The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

A possible regulatory role of 17β-estradiol and tamoxifen on glyoxalase I and glyoxalase II genes expression in MCF7 and BT20 human breast cancer cells

Summary The glutathione-dependent glyoxalases system, composed of glyoxalase I (GloI) and glyoxalase II (GloII) enzymes, is involved in the detoxification of methylglyoxal, a by-product of cell metabolism. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Sometimes, these aberrations seem to differ depending on the organs and on the sensitivity of the tumours to estrogens, as we previously detected in the hormone-responsive breast cancer compared to the hormone-independent bladder cancer. To investigate a possible regulatory role of estrogens, as well as antiestrogens, on glyoxalases system, estrogen receptor (ER)-positive MCF7 and ER-negative BT20 human breast cancer cells were cultured in the presence of 17β-estradiol (E2) and tamoxifen (TAM) performing two independent experiments. After a 24 h or 4 days treatment, we evaluated GloI and GloII mRNA levels, by Ribonuclease Protection Assay (RPA), enzymatic activities spectrophotometrically and cell proliferation by [³H]thymidine incorporation. We found that both steroid molecules affected glyoxalases gene expression and proliferation in a different manner in the cell lines. The modifications in mRNA levels were accompanied by parallel changes in the enzymatic activities. The possibility that modulation of glyoxalase genes by E2 and TAM are due to the presence of estrogen response elements (ERE) or cross-talk mechanisms by proteins of the estrogen signal transduction pathways are discussed. Knowledge regarding the regulation of glyoxalases by E2 and TAM may provide insights into the importance of this enzymes in human breast carcinomas in vivo. Antonio Rulli and Cinzia Antognelli made equal contributions to this study.     

Effect of α-difluoromethyl-ornithine on the expression and function of the epidermal growth factor receptor in human breast epithelial cells in culture

We have previously shown that ornithine decarboxylase (ODC) overexpression enhances the transforming effects of HER-2neu and epidermal growth factor (EGF) in normal MCF-10A human breast epithelial cells. Our data suggest that such potentiation may be mediated by activation of the mitogen-activated protein kinase (MAPK) pathway and, possibly, STAT signalling. To further explore the interaction between the polyamine pathway and EGF/HER-2neu signalling in this system, we inhibited endogenous ODC activity with -difluoromethylornithine (DFMO) and assessed the effects of this blockade on the expression of EGF receptors (EGFR) and HER-2neu as well as activation of downstream EGF target genes. We found that DFMO administration to MCF-10A cells increased EGF-R mRNA and protein levels in a dose-response fashion, while HER-2neu expression was not affected. The effect of DFMO was mediated through polyamine depletion since it could be reversed by exogenous putrescine administration. Our results also indicated that the increase in EGFR induced by DFMO was not a non-specific consequence of inhibition of cell proliferation. The upregulated EGFRs were functional since they could be phosphorylated by EGF and they were able to promote phosphorylation of downstream signalling molecules including ERK, STAT-3, and STAT-5. We propose that physiologic levels of ODC activity may be critical for regulation of a yet undefined signalling pathway, whose blockade by DFMO leads to a compensatory increase in functional EGFR.     

17β-estradiol regulates giant vesicle formation via estrogen receptor-alpha in human breast cancer cells

A significant proportion of the genes regulated by 17-beta-estradiol (E2) via estrogen receptor alpha (ERα) have roles in vesicle trafficking in breast cancer. Intracellular vesicle trafficking and extracellular vesicles have important roles in tumourigenesis. Here we report the discovery of giant (3-42μm) intracellular and extracellular vesicles (GVs) and the role of E2 on vesicle formation in breast cancer (BC) cell lines using three independent live cell imaging techniques. Large diameter vesicles, GVs were also identified in a patient-derived xenograft BC model, and in invasive breast carcinoma tissue. ERα-positive (MCF-7 and T47D) BC cell lines demonstrated a significant increase in GV formation after stimulation with E2 which was reversed by tamoxifen. ERα-negative (MDA-MB-231 and MDA-MB-468) BC cell lines produced GVs independently of E2 and tamoxifen. These results indicate the existence of both intracellular and extracellular vesicles with considerably larger dimensions than generally recognised with BC cells and suggest that the GVs are regulated by E2 via ERα in ERα-positive BC but by E2-independent mechanisms in ER-ve BC.     

Effect of hyperthermia on invasion ability and TGF-β1 expression of breast carcinoma MCF-7 cells

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is still disappointing despite the much improved technologies in its treatment. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Latent membrane protein 1 (LMP1) is a major oncogene of the human DNA tumor virus Epstein-Barr virus (EBV) and is associated with the development of NPC and the emergence of chemo-resistance in NPC. In this study, we investigated the potential role of LMP1 in TRAIL resistance in CNE-1 NPC cells. Results show that overexpression of LMP1 could induce TRAIL resistance in NPC cells without influencing death receptors. The LMP1-induced TRAIL resistance is associated with increased expression of FLIP and elevated cleavage of caspase-8 without altering the TRAIL-mediated mitochondrial events and Bid cleavage. Knockdown of the FLIP gene with siRNA prevented the LMP1-induced TRAIL resistance. Furthermore, we found that overexpression of LMP1 activated Akt. Inhibition of Akt with LY294002 completely prevented the LMP1-induced FLIP expression and TRAIL resistance. Together, these results show that LMP1 can inhibit the TRAIL-mediated apoptosis through activation of PI3K/Akt and expression of FLIP in CNE-1 NPC cells, and may provide new methods to prevent and reverse drug resistance in NPC.     

Anti-apoptotic effect of claudin-1 on TNF-α-induced apoptosis in human breast cancer MCF-7 cells

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in human breast cancer MCF-7 cells under tumor necrosis factor (TNF)-?? treatment. Interestingly, the increased expression of claudin-1 contributes to an anti-apoptotic role in TNF-??-induced apoptosis. In line with this, upon TNF-?? stimulus, downregulation of claudin-1 by siRNA knockdown results in a significant increase in cleavage of caspase-8 and poly (ADP-ribose) polymerase, a decrease of cyclinD1 expression, and DNA fragmentation. Consistently, TdT-mediated dUTP nick end labeling assay also shows that loss of claudin-1 increases the susceptibility of MCF-7 cells to TNF-??-induced apoptosis. However, there is no obvious effect on the expression of Bax and p53 after the treatment aforementioned. In addition, TNF-?? increases the amount of claudin-1 and the cytoplasmic accumulation of ??-catenin, while claudin-1 siRNA increases the amount of ??-catenin in the cell membrane as well as the amount of E-cadherin in the cytoplasm. In conclusion, our data reveal a novel role of claudin-1 in regulating apoptosis in MCF-7 cells.     

Regulatory effects of ΔFosB on proliferation and apoptosis of MCF-7 breast cancer cells

Tumor Biology - Matrix metalloproteinase-9 (MMP-9) plays a vital role in tumor angiogenesis, cell migration, and invasiveness because it can degrade almost all basement membrane and extracellular...     

Role of β1-integrin in promoting cell motility and tamoxifen resistance of human breast cancer MCF-7 cells

Background

The mechanism of acquired resistance of tamoxifen in endocrine therapy of breast cancer is not fully understood. In this study, we investigated the genomic changes in acquired tamoxifen-resistant cell lines.

Methods

Tamoxifen-resistant subclones (MCF-7R) derived from parent MCF-7 cells, which is an ER(+) breast cancer cell line, cultured with 4-hydrotamoxifen more than 6 months were used to obtain genomic alterations. Cell growth, microarray, and quantitative real-time PCR (q-RTPCR) assays were conducted. Additionally, the ITGB1 function was investigated in MCF-7R cells and MCF-7R ITGB1-silenced subclones using MTT and Transwell assays. Online pathway analysis was performed to assess the genetic characteristics of tamoxifen resistance.

Results

The gene expression profile of the tamoxifen-resistant cell line was considerably changed compared to the tamoxifen-sensitive cell line. Of 4102 genes with altered expressions, 1986 genes were upregulated, whereas 2116 were downregulated. The ITGB1 expression in MCF-7R cells was higher than that in MCF-7 cells. Interestingly, ITGB1 silencing partially rescued the sensitivity of MCF-7R cells to tamoxifen and reduced their motility. The activation of the β1-integrin signaling pathway was probably responsible for this phenomenon.

Conclusions

Our data confirm the presence of alterations in the genes of tamoxifen-resistance breast cancer cells. ITGB1 probably partially contributes to tamoxifen resistance and cell motility via the β1-integrin signaling pathway. Thus, ITGB1 may be a potential target for the improvement of anti-hormone therapy reaction in ER(+) breast cancer patients.     

Adipose tissue-derived mesenchymal stem cells cultured at high density express IFN-β and suppress the growth of MCF-7 human breast cancer cells

Although it has been reported that mesenchymal stem cells (MSCs) suppress tumor growth in vitro and in vivo, little is known about the underlying molecular mechanisms. We found that type I interferon is expressed in adipose tissue-derived stem cells (ASCs) cultured at high density, and ASCs and their conditioned medium (ASC-CM) suppress the growth of MCF-7 cells in vitro. Growth inhibition was amplified by glucose deprivation that resulted from high density culture of ASCs after 3 days. The cytotoxic effect of the ASC-CM obtained from high density culture of ASCs was neutralized by anti-IFN-β antibody. STAT1 was phosphorylated in MCF-7 cells treated with ASC-CM, and JAK1/JAK2 inhibitor treatment decreased STAT1 phosphorylation. The cytotoxic effect of ASC-CM was reduced especially by JAK1 inhibitors in MCF-7 cells. Our findings suggest that ASCs cultured at high density express type I interferons, which suppresses tumor growth via STAT1 activation resulting from IFN-β secretion in MCF-7 breast cancer cells.     

Cell cycle synchronization induced by tamoxifen and 17β-estradiol on MCF-7 cells using flow cytometry and a monoclonal antibody against bromodeoxyuridine

Summary Cell cycle synchronization of MCF-7 hormone-sensitive human breast cancer cells has been evaluated after sequential treatment with tamoxifen and 17-estradiol. The analysis was performed by flow cytometry. Two methods were used, one for single-parameter DNA content analysis, and one for bivariate analysis of DNA content and amount of incorporated bromodeoxyuridine (BrdUrd) into DNA using a specific monoclonal antibody. According to the BrdUrd method, tamoxifen was found (over a 30h period) to decrease (with respect to cells grown in control medium) the fraction of cells in S phase from 45% to 20%, to increase cells in G0 + G1 from 47% to 68%, and to induce a slight build-up of cells in G2 + M. Subsequent addition of estradiol resulted in partial synchronous recruitment of the cells from G0+G1 to progress through the S phase; after 6–8 h delay time, the percentage of cells in G0+G1 decreased by 50% and cells in S increased by 175%. The bivariate BrdUrd technique offered more reliable and detailed information than the single-parameter DNA analysis for differentiating and measuring the time course of estrogen-recruited cells as they progressed through early and late S phase, and has the potential for a very detailed cell kinetic analysis of bothin vitro andin vivo hormone-sensitive cells.     

Effects of human recombinant interferons-α2, -β and -γ on growth and survival of human cancer nodules maintained in continuous organotypic culture

Alveolar II pulmonary tumor cells (A549 cells) maintained in continuous tri-dimensional organotypic culture were used to test the effects of recombinant human interferons -α₂, -β and -γ on growth inhibition and survival of the tumor nodules. The organotypic culture method has several advantages: the three-dimensional structures of the cells as well as some cell differentiation are maintained and the extremely low traumatizing culture conditions offer injured cells the maximum chance of survival. A continuous treatment lasting 65 days (three weekly interferon changes) with 10, 10², and 10³ and 10⁴U/ml doses of the three interferons led to growth inhibition and necrosis only in the presence of the two highest doses (10³ and 10⁴U/ml) of IFN-α₂ and -γ, IFN-β had no inhibitory effect. Some nodules, especially at the lower dose levels (10²U/ml), showed enhanced growth in presence of the three types of interferons. After stopping the treatments, all the necrotic and disintegrating nodules resumed growth. Growth of the recovered nodules was followed in the absence of interferon for another period of 70 days. The growth rate of IFN-β and -γ-treated nodules was similar to that of the controls, but was slowed down for the regenerated IFN-α₂-treated nodules. Hence, in A549 organotypic cancer nodules and under our experimental conditions, only high doses of IFN-α₂ and -γ appeared to have a partial cytolytic, but finally no tumoridical action; IFN-β was inactive. At the lower doses growth stimulation was found during the treatments with the three interferons.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

Enzymatic regulation of estradiol-17β concentrations in human breast cancer cells

Summary Estradiol-17 is known to be involved in both the etiology and maintenance of growth of breast cancer. However, blood levels of the hormone do not reflect those found within the cells due to a number of transformations catalysed by enzymes which may be under metabolite and/or hormonal regulation. Recognition of the importance of the hormone microenvironment within the cell focuses attention on these enzymes and provides the subject for this review. An interplay between the sex hormones, estrogen and progestin, can control estradiol-17 concentrations in breast cancer cells at the level of key transforming enzymes. In addition, some enzymes catalyse production of biologically inert derivatives which are rapidly eliminated from the cell. Other enzymes catalyse the formation of derivatives which are exclusively intracellular and can act as reserve forms of the hormone. Yet others lead to estradiol-17 metabolites which are cytotoxic. An improved understanding of the enzymes and the role of the related metabolites can provide the opportunity for the development of new therapeutic agents.     

Trilostane,an inhibitor of 3β-hydroxysteroid dehydrogenase,has an agonistic activity on androgen receptor in human prostate cancer cells

The intracellular androgen metabolism and cell activity in prostate cancer cells with mutated (LNCaP-FGC) or wild-type (VCaP) androgen receptors in the presence of trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, were examined. Trilostane suppressed the intracellular production of androstenedione, testosterone, and dihydrotestosterone from dehydroepiandrosterone in LNCaP-FGC cells. In both LNCaP-FGC and VCaP cell types, the prostate-specific antigen (PSA) levels in media were increased by trilostane alone in a concentration-dependent manner. Both cells pretreated with trilostane showed a dose-dependent decrease in PSA production with bicalutamide (P < 0.001). Trilostane should be used with particular concern when treating prostate cancer.     

Human recombinant interferon-βSER and tamoxifen: growth suppressive effects for the human breast carcinoma MCF-7 grown in the athymic mouse

Summary Tamoxifen is the endocrine treatment of choice for breast cancer. However, resistance to therapy and patient relapse inevitably occurs. In future treatment schedules, interferons could be administered with tamoxifen, in an attempt to prevent disease recurrence.Human recombinant interferon-_SER (rIFN-_SER) inhibited the growthin vitro of the estrogen receptor (ER) positive breast cancer cell line MCF-7 and the ER negative breast cancer cell line MDA-MB-231. This inhibitory effect was achieved at doses of 50U/ml and above.The growth of MCF-7 tumors in estradiol-stimulated athymic mice was greatly inhibited by high dose rIFN-_SER treatment (10⁶U/day). In spite of the impressive antitumor effects upon MCF-7 tumors, rIFN-_SER had no effect upon ER levels within the tumors at either the RNA or protein level, as measured by Northern blotting and ER-EIA respectively. High dose rIFN-_SER (10⁶U/day) did result in some inhibition in the growthin vivo of the tamoxifen-stimulated MCF-7 variant MCF-7 TAM, although not to the same extent as was observed with the estradiol-stimulated MCF-7 tumors.rIFN-_SER was also administered to animals bearing MCF-7 tumors and treated with estradiol and tamoxifen. In the animals undergoing high dose therapy (10⁶U/day), tumor growth was completely suppressed. Furthermore, tumor growth continued to be suppressed in those animals in which the rIFN-_SER therapy was halted and the tamoxifen capsule removed. No tumors were observed in spite of the environment of estradiol stimulation. Thus, the combination of interferon and tamoxifen was totally growth suppressive for MCF-7 xenografts in nude mice.Abbreviations used ER Estrogen Receptor - R Recombinant - IFN Interferon - EIA Enzyme Immobilized Assay - TAM Tamoxifen - MEM Minimal Essential Medium - HBSS-CMF Hanks Balanced Salt Solution-Calcium and Magnesium Free - EDTA Ethylenediamine-tetraacetic acid - i.p. Intra-Peritoneal - SEM Standard Error of the Mean - DEPC Diethylpyrocarbonate - PR Progesterone Receptor - kb Kilobase     

17β‐hydroxysteroid dehydrogenases in normal human mammary epithelial cells and breast tissue

17hydroxysteroid dehydrogenase activity represents a group of several isoenzymes (17HSDs) that catalyze the interconversion between highly active 17hydroxy and low activity 17ketosteroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize the expression of 17HSD isoenzymes in human mammary epithelial cells and breast tissue. In normal breast tissues 17HSD types 1 and 2 mRNAs were both evenly expressed in glandular epithelium. In two human mammary epithelial cell lines, mRNAs for 17HSD types 1, 2 and 4 were detected. In enzyme activity measurements only oxidative 17HSD activity, corresponding to either type 2 or type 4 enzyme, was present. The role of 17HSD type 4 in estrogen metabolism was further investigated, using several cell lines originating from various tissues. No correlation between the presence of 17HSD type 4mRNA and 17HSD activity in different cultured cell lines was detected. Instead, oxidative 17HSD activity appeared in cell lines where 17HSD type 2 was expressed and reductive 17HSD activity was present in cells expressing 17HSD type 1. These data strongly suggest that in mammary epithelial cell lines the oxidative activity is due to type 2 17HSD and that oxidation of 17hydroxysteroids is not the primary activity of the 17HSD type 4 enzyme.     

Essiac® and Flor-Essence® herbal tonics stimulate the in vitro growth of human breast cancer cells

SummaryBackground People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence^? and Essiac^? Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence^? and Essiac^? for their effects on the growth of human tumor cells in culture.Methods The effect of Flor-Essence^? and Essiac^? herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors. Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay.Results Flor-Essence^? and Essiac^? herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls in both estrogen receptor positive (MCF-7 and T47D) and estrogen receptor negative (MDA-MB-231 and MDA-MB-436) cell lines. Exposure to the tonics also produced a dose-dependent increase in ER dependent luciferase activity in MCF-7 cells. A 10⁻⁷ M concentration of ICI 182,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence^? and Essiac^?, but did not affect cell proliferation.Conclusion Flor-Essence^? and Essiac^? Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action.