Estrogen Receptor-negative Breast Cancer Cells Research Articles

Estrogen Receptor-Positive and Estrogen Receptor-Negative Human Breast Cancer Cells: Regulation of Expression of Cancer-Related Genes by Estradiol and Tamoxifen

Breast cancer is one of the most prevalent forms of carcinomas among women world-wide. The oncogenes, tumor suppressor genes and antioxidant enzyme genes are cancer-related genes which play an important role in the initiation, metastasis and malignancy of many cancers, including breast cancer. Estrogen which is a key steroid hormone in the regulation and differentiation of the normal breast also appears to be involved in the carcinogenesis of this tissue. Antiestrogen therapy, such as the use of tamoxifen, targets the estrogen receptors (ER) through which estrogen exerts both its normal biological function as well as the molecular processes leading to cancer formation. The objectives of the present study were to characterize and quantify the expression of genes related to human breast carcinogenesis, using ER- positive (ZR-75-1) and ER-negative (MDA-MB-231) cells as in vitro cellular models of breast cancer. We have examined the expression of a number of genes which have an important clinical significance in human breast cancer, when expressed at levels below or above the normal range. Among these are the receptor oncogenes (EGFR, c-erbB2), other oncogenes (pS2, hMAM, MUC1 and CK-19), the tumor suppressor gene, p53, as well as four of the major antioxidant enzyme genes. These are superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), Se-glutathione peroxidase (GPx1) and catalase (CAT), all of which have been well characterized in human cells, and whose activities have been localized to different cellular compartments, such as the cytoplasm, mitochondria and peroxisomes. Gene expressions were determined using real-time PCR, in control untreated cells as well as when these two cell types were exposed to 17β-estradiol (E), or tamoxifen (TAM) or a combination of E and TAM. Our results show that basal level expressions of receptor oncogenes (EGFR and c-erbB2) as well as two of the four antioxidant enzyme genes (SOD2 and GPx1) were several folds lower in ER-positive breast cancer cells (ZR-75-1) as compared to ER-negative cells (MDAMB- 231). However, treatment with E and TAM, either individually or in combination, produced much greater effects on the ER-positive cells than on the ER-negative cells, with the result that these genes were greatly overexpressed in the ER-positive cells as compared to the ER-negative cells. Of these, the receptor oncogene, c-erbB2, and the antioxidant enzyme gene, SOD2, were the most affected, resulting in striking upregulations ranging over several hundred folds. This, and other observations, would suggest that the carcinogenic effects of estradiol and the anticarcinogenic effects of tamoxifen are not only mediated by the presence of estrogen receptors, but may also be regulated, either directly or indirectly, through other mechanisms, including the mitochondrial antioxidant system. Keywords: Breast cancer, estrogen and antiestrogens, tamoxifen, oncogenes, tumor suppressor genes, antioxidant enzyme genes, oxidative stress and antioxidants, quantitative real-time PCR

Aberrant Expression of Fra-1 in Estrogen Receptor-negative Breast Cancers and Suppression of their Propagation In Vivo by Ascochlorin, an Antibiotic that Inhibits Cellular Activator Protein-1 Activity

Estrogen receptor-negative breast cancers generally are highly malignant, resistant to chemotherapy and poorly prognostic. Here we demonstrate that estrogen receptor-negative human breast cancer cell lines highly express Fra-1, c-Fos and c-Jun, components of the transcription factor, activator protein-1 (AP-1). Retrospective observation of breast cancer tissues obtained by core needle biopsy before surgery from stages II and III patients demonstrates that Fra-1 expression is high in estrogen receptor-negative human breast cancers, and negatively correlated to paclitaxel sensitivity. Ascochlorin, which suppresses cellular AP-1 activity, selectively kills estrogen receptor-negative human and mouse breast cancer cell lines, and prolongs the survival time of mice implanted with an estrogen receptor-negative mammary carcinoma. These results suggest that chemotherapy targeting AP-1 activity is a potent strategy for estrogen receptor-negative human breast cancers.

Estrogen-dependent regulation of Eg5 in breast cancer cells

HsEg5 (Eg5) is a kinesin required for proper execution of mitosis. Several compounds that specifically block Eg5 are in clinical development and have the potential to be used in the treatment of breast cancer. In this study, we investigated the interaction between Eg5 and estrogen receptor signaling. We observed decreased Eg5 expression after treatment of estrogen receptor-positive human breast cancer MCF-7 cells with the estrogen receptor downregulator fulvestrant. Downregulation of Eg5 expression in response to fulvestrant was also observed in another estrogen receptor-positive cell line ZR-75, but not in the estrogen receptor-negative breast cancer cell line MDA-231. Moreover, in MCF-7 cells previously arrested in the G0/G1 phase of the cell cycle by fulvestrant, addition of estrogen increased Eg5 expression. This upregulation correlated with progression through S-phase. Nevertheless, the effect of fulvestrant in Eg5 expression could not be explained solely by cell cycle arrest, because treatments that blocked cell cycle progression did not consistently decrease Eg5 expression. Pharmacological inhibition of Eg5 function, with either S-trityl-L-cysteine or monastrol, prevented growth of estrogen-treated MCF-7 cells with an IC50 of 0.46 and 29.71 micromol/l, respectively. Simultaneous inhibition of estrogen receptor function with fulvestrant increased the IC50 for S-trityl-L-cysteine to 2.30 micromol/l and for monastrol to 112.69 micromol/l. Our results suggest that pharmacological inhibition of Eg5 may be an effective treatment for estrogen receptor-positive breast cancer, even without concomitant hormonal therapy.

Myc stabilization in response to estrogen and phospholipase D in MCF-7 breast cancer cells

Estrogen, which has been strongly implicated in breast cancer, suppresses apoptosis in estrogen receptor (ER) positive MCF-7 breast cancer cells. Phospholipase D (PLD), which is commonly elevated in ER negative breast cancer cells, also suppresses apoptosis. Survival signals generated by both estrogen and PLD are dependent upon elevated Myc expression. We report here that estrogen- and PLD-induced increases in Myc expression are due to reduced turnover of Myc protein. Estrogen and PLD suppressed phosphorylation of Myc at Thr58 – a site that targets Myc for degradation by the proteasome. The data provide a mechanism for elevated Myc expression in hormone-dependent and hormone-independent breast cancer.

Induction of Apoptosis in Estrogen Receptor-Negative Breast Cancer Cells by Natural and Synthetic Cyclopentenones: Role of the IκB Kinase/Nuclear Factor-κB Pathway

Nuclear factor-kappaB (NF-kappaB), a transcription factor with a critical role in promoting inflammation and cell survival, is constitutively activated in estrogen-receptor (ER)-negative breast cancer and is considered a potential therapeutic target for this type of neoplasia. We have previously demonstrated that cyclopentenone prostaglandins are potent inhibitors of NF-kappaB activation by inflammatory cytokines, mitogens, and viral infection, via direct binding and modification of the beta subunit of the IkappaB kinase complex (IKK). Herein, we describe the NF-kappaB-dependent anticancer activity of natural and synthetic cyclopentenone IKK inhibitors. We demonstrate that the natural cyclopentenone 15-deoxy-Delta(12,14)prostaglandin J(2) (15d-PGJ(2)) is a potent inhibitor of constitutive IkappaB-kinase and NF-kappaB activities in chemotherapy-resistant ER-negative breast cancer cells. 15d-PGJ(2)-induced inhibition of NF-kappaB function is rapidly followed by down-regulation of NF-kappaB-dependent antiapoptotic proteins cIAPs 1/2, Bcl-X(L), and cellular FLICE-inhibitory protein, leading to caspase activation and induction of apoptosis in breast cancer cells resistant to treatment with paclitaxel and doxorubicin. We then demonstrate that the cyclopentenone ring structure is responsible for these activities, and we identify a new synthetic cyclopentenone derivative, 3-tert-butyldimethylsilyloxy-5-(E)-iso-propylmethylenecyclopent-2-enone (CTC-35), as a potent NF-kappaB inhibitor with proapoptotic activity in ER-negative breast cancer cells. The results open new perspectives in the search for novel proapoptotic molecules effective in the treatment of cancers presenting aberrant NF-kappaB regulation.

PPARr agonist, troglitazone induce growth arrest and apoptosis in MDA-MB-231 human breast cancer cell

13155 Background: Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. It is recently reported that peroxisome proliferator-activated receptor-gamma (PPAR-γ) has become a potential target for the prevention and treatment of human cancer. Therefore, PPAR-γ agonist, troglitazone has been extensively studied as a chemopreventive or therapeutic agent in several types of cancer. The purpose of this study was to assess whether troglitazone (TRO) would induce cell growth arrest and apoptosis in MDA-MB-231 (ER-negative) human breast cancer cells. Methods: Cytostatic/cytotoxic effects of troglitazone were measured with MTT assay. The cell cycle distribution was evaluated with flow cytometer. The expression of cyclin-dependent kinase inhibitors, p21, p27, and cell cycle dependent kinases was measured with Western blotting. Apoptotic cells were determined by Hoechst staining and TUNEL assay. Results: Troglitazone inhibited cell proliferation by inducing 65% G0/G1 arrest after 48 h. Accumulation of cells in G0/G1 was accompanied by a decrease of Rb protein phosphorylation associated with reduced activities of cell cycle dependent kinases (CDKs) such as cdk2 and cyclin D. Troglitazone increased the expression cyclin kinase inhibitors, p21 and p27 as MDA-MB-231 cell underwent G0/G1 arrest. Apoptotic effect by troglitazone demonstrated that apoptotic cells elevated from 2.5-fold of the control level at 10 uM, to 3.6-fold at 50 uM and to 4.7-fold at 75 uM. Conclusion: These results indicate that activation of PPAR-gamma with troglitazone induces apoptosis through cell cycle inhibition in estrogen receptor-negative breast cancer cell MDA-MB-231. PPAR-gamma therefore represents a putative molecular target for chemopreventive therapy or inhibition of breast cancer growth. No significant financial relationships to disclose.

The synergistic effect between PDK1 inhibitor (OSU‐03012) and tamoxifen in ER positive and ER negative breast cancer

Tamoxifen is being used widely in ER positive breast cancer patients for over a decade. Although it is believed that tamoxifen induces breast cancer cell lines’ growth arrest and apoptosis via estrogen receptor (ER) inhibition, the non-ER mediated mechanism has been reported in recent studies. On the other hand, the importance of phosphatidylinositol 3-kinase (PI3K)/PDK-1/Akt signaling cascade in breast cancer cell lines is well documented. Our study purpose is using the synergistic effect of Tamoxifen and the PDK1 inhibitor (OSU03012) to inhibit the cell proliferation on both ER positive and ER negative breast cancer cells through the cross talk between Akt inhibition and tamoxifen's non-ER target. Our results showed that tamoxifen induced apoptotic cell death by IC50 values of 13 μM and 15 μM in ER-positive MCF-7 cell line and ER-negative MDA-MB-231 breast cancer cells. Moreover, by adding 5 μM of OSU03012, the sensitivity to tamoxifen-induced apoptotic death was increased by around ten folds of magnitude. OSU03012 decreased the phospho-Akt level, which was transiently increased by tamoxifen and caused the down regulation of PDK-1/Akt machinery, such as GSK-3, and P27. The fluorescent microscope data also demonstrated that the FOXO3a nuclear translocation was increased with OSU03012 treatment. These findings indicate that tamoxifen and OSU03012 combination induces breast cancer apoptosis through the synergistic interactions between ER-independent and PI3K/PDK-1/Akt pathway. This study could be clinically exploited for ER-independent breast cancer therapy.

Tissue-Type Plasminogen Activator Is Upregulated in Metastatic Breast Cancer Cells Exposed to Insulin-Like Growth Factor—I

The insulin-like growth factor (IGF) system plays an important role in breast tumorigenesis. Breast cancer cells express the type I IGF receptor (IGF-IR) and respond to IGFs in the environment. Tissue-type plasminogen activator (tPA) has been shown to be associated with neoplastic transformation and the invasive phenotype for highly aggressive tumors; however, its role in breast cancer remains unclear. We asked whether there is a relationship between the IGF system and tPA in estrogen receptor—negative breast cancer cells that could contribute to invasion. When MDA-MB-435s breast cancer cells were exposed to IGF-I, tPA messenger RNA (mRNA) was upregulated in a time-dependent fashion. Tissue-type plasminogen activator protein accumulation was also increased in a similar manner. The invasiveness of MDA-MB-435s cells was enhanced in the presence of IGF-I. When the MDA-MB-435s cells were stably transfected with an antisense IGF-IR expression construct, the transfectants expressed high levels of IGF-IR antisense, dramatically reduced levels of endogenous IGF-IR, and a decrease in relative staining intensity for IGF-IR protein. A marked suppression in tPA mRNA expression occurred in MDA-MB-435s cells accompanying inhibition of IGF-IR. When cells carrying the antisense IGF-IR expression construct were exposed to IGF-I, tPA protein accumulation was significantly lower than that of control transfected cells. To our knowledge, this study is the first to show a relationship between the IGF system and tPA. Strategies that target the IGF/tPA pathway could provide alternative treatments for patients with certain types of metastatic breast cancer.

Perillyl Alcohol Inhibits a Calcium-Dependent Constitutive Nuclear Factor-κB Pathway

The cell death induced by the monoterpene anticancer agent perillyl alcohol correlates to the increased expression of certain proapoptotic genes known to influence cell survival. Whereas sequence-specific DNA-binding factors dictate the expression patterns of genes through transcriptional regulation, those transcriptional factors influencing constitutive cell survival with perillyl alcohol treatment are not well studied. Here, we investigated whether the monoterpenes can regulate the activity of nuclear factor-kappaB (NF-kappaB), a calcium-dependent transcription factor necessary for survival in the WEHI-231 B-lymphoma cells. Unique among the monoterpenes, perillyl alcohol short-term treatment induced a persistent decrease of calcium levels, whereas other various monoterpenes caused transient reductions in calcium levels. Perillyl alcohol treatment also rapidly elicited reductions of NF-kappaB DNA-binding activity and target gene induction, which was associated with an increase in apoptosis in these B-lymphoma cells. This apoptosis was directly due to NF-kappaB because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-kappaB function to modulate gene expression patterns and cell survival of certain B-lymphoma cells. The effects of perillyl alcohol were not limited to these B-lymphoma cells but were also observed in MDA-MB 468 cells, an estrogen receptor-negative breast cancer cell line. These results identify a calcium-dependent NF-kappaB pathway as a molecular target of perillyl alcohol activity in different cancer cell types.

Function and prognostic value of tumor suppressor gene LKB1 in human breast carcinoma

To study the function and prognostic value of the recently identified tumor suppressor gene LKB1 in human breast cancer. Estrogen receptor-positive human breast cancer cells of MCF-7 line and estrogen receptor-negative human breast cancer cells of the lines MDA-MB-435 and MDA-MB-231 were cultured and tested for the LKB1 expression. Plasmid pcDNA4 TOPO HisMAX/LKB1 expressing LKB1 was constructed and transfected into the MDA-MB-435 cells. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein. Flow cytometry was used to determine the cell cycle. PCR and Western blotting were used to detect the expressions of LKB1 mRNA and protein in 121 human breast cancer samples. The association with relapse free survival (RFS) and overall survival (OS) was assessed by univariate analysis (log-rank test and Kaplan-Meier method). Human breast cells of MCF-7 line expressed LKB1 and the cells of MDA-MB-435 and MDA-MB-231 lines did not. Flow cytometry showed that 40.9% of the MDA-MB-435 transfected with pcDNA4 TOPO HisMAX/LKB1 was at the G(1) stage, a proportion significantly higher than that of the control (18.3%). The expressions of cyclin D1 and cyclin E were significantly down-regulated and the expression of p21(WAF1/CIP1) was significantly up-regualed. LKB1 was lowly expressed in 43 out of the 121 cases of breast cancer (35.4%) and highly expressed in 78 cases (64.6%). Low expression of LKB1 was correlated with pathological grade (P = 0.013), cancer size (P = 0.001), status of estrogen receptor (P = 0.048), and status of lymph nodes metastasis (P = 0.003). Univariate analysis showed that low expression of LKB1 was significantly correlated with shorter RFS (P = 0.048) and low OS (P = 0.003). Reintroducing LKB1 into breast cell of the lines lacking LKB1 expression restores LKB1 activity and induces growth suppression by G(1) cell cycle block. The LKB1 mediated G(1) cell cycle arrest is caused by up-regulation of the expression of p21(WAF1/CIP1) in MDA-MB-435 breast cancer cells.

A Novel RARβ Isoform Directed by a Distinct Promoter P3 and Mediated by Retinoic Acid in Breast Cancer Cells

Retinoids regulate gene transcription through activating retinoic acid receptors (RARs)/retinoic X receptors (RXRs). Of the three RAR receptors (alpha, beta, and gamma), RARbeta has been considered a tumor suppressor gene. Here, we identified a novel RARbeta isoform-RARbeta5 in breast epithelial cells, which could play a negative role in RARbeta signaling. Similar to RARbeta2, the first exon (59 bp) of RARbeta5 is RARbeta5 isoform specific, whereas the other exons are common to all of the RARbeta isoforms. The first exon of RARbeta5 does not contain any translation start codon, and therefore its protein translation begins at an internal methionine codon of RARbeta2, lacking the A, B, and part of C domain of RARbeta2. RARbeta5 protein was preferentially expressed in estrogen receptor-negative breast cancer cells and normal breast epithelial cells that are relatively resistant to retinoids, whereas estrogen receptor-positive cells that did not express detectable RARbeta5 protein were sensitive to retinoid treatment, suggesting that this isoform may affect the cellular response to retinoids. RARbeta5 isoform is unique among all of the RARs, because a corresponding isoform was not detectable for either RARalpha or RARgamma. RARbeta5 mRNA was variably expressed in normal and cancerous breast epithelial cells. Its transcription was under the control of a distinct promoter P3, which can be activated by all-trans-retinoic acid (atRA) and other RAR/RXR selective retinoids in MCF-7 and T47D breast cancer cells. We mapped the RARbeta5 promoter and found a region -302/-99 to be the target region of atRA. In conclusion, we identified and initially characterized RARbeta5 in normal, premalignant, and malignant breast epithelial cells. RARbeta5 may serve as a potential target of retinoids in prevention and therapy studies.

Digitalis-Induced Signaling by Na+/K+-ATPase in Human Breast Cancer Cells

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.

Ethanol stimulates proliferation, ERα and aromatase expression in MCF-7 human breast cancer cells

It is well documented that alcohol is associated with an increased risk factor for breast carcinogenesis although the underlying mechanisms are not clearly understood. It has been reported that in vitro, the culture of estrogen receptor (ER) expressing breast cancer cells in ethanol containing medium was associated with an increase in the proliferation rate, in the ERalpha content as well as in ER transcriptional activity. Since these changes are not observed in ER negative breast cancer cells, and since alcohol intake has been associated to an increased level of circulating estrogens, we have postulated that aromatase expression could be increased following ethanol exposure. The results of our studies show a 1.3-fold increase in cell proliferation after 6 days of culture of MCF-7 cells in the presence of 0.1% ethanol. This enhanced proliferation is confirmed by the use of clonogenic assays which show a 1.5-fold increase in clonal growth in the presence of 0.1% ethanol. No statistically significant changes were observed in the presence of higher ethanol concentration (0.3%). After a 6-day exposure to 0.1% ethanol, RT-PCR analyses reveal a 1.7-fold increase in ERalpha mRNA that was not significant, whereas western blot analyses show a significant 3.3-fold increase in ERalpha content. At the same stage, RT-PCR studies demonstrate a 2.4-fold increase in aromatase mRNA level which is confirmed at the protein level by western blots performed after immuno-precipitation of the enzyme. Taken together, these results are in agreement with the involvement of ER signalling in ethanol-induced stimulation of breast cancer cell proliferation and could help to understand why alcohol consumption is associated with breast cancer risk.

Indoleamine 2,3-dioxygenase activity and l-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with l-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-γ (1000 units/ml). Accordingly, l-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-γ. 1-Methyl-dl-tryptophan (1 mM) inhibited interferon-γ induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-γ. l-Tryptophan transport into MDA-MB-231 cells was via a Na+-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-d,l-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, l-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.

Effect of His-Gly-Lys Motif Derived from Domain 5 of High Molecular Weight Kininogen on Suppression of Cancer Metastasis Both in Vitro and in Vivo

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.

Differential Expression of Metallothionein 1 and 2 Isoforms in Breast Cancer Lines with Different Invasive Potential: Identification of a Novel Nonsilent Metallothionein-1H Mutant Variant

Metallothionein (MT), a low-molecular weight protein with pleiotropic functions, is believed to play an important role in tumorigenesis. The aim of this study was to compare the expression of functional MT-1 and MT-2 mRNA isoforms in five breast cancer cell lines ranging from noninvasive MCF7 breast cancer cells to highly aggressive MDA-MB-231 breast cancer cells together with breast myoepithelial cells in vitro by conventional semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The MT-2A isoform was observed to be differentially upregulated in the invasive phenotype. The MT-1E isoform was found to be present in estrogen receptor-negative breast cancer cell lines (MDA-MB-231 and Hs578T) but not detectable in the estrogen receptor-positive cell lines (T47D, MCF7, and ZR75-1 cells). Only the myoepithelial cells exhibited the presence of the MT-1G transcript. Direct sequencing of the RT-PCR products revealed the occurrence of a variant MT-1H isoform with changes in amino acid residues in the protein sequence and notable differences in the predicted secondary protein structure. The observations in this study are relevant to the development of novel approaches to metastatic breast cancer disease, and may herald the search for novel MT mutants and the elucidation of their biological roles.

Biologic activity of spores and dried powder from Ganoderma lucidum for the inhibition of highly invasive human breast and prostate cancer cells.

Ganoderma lucidum has been used in East Asia as a home remedy to prevent or cure cancer. Furthermore, Ganoderma lucidum is one of the herbs in the herbal mixture PC-SPES that has become an alternative herbal therapy for prostate cancer. Because the dried powder of ganoderma is commercially available as a dietary supplement itself, the purpose of this study was to evaluate the biologic activity of samples of Ganoderma lucidum from different sources. Samples of Ganoderma lucidum were characterized morphologically and evaluated for their ability to inhibit cell migration of highly invasive breast cancer MDA-MB-231 cells and prostate cancer PC-3 cells. Because the inhibition of cell motility is directly linked to the inhibition of the signaling pathway for constitutively active NF-kappaB in breast and prostate cancer cells, we determined how different samples of Ganoderma lucidum inhibit constitutively active NF-kappaB in a reporter gene assay. Some of the samples of Ganoderma lucidum demonstrated strong inhibition of cancer cell migration comparable to the inhibition of constitutively active NF-kappaB, whereas other samples showed less or no activity in highly invasive estrogen receptor-negative breast cancer cells or androgen receptor-negative prostate cancer cells, respectively. Interestingly, we did not find any correlation between the purity and composition (spores versus powder) of Ganoderma lucidum and biologic activity. Ganoderma lucidum has demonstrated strong activity against breast and prostate cancer cells. Nevertheless, the composition of samples did not correlate with their ability to inhibit cell migration and activation of NF-kappaB in vitro.

Estradiol enhances osteolytic lesions in mice inoculated with human estrogen receptor-negative MDA-231 breast cancer cells in vivo.

The effect of 17-beta-estradiol (E2) on the induction of osteolytic lesions by estrogen receptor (ER)-negative breast cancer cells was investigated in 4-week-old female nude mice. Exposure to exogenous E2 was found to increase osteolytic areas on radiographs up to 5.3 times in mice inoculated intracardially with MDA-231 human breast cancer cells. The MDA-231 cells were ER-negative, both before inoculation, and after isolation from osteolytic lesions, and the corresponding cell cultures were insensitive to E2. The induction of skeletal lesions by E2 in this mouse model was mainly effectuated at the early development of bone metastases, since exposure to E2 for 8 days around MDA-231 inoculation increased osteolysis to the same level, as did E2 given throughout the entire 31-day experimental period, and because E2-exposure for just the final 14 days had no effect. Independently of exposure to E2, histology revealed cancer cells in hind limp long bones of approximately 80% of the mice, and tumors were absent in non-skeletal organs. In vitro studies showed that the number and activity of osteoclasts generated from mouse bone marrow cells were increased 5-6 times when co-cultured with MDA-231 cells. Addition of 0.1-10 nM E2 further dose-dependently increased the osteoclastogenesis and associated bone resorption in these co-cultures. In conclusion, E2 was found to increase the morbidity in mice inoculated with ER-negative MDA-231 cells, and to stimulate osteoclast formation and bone resorption in co-cultures of bone marrow cells and MDA-231, suggesting that the progression of osteolytic metastases by ER-negative breast cancer cells can be induced by E2 due to stimulation of osteoclastogenesis.

Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation

Endocrine therapy is widely accepted for the treatment of hormone receptor-positive breast cancer. However, in many cases eventually resistance will develop and tumor regrows. Combination therapy may be one way to resolve this problem. In the present study we investigated the effect of a combination of the widely used antiestrogen tamoxifen with the endogenous estradiol metabolite 2-methoxyestradiol (2-ME) on the proliferation of human estrogen receptor-positive and receptor-negative breast cancer cells. The receptor-positive cell line MCF-7 and the receptor-negative cell line BM were treated with 4-hydroxytamoxifen (4-OHTam) and 2-methoxyestradiol in the concentration range of 0.8–25 μM alone and equimolar combinations for 4 days. The proliferation of the cells was determined using the ATP-chemosensitivity test. 4-Hydroxytamoxifen inhibited proliferation of MCF-7 and BM cells with IC 50 values of 31 and 10 μM, the corresponding figures for 2-methoxyestradiol were 52 and 8 μM. The combination showed IC 50 values of 6 μM and 4 μM. These results indicate that a combination of tamoxifen with 2-methoxyestradiol showed an additive inhibitory effect concerning the proliferation of estrogen receptor-positive and receptor-negative breast cancer cell lines. Thus a combination of these substances may allow ameliorating of adverse events of tamoxifen by reducing its concentrations and probably also drug resistance and should be tested in clinical trials.

Resistance to chemotherapy via Stat3-dependent overexpression of Bcl-2 in metastatic breast cancer cells.

Disruption of apoptosis may allow metastatic cell survival and confer resistance to chemotherapeutic drugs. We have analysed the molecular pathways that activate these survival genes in specific sites of metastasis. Estrogen receptor-negative breast cancer cell line MDA-MB435 and two metastatic sublines derived from lung (435L) and brain (435B) were analysed for the expression of members of the Bcl-2 family of apoptosis regulators. The levels of Bcl-2 were higher in the metastatic sublines than in parental cells, which correlated with the activation of Stat3, but not with the expression and/or activation of known bcl-2 transcription factors (CREB and WT1). In the brain subline, both expression of Bcl-2 and Stat3 activation were induced by epidermal growth factor and abrogated after treatment with kinase inhibitors specific for epidermal growth factor receptor or Jak2. Furthermore, transfection of 435B with a dominant-negative Stat3 markedly reduced the expression of Bcl-2 protein, whereas transient expression of a constitutively active Stat3 increased Bcl-2 in parental 435 cells. In addition, blockade of Stat3 activation by treatment with epidermal growth factor receptor and Jak2 kinase inhibitors or transfection with a dominant negative Stat3, sensitizes 435B cells to chemotherapy-induced apoptosis. Our data suggest that an increased activation of the Stat3-Bcl-2 pathway in estrogen receptor-negative metastatic breast cancer cell lines confer a survival advantage to these cells and contribute to their chemoresistance.