Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

Interaction of retinoids and tamoxifen on the inhibition of human mammary carcinoma cell proliferation

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).     

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H₂O₂ levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.     

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.     

Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line.

Differences in gene expression are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast cancer. We have identified differentially expressed cDNAs in the estrogen receptor (ER)-positive MCF7 breast carcinoma cell line compared with the ER-negative MDA-MB-231 breast carcinoma cell line. Differential screening isolated four differentially expressed genes: cytokeratin 8, cytokeratin 18, Hsp27 and GPCR -Br. To identify differentially expressed genes of lower abundance, suppression subtractive hybridization was utilized and 29 differentially expressed clones were isolated. Sequence analysis revealed that 11 clones were from previously described genes: HEK8, neuropeptide Y receptor Y1, p21 WAF-1, p55 PIK, cytokeratin 18 (cloned twice), fructose-1,6-biphosphatase, cytokeratin 8, TGFbeta1 binding protein, elongation factor 1alpha2 and pS2. The remaining 18 clones did not match sequences in the GenBank/EMBL database, indicating that they may be novel genes. Expression of pS2, neuropeptide Y receptor Y1 and three novel clones was induced by estradiol, indicating estrogen-responsiveness. The expression pattern of one novel gene, DEME -6, correlated with expression of ER and ERF -1/ AP -2gamma in a panel of breast carcinoma cell lines. A 2.6 kb cDNA of DEME -6 was sequenced and contains an open reading frame of 574 amino acids that demonstrates 62.4% similarity with a gene from Caenorhabditis elegans chromosome III. Expression of DEME -6 was also detected in primary breast carcinomas but not in normal breast tissue, as determined by RT-PCR. These findings support the hypothesis that a set of genes coordinately regulated with ER , but not necessarily estradiol-responsive, are characteristic of the hormone-responsive breast cancer phenotype.     

Selenium Nanoparticles Induce the Chemo-Sensitivity of Fluorouracil Nanoparticles in Breast and Colon Cancer Cells

Drug resistance is a major challenge of breast and colon cancer therapies leading to treatment failure. The main objective of the current study is to investigate whether selenium nanoparticles (nano-Se) can induce the chemo-sensitivity of 5-fluorouracil (FU)-encapsulated poly (D, L-lactide-co-glycolide) nanoparticles (nano-FU) in breast and colon cancer cell lines. Nano-Se and nano-FU were synthesized and characterized, then applied individually or in combination upon MCF7, MDA-MB-231, HCT 116, and Caco-2 cancerous cell lines. Cytotoxicity, cellular glucose uptake, and apoptosis, as well as malondialdehyde (MDA), nitric oxide (NO), and zinc (Zn) levels, were investigated upon the different treatments. We have resulted that nano-FU induced cell death in MCF7 and Caco-2 more effectively than MDA-MB-231 and HCT 116 cell lines. Moreover, nano-FU plus nano-Se potentiate MCF7 and Caco-2 chemo-sensitivity were higher than MDA-MB-231 and HCT 116 cancerous cell lines. It is relevant to note that Se and FU nano-formulations inhibited cancer cell bioenergetics via glucose uptake slight blockage. Furthermore, nano-FU increased the levels of NO and MDA in media over cancer cells, while their combinations with nano-Se rebalance the redox status with Zn increment. We noticed that MCF7 cell line is sensitive, while MDA-MB-231 cell line is resistant to Se and nano-Se. This novel approach could be of great potential to enhance the chemo-sensitivity in breast and colon cancer cells.

     

Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells

We compared in this study the effects of 2-methoxyestradiol (2-MeO-E(2)) on the growth of two estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and two ER-positive human breast cancer cell lines (MCF-7 and T-47D). 2-MeO-E(2) exerted a concentration-dependent antiproliferative action in the ER-negative MDA-MB-231 and MDA-MB-435s cells. The presence or absence of exogenous 17beta-estradiol (E(2)) in the culture medium did not affect the potency and efficacy of 2-MeO-E(2)'s antiproliferative action in these ER-negative cells. When the ER-positive MCF-7 and T-47D cells were cultured in a medium supplemented with 10nM of exogenous E(2), 2-MeO-E(2) at 750 nM to 2 microM concentrations exerted a similar antiproliferative effect. However, when the ER-positive cell lines were cultured in the absence of exogenous E(2), 2-MeO-E(2) at relatively low concentrations (10-750 nM) had a moderate mitogenic effect, with its apparent efficacy 75-80% of that of E(2). This mitogenic effect of 2-MeO-E(2) was ER-mediated and largely attributable to 2-MeO-E(2)'s residual estrogenic activity on the basis of our following findings: (i) its effect was only manifested in the ER-positive cells but not in the ER-negative cells; (ii) its effect in the ER-positive cells was partially or fully abolished when exogenous E(2) was concomitantly present in the culture medium; (iii) 2-MeO-E(2) retained 1-2% of E(2)'s binding affinity for the human ERalpha and ERbeta, and its mitogenic effect was inhibited in a concentration-dependent manner by ICI-182,780, a pure ER antagonist; and (iv) its effect was not due to its metabolic conversion to 2-hydroxyestradiol. Our timely findings are of importance to the on-going clinical trials designed to evaluate 2-MeO-E(2)'s effectiveness for the treatment of different types (ER-positive or ER-negative) of human breast cancer. This knowledge will improve the design of clinical trials as well as the interpretation of clinical outcomes when 2-MeO-E(2) is used as a single agent therapy or as part of a combination therapy for human breast cancer.     

The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer.The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line.CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the β-catenin/[phospho]GSK-3β complex as well as the E-cadherin/β-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status.These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.     

Assessment of Interactions between Cisplatin and Two Histone Deacetylase Inhibitors in MCF7, T47D and MDA-MB-231 Human Breast Cancer Cell Lines – An Isobolographic Analysis

Histone deacetylase inhibitors (HDIs) are promising anticancer drugs, which inhibit proliferation of a wide variety of cancer cells including breast carcinoma cells. In the present study, we investigated the influence of valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), alone or in combination with cisplatin (CDDP) on proliferation, induction of apoptosis and cell cycle progression in MCF7, T47D and MDA-MB-231 human breast carcinoma cell lines. The type of interaction between HDIs and CDDP was determined by an isobolographic analysis. The isobolographic analysis is a very precise and rigorous pharmacodynamic method, to determine the presence of synergism, addition or antagonism between different drugs with using variety of fixed dose ratios. Our experiments show that the combinations of CDDP with SAHA or VPA at a fixed-ratio of 1:1 exerted additive interaction in the viability of MCF7 cells, while in T47D cells there was a tendency to synergy. In contrast, sub-additive (antagonistic) interaction was observed for the combination of CDDP with VPA in MDA-MB-231 “triple-negative” (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) human breast cancer cells, whereas combination of CDDP with SAHA in the same MDA-MB-231 cell line yielded additive interaction. Additionally, combined HDIs/CDDP treatment resulted in increase in apoptosis and cell cycle arrest in all tested breast cancer cell lines in comparison with a single therapy. In conclusion, the additive interaction of CDDP with SAHA or VPA suggests that HDIs could be combined with CDDP in order to optimize treatment regimen in some human breast cancers.     

Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction in breast cancer.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Characterization of Catecholestrogen Membrane Binding Sites in Estrogen Receptor Positive and Negative Human Breast Cancer Cell-Lines

Abstract

A specific membrane-binding of an estradiol metabolite, the catecholestrogen (CE) 2 hydroxyestrone (2OH-E1), was demonstrated in two receptor-positive (MCF7 and VHB1) and one receptor-negative (MDA-MB-231) human mammary carcinoma cell lines. The three cell lines were found to be able to synthesise and inactivate CE. Solubilization of membrane bound CE results in a high molecular weight component whose specificity differs from that of the classical estrogen receptor. Apparent dissociation constants were 6-10. 10^?9 M and binding capacities were higher in the receptor positive cell lines than in the receptor-negative one. Since CE are susceptible to rapid degradation, the presence of such a site may be relevant in the protection and concentration of 2OH-E1 which has been shown to have “in vitro” anti-estrogenic properties in MCF7 breast tumor cells.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.