Estrogen Receptor-positive Breast Cancer Cells Research Articles

WW domain containing E3 ubiquitin protein ligase 1 (WWP1) expression in breast cancer and its correlation with estrogen receptor (ER) and insulin-like growth factor receptor 1 (IGF-1R) status

22076 Background: WWP1, a HECT type E3 ubiquitin ligase, is frequently amplified and overexpressed in breast cancer and may associated with positive ER status. IGF-1R is a tyrosine kinase growth factor receptor that has been linked to prognosis in a variety of malignancies. Activation of IGF-1R by autocrine, paracrine and endocrine stimulation via exposure to its activating ligand, insulin, leads to cell growth. Methods: Formalin-fixed, paraffin-embedded tissue sections from 122 cases of invasive mammary carcinoma (90 ductal carcinomas (IDC) and 32 lobular carcinomas (ILC) were immunostained by automated methods (Ventana Medical Systems Inc., Tucson, AZ) using mouse anti-human WWP1 (Novus Biologicals, Littleton, CO) and mouse anti-human IGF-1R antibody (sc-462; Santa Cruz Biotechnology, Santa Cruz, CA). Cytoplasmic immunoreactivity was semiquantitatively scored based on staining intensity and distribution for each protein and the results were correlated with morphologic and prognostic variables. The correlations between WWP1 and ER/IGF-1R were also analyzed in a panel of breast cancer cell lines by Western blot. WWP1 knockdown studies were used to test WWP1 regulation of ER and IGF-1R in ER positive breast cancer cell lines. Results: The adjacent benign epithelium was essentially negative for both proteins. Cytoplasmic WWP-1 immunoreactivity was observed in 53/122 (43%) tumors and showed a positive correlation with ER status (p=0.038). Cytoplasmic IGF-1R positivity was observed in 66/122 (54%) tumors and correlated with tumor subtype (61% IDC vs. 34% ILC, p=0.011) and ER status within the IDC subgroup (p=0.036). There was a significant co-expression of both proteins (p=0.001). The positive correlations between WWP-1 and ER/IGF-1R were also observed in a panel of breast cancer cell lines assessed by Western blot. WWP1 knockdown decreased the expression levels of both ER and IGF-1R in MCF7 and T47D cell lines. Conclusions: WWP-1 and IGF-1R proteins are overexpressed and are associated with each other and the ER+ phenotype in breast carcinoma. Further development of WWP-1 and IGF-1R as biomarkers for breast cancer management appears warranted. No significant financial relationships to disclose.

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

Choline Kinase Down-regulation Increases the Effect of 5-Fluorouracil in Breast Cancer Cells

Identifying strategies to increase cancer cell kill while sparing normal tissue is critically important in cancer chemotherapy. Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), is elevated in cancer cells and presents a novel target for increasing cell kill. Here, we have examined the effects of transiently down-regulating Chk by small interfering RNA against Chk (siRNA-chk) on PC and total choline-containing compound (tCho) levels and on the viability/proliferation of estrogen receptor-negative and estrogen receptor-positive breast cancer cell lines and a nonmalignant mammary epithelial cell line. We investigated the effects of combination treatment with transient siRNA-chk transfection and the anticancer drug 5-fluorouracil (5-FU) in those cell lines. Microarray analysis of the invasive estrogen receptor-negative MDA-MB-231 cell line was done to characterize molecular changes associated with Chk down-regulation. Chk down-regulation decreased PC and tCho levels in the malignant cell lines, whereas the cell viability/proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost negligible effect on PC and tCho levels as well as cell viability/proliferation in the nonmalignant cell line. A combination of siRNA-chk with 5-FU treatment resulted in a larger reduction of cell viability/proliferation in the breast cancer cell lines; this reduction was evident to a much lesser degree in the nonmalignant cells. Microarray analysis showed that Chk down-regulation affected 33 proliferation-related genes and 9 DNA repair-related genes. Chk down-regulation with siRNA-chk may provide a novel alternative to enhance the effect of anticancer drugs in malignant cells.

Oestrogen- and anti-oestrogen-regulated genes in human breast cancer.

The study of several human breast cancer cell lines containing oestrogen receptors has allowed characterization of a number of oestrogen-induced proteins (e.g. progesterone receptor, cathepsin D, pS2, Hsp27, c-Myc). In primary tumours these markers have different prognostic significance for predicting whether the tumour will be hormone responsive (e.g. pS2, progesterone receptor) and whether it will metastasize (e.g. cathepsin D). The mechanism of regulation of gene expression by oestrogens and anti-oestrogens in breast cancer is complex and varies according to the nature of both the gene and the cell in which it is transcribed. Our laboratory has identified the sequences mediating oestrogen activity in the proximal region of cathepsin D, including a non-consensus oestrogen-responsive element located at -260 which acts in synergy with other regulatory elements. In addition to the classical effect of oestrogen receptor in stimulating transcription of genes controlled by the oestrogen-responsive element, we found that estrogen receptor is able to modulate transcription of AP-1-responsive genes without interacting directly with DNA. Cross-talk between oestrogen receptor and members of the Fos/Jun family via protein-protein interactions may explain how anti-oestrogens inhibit the mitogenic effect of growth factors in the apparent absence of oestrogens and why tamoxifen is able to stimulate cathepsin D gene expression and induce apoptosis in certain oestrogen receptor-positive breast cancer cells. The nature and degree of this cross-talk appears to vary according to the gene, the cell type and the type of oestrogen receptor ligand involved. Studies of oestrogen-regulated genes are not only useful for classifying breast cancers according to their ability to metastasize and respond to therapies, but also should lead to new therapeutic approaches for hormone-dependent and hormone-resistant cancers.

Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation

BackgroundDe-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target β-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that, in addition to the canonical WNT/β-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation.MethodsUsing the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; β-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors, or blocking antibodies.ResultsPhospho-DVL and stabilized β-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active β-catenin levels, lowers ERK1/2 activity, blocks proliferation, and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1, and MCF-7 cells. The effects of WNT signaling are mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor.ConclusionOur data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments.

Effects of a phytoestrogen-containing soy extract on the growth-inhibitory activity of ICI 182 780 in an experimental model of estrogen-dependent breast cancer

The study reported here was designed to determine whether a phytoestrogen-containing soy extract (SSE) could negate/overwhelm the inhibitory effects of ICI 182 780 on the growth of estrogen-sustained human breast cancer xenografts (MCF-7), in ovariectomized athymic mice. As expected, estradiol-supplemented tumors did not grow over the study period in ICI 182 780-treated females; concomitant administration of 50 mg/kg per day SSE slightly potentiated the inhibitory activity of the drug, while at 100 mg/kg per day, SSE partially negated ICI 182 780 activity. In keeping with these in vivo outcomes, we observed that the level of cyclin D1 (and progesterone receptor) in MCF-7 xenografts was considerably reduced by ICI 182 780, an effect enhanced by concomitant treatment with 50 SSE, but reduced by the higher dosage (i.e. 100 mg/kg per day). Thrombospondin-1 (TSP-1) and kallikrein 6 (KLK6) levels were also reduced following ICI 182 780, although to a lesser degree; again, combined anti-estrogen and SSE produced a dose-dependent regulation in TSP-1 and KLK6 tumor level, with a further reduction in the mRNA gene expression at 50 SSE (compared with ICI 182 780) and a partial reversion of the drug-induced down-regulation at 100 mg/kg per day. No modulation was detected in the serum concentration of IGF-1 (a potent mitogen for estrogen receptor-positive breast cancer cell lines) either upon treatment with ICI 182 780 or concomitant administration of the anti-estrogen with SSE. In conclusion, results from this study raise concerns about the consumption of isoflavone supplements in conjunction with ICI 182 780 therapy, in postmenopausal women with estrogen-dependent breast cancer.

Secretion of Cytokines in Breast Cancer Cells: The Molecular Mechanism of Procathepsin D Proliferative Effects

Procathepsin D (pCD) is a major secreted protein in estrogen receptor-positive (ER+) breast cancer cell lines. Several independent studies have documented pronounced mitogenic effect of secreted pCD on cancer tissue-derived cell lines, including those from breast, lung, and prostate cancer. It has also been shown that the proliferative effect of pCD involves both autocrine and paracrine modes of action. Recent studies have suggested that pCD could act as a key paracrine communicator between cancer and stromal cells. We have shown earlier that the proliferative activity of pCD depends on the activation peptide sequence of pCD. The present study casts light on the mechanism by which pCD influences the proliferation of cancer cells expressing the ER. Results described in the current paper clearly show that pCD initiates secretion of cytokines interleukin-4 (IL-4), IL-8, IL-10, IL-13, macrophage inflammatory protein-1beta and (MIP-1beta) from such tumor cells. Secreted cytokines take part in the proliferation of the cancer cells, as proven by selective inhibition using antibodies. In addition, expression of cytokine receptors on tested cell lines corresponded to the effects of individual cytokines. An analogous pattern was also observed for fibroblasts, which, under physiologic conditions, are the cells in closest contact with the tumor tissue and play a role in tumor growth and invasion. Our observations were further supported by coculture experiments that are in agreement. Although very similar in response to addition of pCD, the invasive ER- cells do not secrete cytokines. Together with previous in vivo results, these data point to pCD as one of key molecules for therapeutic attack in breast cancer.

Inhibition of estrogen-induced mammary tumor formation in MMTV-aromatase transgenic mice by 4-chlorophenylacetate

Treatment of estrogen-sensitive breast cancer with selective estrogen selective modulators (SERMs) and, more recently, aromatase inhibitors has met with wide success. However, antagonism of estrogen receptor (ER) activity in breast carcinomas by SERMs such as tamoxifen has been associated with increased risk of cancer in other tissue such as the endometrium. Furthermore, current therapies using aromatase inhibitors have side effects on bone resulting in development of osteoporosis in some patients. We present in this paper the results of a study using 4-chlorophenylacetate (4-CPA), a compound which belongs to a family of small aromatic fatty acids that has been shown to possess anticancer properties, to treat DMBA exposed MMTV-aromatase mice. These animals exhibit elevated levels of estrogen in their mammary glands and develop estrogen-responsive tumors. Consistent with our earlier findings showing that 4-CPA inhibited the growth of ER positive breast cancer cells in vitro, we now demonstrate that this compound inhibits tumor formation in MMTV-aromatase mice. This effect was not associated with reduction of ER expression in their mammary tissue, or to alteration of aromatase levels or activity. The data suggest that 4-CPA is a novel therapeutic agent that could be used in the prevention or treatment of estrogen-sensitive breast cancer.

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor– positive breast cancer cells

Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.

Methyl donor nutrients inhibit the growth of human breast cancer cells

Lipotropes (methyl donor nutrients) are involved in one-carbon metabolism including DNA methylation. Hypermethylation of CpG islands within promoter regions of genes is associated with transcriptional inactivation of the gene. Methyl donors may alter the expression of apoptosis-related genes via alterations in DNA methylation and consequently increase the sensitivity of cancer cells to apoptosis. To investigate the effects of lipotropes on breast cancer cell growth and apoptosis, two estrogen receptor-positive breast cancer cell lines, MCF-7 and T47D, as well as a normal mammary cell line, MCF-10A, were cultured in control, lipotrope-supplemented, tamoxifen (TAM), and lipotrope-supplemented plus TAM treatment media. Cell proliferation was measured by a tetrazolium salt reduction assay. Lipotrope supplementation inhibited the growth of both T47D (P < 0.01) and MCF-7 (P < 0.01) cell lines. All treatment groups, except the control groups in all cell lines, demonstrated morphological changes, as assessed by DAPI staining, consistent with apoptotic cell death. Lipotropes significantly decreased the Bcl-2 protein level, quantified by an enzyme immunometric assay, in the T47D cell line (P = 0.01) but not in the MCF-7 cell line. These results show that lipotrope supplementation may be useful in the development of a nutritional strategy for human breast cancer therapy. Supported by NIH-NCI (# R01CA78179-01).

Effect of Antiestrogens on EGF-Mediated Movement of Human Breast Cancer Cells

In a previous study we compared the influence of several growth factors on breast cancer cells in culture and observed that epidermal growth factor (EGF) enhanced the invasiveness of estrogen receptor-positive breast cancer cells. The objective of the present study was to determine the influence of three unique antiestrogens on EGF-mediated movement of human breast cancer cells. The rate of movement of MCF-7 breast cancer cells was measured using time-lapse videomicroscopy (TLVM). The MCF-7 cells were pretreated with antiestrogen (either tamoxifen, ICI-182-780 (ICI) or 1,1-dichloro-cis-2,3-diarylcyclopropane (AII)) at 10^–6 mol/l for 4 days, and then treated with EGF (10^–10 mol/l) immediately prior to TLVM. EGF enhanced the motility of the MCF-7 cells at 30–90 min post-administration. However, EGF-mediated motility of the MCF-7 cells was inhibited by antiestrogen pretreatment, with TAM and ICI producing complete inhibition of EGF-induced motility. In conclusion, this study demonstrates that EGF enhances the rate of movement of MCF-7 breast cancer cells and that antiestrogen pretreatment inhibits EGF-mediated motility.

Molecular cloning and characterization of the human WISP-2/CCN5 gene promoter reveal its upregulation by oestrogens

Wnt-1-induced signalling pathway protein-2 (WISP-2)/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN)5 is a member of the CCN family of growth factors and was identified as an oestrogen- inducible gene in the MCF-7 cell line. However, the role of WISP-2/CCN5 in breast carcinogenesis remains unclear. In this study, we examined the mechanism by which oestrogens regulate the expression of human (h) Wnt-1 induced signalling pathway protein (WISP-2)/CCN5. Real-time RT-PCR showed that hWISP-2/CCN5 mRNA transcripts level is upregulated by oestrogens in the oestrogen receptor-positive human breast cancer cell lines MCF-7, T47D and ZR-75.1. Cloning of a 1.9 kb fragment of the hWISP-2/CCN5 5'-flanking sequence and subsequent analysis of potential transcription factor-binding sites identified a functional oestrogen response element site located between - 581 and - 569 upstream from the oestrogen-induced transcription start site. Transient transfections of MCF-7 cells with the cloned fragment showed that oestradiol caused an increase in reporter gene activity, which was inhibited by anti-oestrogens ICI 182 780 and 4-hydroxytamoxifen. Chromatin immunoprecipitation analysis revealed an oestradiol-dependent recruitment of the oestrogen receptor alpha to the oestrogen- responsive region of the hWISP-2/CCN5 gene promoter. We also showed that endogenous CREB-binding protein (CBP) and p21(WAF1/CIP1) are recruited to the chromosomal hWISP-2/CCN5 promoter in MCF-7 cells in an oestrogen-dependent manner, suggesting that CBP and p21(WAF1/CIP1) participate in the oestrogen receptor alpha-mediated transcriptional control of the hWISP-2/CCN5 gene.

The Forkhead Box M1 Protein Regulates the Transcription of the Estrogen Receptor α in Breast Cancer Cells

In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor alpha (ERalpha) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ERalpha at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75-30, led to up-regulation of ERalpha expression at protein and transcript levels. Furthermore, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ERalpha expression. Consistent with this, silencing of FoxM1 expression in MCF-7 cells using small interfering RNA resulted in the almost complete abrogation of ERalpha expression. We also went on to show that FoxM1 can activate the transcriptional activity of human ERalpha promoter primarily through two closely located Forkhead response elements located at the proximal region of the ERalpha promoter. Chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays have allowed us to confirm these Forkhead response elements as important for FoxM1 binding. Further co-immunoprecipitation experiments showed that FoxO3a and FoxM1 interact in vivo. Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ERalpha gene transcription.

Additive antitumor effect of concurrent treatment of 4-hydroxy tamoxifen with 5-fluorouracil but not with doxorubicin in estrogen receptor-positive breast cancer cells

The sequential addition of tamoxifen (TAM) to chemotherapy seems superior to its concurrent addition in patients with breast cancer. This study was conducted to clarify the hypothesis that there are differential interactions among TAM and chemotherapeutic agents. Estrogen receptor (ER)-alpha-positive or -negative breast cancer cells were treated with 4-hydroxy TAM (4OHT), 5-fluorouracil (FU) and/or doxorubicin (Dox). Changes in the expression levels of genes related to sensitivity and resistance to TAM, 5-FU or Dox were tested. Concurrent treatment of 4OHT with 5-FU but not with Dox additively inhibited the growth of ER-alpha-positive cells. 5-FU did not change the expression levels of any tested genes related to either sensitivity or resistance to TAM. Although Dox did not change the expression levels of any genes related to the sensitivity to TAM, Dox significantly increased the expression levels of some genes related to TAM resistance, Eph A-2, ER-beta, Fos and vascular endothelial growth factor. 4OHT significantly decreased thymidilate synthase (TS) activity. Although the antitumor effect of concurrent 4OHT and 5-FU was additive, that of concurrent 4OHT and Dox was less than additive in ER-alpha-positive cells. The increased expression of genes related to TAM resistance by Dox might be responsible for the interaction. Decreased TS activity by 4OHT might increase the antitumor activity of 5-FU. These findings may provide a preclinical rationale for concurrent use with 5-FU and TAM.

Estrogen enhances the efficacy of an oncolytic HSV-1 mutant in the treatment of estrogen receptor-positive breast cancer

Oncolytic herpes simplex virus-1 (HSV-1) mutants selectively replicate in and lyse tumor cells. Viral replication is dependent on the cellular proliferative mechanism. Estrogen increases cellular proliferation and decreases apoptosis in estrogen receptor-positive (ER+) human breast cancer cells. We hypothesize that the cellular changes produced by estrogen may enhance oncolytic viral replication and improve the treatment of ER+ breast cancer cells. Estrogen increased proliferation and replication of the HSV-1 mutant, NV1066, in ER+ breast cancer cells. Additionally, cells grown with estrogen had lower rates of apoptosis and higher bcl-2 levels at baseline and after infection. Estrogen enhanced the oncolytic effect of NV1066, with cell kills of 95% and 97% at MOIs of 0.1 and 0.5, compared to 53 and 87% respectively without estrogen (p<0.001). Therapy of ER+ human breast cancer cells with a replication-competent HSV-1 mutant is improved in the presence of estrogen, in contrast to more standard therapies, such as chemotherapy and radiation, which demonstrate decreased efficacy in similar conditions. These data provide the mechanistic basis for the use of oncolytic HSV-1 in patients with hormone receptor-positive breast cancer, particularly if the disease progresses with conventional therapies.

Involvement of IL-8 in COX-2-Mediated Bone Metastases from Breast Cancer

Cyclooxygenase-2 (COX-2) overexpression by a primary tumor correlates with poor prognosis in breast cancer, including early spread to bone. Interleukin-8 (IL-8) stimulates osteoclastogenesis and resorption of bone, and elevated IL-8 levels predict early metastatic spread of breast cancer. The purpose of this study was to test our hypothesis that tumors that overexpress COX-2 induce IL-8 production. We cotransfected MCF-10A (nonmalignant breast epithelial) cells, as well as MDA-231 (highly metastatic human breast cancer) cell lines with a pSG5-COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). COX-2 overexpression was confirmed by Western blot and PGE2 (a product of the COX-2 pathway) immunoassay. IL-8 production was measured by immunoassay. In vivo testing used a nude mouse model to measure COX-2 and IL-8 production from breast cancer cells that had metastasized to bone (bone-seeking clones (BSCs)). Long bone metastases were localized and quantified by luciferase imaging (Xenogen IVIS system) and X-ray. BSCs were isolated and cultured and then tested for the production of PGE2 and IL-8. COX-2 overexpression caused a 4- to 5-fold increase in IL-8 production in both MCF-10A and MDA-231 cells in vitro. In vivo, we observed that the MDA-231-BSC (metastatic cells isolated from bone metastases) produced significantly greater levels of both PGE2 and IL-8 compared to the parental MDA-231 cells (P < 0.01). In contrast to the results obtained with these estrogen receptor-negative cell lines, COX-2 expression failed to induce IL-8 in the MCF-7 estrogen receptor-positive breast cancer cell line. Treatment with the COX-2 inhibitor NS-398 at a low 1-mu[scap]M dose reduced the production of IL-8 in COX-2-transfected MDA-231 cells by 30%, thus confirming the involvement of COX-2 in IL-8 induction. COX-2 expression induced formation of PGE2 and IL-8 in breast cancer cells. Since PGE2 and IL-8 stimulate osteoclasts to resorb bone, COX-2 inhibition is a potential target for treatment to prevent bone metastases.

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.

Differential Regulatory Effects of Nitric Oxide on Estrogen Stimulated MCF7 Breast Cancer Cell Growth

17-β estradiol (E2) stimulates the growth of estrogen receptor (ER)positive cells such as MCF7 line of human breast cancer cells. There have been reports showing E2 affects the level of endothelial nitric oxide synthase (eNOS) expression and its activity in vitro. Using ER positive MCF7 breast cancer cells as a model, we have studied the relationship between E2 and NO in controlling cell growth. MCF7 cells were cultured in medium containing 5% charcoal-stripped fetal bovine serum (SFBS) or serum-free medium (SFM) respectively. Using [methyl-3H]thymidine incorporation as the growth assay, E2 stimulated cell growth in a dose-response manner under both conditions. In the presence of 5 mM N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, an enhancement of E2 stimulation on MCF7 cell growth of up to 2 folds was observed when cultured with 5% SFBS, however, it completely blocked the stimulatory effect of E2 on cell growth under serum-free condition. Fluorescent agent DAF-2A was used to measure NO production by MCF7 cells under different treatments. We found 1 nM E2 decreased NO production by 50% compared with untreated cells in 5% SFBS but did not significantly change NO production under serum-free condition. 5 mM L-NAME suppressed NO production in all conditions below their respective untreated control level. Our data show that in spite of the stimulatory effect of E2 on MCF7 cell growth, there is a differential regulation of NO production by E2 under different culture conditions and NO in turn differentially regulates the growth activity of breast cancer cells.

Effect of chlorination on estrogenicity in chlorinated treated effluent

Effluents from sewage treatment plants can be discharged into rivers with estrogenic contaminants at levels sufficient to induce adverse reproductive and fertility developments in humans and wildlife. Of great concern in recent years are the estrogenic activities of chlorinated by-products (CBPs) in effluents. Simplified cell proliferation tests using the human estrogen receptor-positive MCF-7 ATCC breast cancer cells (E-SCREEN) were performed to investigate the influence of chlorination on the estrogenicity in effluents. It was found that the increase of chlorine dosages from 0 mg/L to 4 mg/L led to the decrease of total content of estrogenic activity: the 17ß-estradiol equivalent concentration (EEQ) from 22.40 to 8.35 ng/L. However, the increase of contact time from 10–30 minutes increased EEQ from 14.04 to 29.97 ng/L. Furthermore, increasing TOC level using humic acid from 5 mg/L to 15 mg/L in effluents correspondingly resulted in an increase of EEQ from 19.69–27.20 ng/L; it was thus confirmed that chlorination of humic acid could produce estrogenic by-products.

Sequence-dependent synergistic effects of ibandronate in combination with antiestrogens on growth inhibition of estrogen receptor-positive breast cancer cells

Sequence-dependent synergistic effects of ibandronate in combination with antiestrogens on growth inhibition of estrogen receptor-positive breast cancer cells