen significantly increases the aggregation of MCF-7/6 cells. This effect is abrogated by a monoclonal antibody against E-cadherin (HECD-1), is fast (within 30 min), and does not require de novo protein synthesis. Tamoxifen was also found to inhibit the invasion of MCF-7/6 cells in organ culture. Our data is the first demonstration that tamoxifen can activate the function of an invasion suppressor molecule and suggest that the restoration of E-cadherin function may contribute to the therapeutic benefit of tamoxifen in breast cancer patients.     

Two-dimensional electrophoresis of proteins from breast cancer cells MCF-7. Changes in synthesis induced by FGF-2

Two-dimensional electrophoresis analysis of proteins from breast cancer cells MCF-7. Modifications of synthesis induced by FGF-2. Using high resolution two-dimensional electrophoresis, we have separated more than 1,000 proteins from the breast cancer cell line MCF-7. Computer assisted analysis of gels allowed us to classify these proteins in function of their isoelectric point, molecular weight and relative quantity. This data-base will now be used as a powerful tool to identified proteins which synthesis is regulated in various experimental or pathological situations. Thus we studied modifications induced by FGF-2. This growth factor induces the synthesis of four polypeptides which are not detected in cells not stimulated by this factor. In addition, intensity of nine other polypeptides was found increased in presence of FGF-2.