Normal Mammary Epithelial Cell Line Research Articles

18β‐glycyrrhetinic acid induces apoptosis through modulation of Akt/FOXO3a/Bim pathway in human breast cancer MCF‐7 cells

Triterpenes found in plants display a multitude of biological activities, including anti-tumor properties. The present study investigates the effect of 18β-glycyrrhetinic acid (GRA) a pentacyclic triterpenoid of the β-amyrin type, isolated from the root of Licorice (Glycyrrhizza glabra) on human breast cancer cells, MCF-7. GRA showed potent inhibitory effects on MCF-7 proliferation in a concentration- and time-dependent manner without affecting immortalized normal mammary epithelial cell line (MCF-10A). Growth inhibition of MCF-7 cells by GRA occurred through apoptosis, as evident from phosphatidyl serine externalization and DNA fragmentation. Apoptosis was primarily mediated through mitochondrial death cascade as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9. GRA induced an increase in Bax:Bcl-2 ratio along with a significant increase in the protein level of the BH3 protein Bim. SiRNA-mediated knock down of Bim markedly attenuated GRA-mediated apoptosis. Profiling of transcriptional regulators of Bim revealed a role of Forkhead box O 3a transcription factor (FOXO3a) as judged by increased expression and nuclear translocation of FOXO3a. Silencing of FOXO3a resulted in marked attenuation in the expression of Bim as well as protection against GRA-mediated apoptosis. Furthermore, GRA-induced activation and nuclear localization of FOXO3a was associated with a reduced activity of Akt kinase. These results suggest that GRA induces apoptosis in human breast carcinoma MCF-7 cells via caspase activation and modulation of Akt/FOXO3a pathway.

Functional elements demarcated by histone modifications in breast cancer cells

Histone modifications are regarded as one of markers to identify regulatory elements which are DNA segments modulating gene transcription. Aberrant changes of histone modification levels are frequently observed in cancer. We have employed ChIP-Seq to identify regulatory elements in human breast cancer cell line, MCF-7 by comparing histone modification patterns of H3K4me1, H3K4me3, and H3K9/14ac to those in normal mammary epithelial cell line, MCF-10A. The genome-wide analysis shows that H3K4me3 and H3K9/14ac are highly enriched at promoter regions and H3K4me1 has a relatively broad distribution over proximity of TSSs as well as other genomic regions. We identified that many differentially expressed genes in MCF-7 have divergent histone modification patterns. To understand the functional roles of distinctively histone-modified regions, we selected 35 genomic regions marked by at least one histone modification and located from 3 to 10kb upstream of TSS in both MCF-7 and MCF-10A and assessed their transcriptional activities. About 66% and 60% of selected regions in MCF-7 and MCF-10A, respectively, enhanced the transcriptional activity. Interestingly, most regions marked by H3K4me1 exhibited an enhancer activity. Regions with two or more kinds of histone modifications did show varying activities. In conclusion, our data reflects that comprehensive analysis of histone modification profiles under cell type-specific chromatin environment should provide a better chance for defining functional regulatory elements in the genome.

Differential control of growth, apoptotic activity, and gene expression in human breast cancer cells by extracts derived from medicinal herbs Zingiber officinale.

The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale) on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A). Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose) polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4). On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT). These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas.

Impact of infertility regimens on breast cancer cells: follicle-stimulating hormone and luteinizing hormone lack a direct effect on breast cell proliferation in vitro

To examine the impact of hormones used for controlled ovarian hyperstimulation (COH) on normal and malignant breast cell growth and proliferation. In vitro study of cultured normal and malignant breast cell lines. Academic medical center. None. Normal and malignant breast cell lines cultured in two- and three-dimensional (2D and 3D) systems and treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), or FSH with LH or human chorionic gonadotropin (hCG). Effects of treatment on cell proliferation in 2D culture using the MTS assay and on colony growth in 3D culture. Compared with untreated cells, normal MCF-10A cells showed a decrease in proliferation and colony size when exposed to a combination of FSH and hCG. The HCC 1937 cells treated with FSH and LH also showed a decrease in colony growth but no change in proliferation. None of the treatments had an effect on the proliferation or colony size of the MCF-7 cells. Follicle-stimulating hormone, LH, and hCG do not appear to cause an increase in cell proliferation or colony growth in either normal or malignant mammary epithelial cell lines. The potential risk for mammary cell transformation associated with these agents may be related to indirect endocrine effects on breast cell physiology.

Repression of cancer cell senescence by PKCι

Senescence is an irreversible growth arrest phenotype adopted by cells that has a key role in protecting organisms from cancer. There is now considerable interest in therapeutic strategies that reactivate this process to control the growth of cancer cells. Protein kinase-Cι (PKCι) is a member of the atypical PKC family and an important downstream mediator in the phosphoinositide-3-kinase (PI-3-kinase) pathway. PKCι expression was found to be upregulated in a subset of breast cancers and breast cancer cell lines. Activation of the PI-3-kinase pathway by introduction of mutant, oncogenic PIK3CA into breast mammary epithelial cells increased both the expression and activation of PKCι. In breast cancer cells lines overexpressing PKCι, depletion of PKCι increased the number of senescent cells, as assessed by senescence-associated β-galactosidase, morphology and bromodeoxyuridine incorporation. This phenomenon was not restricted to breast cancer cells, as it was also seen in glioblastoma cells in which PKCι is activated by loss of PTEN. Senescence occurred in the absence of a detectable DNA-damage response, was dependent on p21 and was enhanced by the aurora kinase inhibitor VX-680, suggesting that senescence is triggered by defects in mitosis. Depletion of PKCι had no effect on senescence in normal mammary epithelial cell lines. We conclude that PKCι is overexpressed in a subset of cancers where it functions to suppress premature senescence. This function appears to be restricted to cancer cells and inhibition of PKCι may therefore be an effective way to selectively activate premature senescence in cancer cells.

The Natural Product NI-07, Is Effective Against Breast Cancer Cells While Showing No Cytotoxicity to Normal Cells

With the exception of a few anticancer agents, most breast cancer chemotherapeutics currently used have devastating effects on normal cells. We investigated the effectiveness of the natural product NI-07, derived from Arctium lappa,� to determine its killing potential on breast cancer cells and its cytotoxic effects in breast normal cells. The breast cancer cell lines HCC1419, MCF7, MDA-MB-231, MDA-MB-468, SKBR3, the normal mammary epithelial cell line HME 50 HT and the normal mammary fibroblast cell line CCD-1074sk were analyzed in concurrently treated cultures of NI-07, Taxol™ or Untreated. NI-07- treated and Taxol™ -treated cells were cultured for two weeks to assess cell resistance/recovery. Cell viability was determined by cell counts, Trypan Blue exclusion and microscopy. Additionally, cell viability and cytotoxicity were measured by XTT. Statistical significance (p � 0.05) of NI-07 to Untreated or Taxol™ was first determined using Two-Way ANOVA. Identified significant differences were further analyzed by One-Way ANOVA and Tukey's test for honestly significant differences. The effect size of NI-07 compared to untreated or Taxol™ was determined using Cohen's d. Our results showed significant declines in cell viability occurring in NI-07- treated cancer cells after 48 hours of treatment in contrast to the more rapid effect of Taxol™ (<24h). Additionally, cancer cell recovery was less effective in NI-07 versus Taxol™. Furthermore, NI-07 showed no cytotoxicity in normal cells. This lack of cytotoxicity, coupled with its killing efficiency in cancer cells, suggests that NI-07 could potentially make a strong anti-cancer compound or neo-adjuvant to current chemotherapy-based treatments.

RNAi Screening Identifies TAK1 as a Potential Target for the Enhanced Efficacy of Topoisomerase Inhibitors

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.

Withaferin A-Induced Apoptosis in Human Breast Cancer Cells Is Mediated by Reactive Oxygen Species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

NJ262 Induces Apoptosis in Breast Cancer Cells

Gene deletion in normal host cells is one of the prevailing causes of breast cancer in humans. In this investigation, breast cancer cell line, MCF-7, was treated with our newly discovered DNA polymorphic biomarker, NJ262, {NIH/NCBI Accession Number DQ676892}. NJ262 is present in normal human mammary epithelial cell line, MCF-10A but absent in breast cancer cell line, MCF-7. MCF-7 cells expressing NJ262 displayed cytoplasmic swelling and DNA laddering that are evidence of cell death by necrosis. Some of the transformed cells exhibit chromatin condensation, membrane blebbing, cytoplasmic condensation and nuclear membrane margination that are typical of cells death by apoptosis. NJ262 expression in MCF-7, therefore, induces cell death by both apoptosis and necrosis. The cell proliferation and apoptotic assays of transformed MCF-7 cells revealed that NJ262 reduced MCF-7 cell proliferation by over 50%, and increased cell death by 200%. This study, for the first time, showed that treatment of breast cancer cells with NJ262 down-regulated cell proliferation and drastically induced cell death in breast cancer cells.

Abstract 5422: Delphinidin dietary anthocyanidin modulates EGFR/HER-2 signaling pathway in breast cancer cells

Abstract Breast cancer is the second leading cause of cancer deaths in women in the United States. The epidermal growth factor receptor (EGFR) and HER-2 are expressed in many breast cancer patients and are associated with poor prognosis. Over-expression of EGFR and HER-2 lead to resistance against tamoxifen, a selective estrogen receptor (ER) modulator. In addition, the efficacy of trastuzumab or lapatinib for the treatment of ER-negative breast cancer as a monotherapy or in combination with chemotherapy is limited in more advanced stages of HER-2 positive breast cancers by development of therapeutic resistance. Considerable attention has been devoted towards the development of dual inhibitors of EGFR and HER-2 for breast cancer treatment. Several of these inhibitors such as lapatinib have been tested preclinically and clinically, but their use is limited because of their unacceptable cytotoxic effects on normal cells or cancer cells acquire resistance to such treatment. Therefore, identification of a natural, nontoxic agent(s) capable of inhibiting both EGFR and HER-2 is of utmost importance. Delphinidin, an anthocyanidin, present in many pigmented fruits and vegetables possesses anti-oxidant and anti-proliferative properties. We showed that delphinidin inhibits constitutive and EGF-induced kinase activities of EGFR (Afaq et. al., Int. J Cancer 2008; 123: 1508-1515) in breast cancer cell lines. Recently, we found that delphinidin treatment also inhibits protein expression of HER-2 in breast cancer cells. The goal of the present study was to determine the antiproliferative and proapoptotic potentials of delphinidin in ER-positive and ER-negative breast cancer cell lines that overexpress EGFR/HER2. We first determined whether blockade of EGFR/HER-2 signaling pathways by delphinidin exhibits growth inhibitory effect. Delphinidin (5-60 μM; 72 hrs) treatment of ER-positive (BT-474 and MCF-7) and ER-negative (MDA-MB-231 and MDA-MB-468) breast cancer cells resulted in a dose-dependent decrease in cell viability but had only minimal effects on normal mammary epithelial cell line, 184A1. The signaling pathways induced by activated EGFR/HER-2 include the PI3K/AKT and MAPK, both of which play a role in the mitogenic and cell survival responses. Therefore, we analyzed the expression of these proteins and found that delphinidin treatment of ER-positive and ER-negative breast cancer cells inhibited the activation of PI3K and phosphorylation of AKT and MAPKs. Treatment of BT-474 and MDA-MB-231 cells with delphindin (5-60 μM; 72 hrs) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and-9, (iii) downregulation of cyclin D1 and Bcl-2, and (iv) increased expression of p27 and Bax. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be useful for the management of both ER-positive and ER-negative breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2011-5422

Abstract LB-131: Decreased nuclear localization and expression of the FRY tumor suppressor gene, a repressor of epithelial mesenchymal transition, are highly correlated with breast cancer phenotype, grade, and progression

Abstract Using Quantitative Trait Locus (QTL) mapping in a backcross between susceptible Fischer 344 and resistant Copenhagen rat strains, we identified Fry, the rat ortholog of the Drosophila melanoganster furry gene, as a mammary carcinoma susceptibility (Mcs) gene. The Fischer 344 Fry allele harbors two single nucleotide polymorphisms that alter amino acid residues that are highly conserved from yeast to humans. One of these SNPs replaces an Alanine with a Serine residue, creating a de novo concensus sequence for several cancer-associated protein kinases. In the lower eucaryoyes, the furry orthologs are implicated in diverse cell processes including epithelial cell polarization, morphogenesis, proliferation, cytokinesis, and regulation of the NDR family of kinases, some of which are putative human tumor suppressor genes. Consistent with its function as a tumor suppressor gene, ectopic expression of the FRY gene was reduced in 3 of 4 human breast cancer cell lines. Ectopic expression of the wildtype allele significantly reduced the tumorigenicity of the human MDA-MB-231 mammary carcinoma cell line. In contrast to the parental MDA-MB-231 cells, which grew randomly in Matrigel® matrix, those expressing Fry (231CFry) formed spherical colonies of differentiated cells, closely resembling those formed by MCF-10A normal mammary epithelial cell line. When xenographed into nude mice, the 231CFry cells demonstrated a dramatic reduction in tumorigenicity (8x reduction in tumor volume at 15 days) and diminished invasiveness into surrounding tissues. Gene expression profiling suggested that in MDA-MB-231 cells, ectopic Fry induced the expression of gene networks involved in cell differentiation, proliferation, and polarization, consistent with a role for FRY in suppressing epithelial-mesenchymal transition (EMT). To investigate the role of human FRY in breast cancer, we examined FRY mRNA expression in &amp;gt;1,500 human breast cancers, representing distinct eight cohorts entered into the Oncomine 3.0 Cancer Profiling Database. Our analysis revealed that decreased FRY mRNA was highly correlated with poorly differentiated breast cancer phenotypes (p&amp;lt;0.0001), increasing Elston tumor grade (p&amp;lt;0.0001) and clinically aggressive cancer phenotypes (p&amp;lt;0.05). Using an anti-FRY peptide antiserum, we examined FRY protein expression in cores comprising the BR208 breast cancer and tissue microarrays from US Biomax, Inc. Immunohistochemical staining patterns were analyzed by quantitative image analysis software and independently by a board certified pathologist. The analyses indicated that decreased nuclear localization and expression of FRY were highly correlated with histopathology, clinical grade and progression, indicating that the FRY tumor suppressor is an important new biomarkers of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2011-LB-131

Abstract 1224: Repression of cancer cell senescence by PKCι

Abstract Senescence is an irreversible growth arrest phenotype adopted by cells that has a key role in protecting organisms from cancer. There is now considerable interest in therapeutic strategies that reactivate this process to control the growth of cancer cells. Protein kinase C iota (PKCι) is a member of the atypical protein kinase C family and an important downstream mediator in the phosphoinositide pathway. PKCι expression was found to be upregulated in a subset of breast cancers and breast cancer cell lines. Introduction of mutant, oncogenic PIK3CA, but not wild-type PIK3CA, into breast mammary epithelial cells increased both the expression and activation of PKCι. In breast cancer cell lines overexpressing PKCι, depletion of PKCι increased the number of senescent cells, as assessed by senescence-associated β-galactosidase, morphology and bromodeoxyuridine incorporation. This phenomenon was not restricted to breast cancer cells, as it was also seen in glioblastoma cells. Senescence induction did not require p53, p16 or Arf and was not associated with activation of the DNA damage response. Depletion of PKCι had no effect on senescence in normal mammary epithelial cell lines. We conclude that PKCι is overexpressed in a subset of cancers where it functions to suppress premature senescence. This function appears to be restricted to cancer cells and inhibition of PKCι may therefore be an effective way to selectively activate premature senescence in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1224. doi:10.1158/1538-7445.AM2011-1224

Abstract 1456: Prostaglandin E2 receptor EP1 as a metastasis suppressor in breast cancer

Abstract The cyclooxygenase-2 enzyme (COX-2) is highly expressed in breast cancer and contributes to the metastatic process. Inhibition of COX-2 can decrease breast tumor growth and metastasis. Prostaglandin E2 (PGE2), the chief COX-2 product in tumors, has been identified as a predominant protumorigenic prostanoid. PGE2 mediates biological effects by binding to each of four EP receptors (EP1-4). EP1 is coupled to calcium mobilization and PKC; whereas, EP2 and EP4 activate PKA/cAMP, PI3K and ERK pathways. EP3 generally inhibits cAMP levels. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Using a combination of immunohistochemistry, pharmacologic and genetic approaches, we studied EP1 expression in human breast tissue and function in a murine model of breast cancer metastasis. Our published studies indicate that EP1 was detected by immunohistochemistry in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression. Additionally, pharmacologic antagonism and reduction of EP1 expression by shRNA increased metastatic capacity in our murine model of metastatic breast cancer. These data suggested that EP1 may function as a metastasis suppressor. We now report that murine mammary tumor cell lines 410, 410.4 and 66.1 have decreased mRNA levels of EP1 compared to normal murine mammary epithelial cell line EpH4. The EP1 receptor is normally coupled to calcium mobilization. Even though EP1 expression is decreased in mammary tumor cell lines, preliminary studies suggest the existence of functional EP1 receptor as PGE2 is capable of inducing calcium mobilization in 410.4 cells. In addition, treatment of 410 and 66.1 cells with 5-azacytidine is capable of increasing EP1 mRNA levels which may identify a clinically relevant strategy to increase expression of this protective receptor. We have also identified the presence of a variant EP1 (EP1v) transcript in this murine metastatic breast cancer model. EP1v has been identified previously in murine mast cell line MC/9 and in rat uterus; however, this is the first report that EP1v has been identified in malignant cells. Like EP1, mRNA expression of EP1v is also decreased in 410, 410.4 and 66.1 cell lines compared to EpH4 cells. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2011-1456

Down-Regulation of FXYD3 Is Induced by Transforming Growth Factor-.BETA. Signaling via ZEB1/.DELTA.EF1 in Human Mammary Epithelial Cells

FXYD3, a regulator of Na, K-ATPase, was identified as an mRNA overexpressed in murine breast cancers induced by neu oncogene, which had inactivated transforming growth factor (TGF)-β signaling due to the defect of TGF-β receptor I (TβRI) expression. To elucidate whether the expression of FXYD3 mRNA was regulated by TGF-β signaling, we used a normal human mammary epithelial cell line, MCF-10A which responds to TGF-β and tumor necrosis factor (TNF)-α, followed by induction of epithelial-to-mesenchymal transition (EMT). Here, we showed that FXYD3 at plasma membrane in epithelial state of MCF-10A cells was decreased by treatment of TGF-β and TNF-α. The repression of FXYD3 mRNA induced by TGF-β and TNF-α in MCF-10A cells was abolished by TβRI inhibitor or Smad3 inhibitor, but not by small interfering RNA (siRNA) for Smad2. In addition, expression level of FXYD3 mRNA was up-regulated by the silencing of ZEB1/δEF1 transcriptional repressor which was a down-stream target gene of TGF-β and an inducer of EMT. On the other hand, expression level and cellular localization of E-cadherin and N-cadherin were not changed by siRNA for FXYD3 in MCF-10A and human breast cancer MCF-7 cells. These results suggest that FXYD3 is a target gene of TGF-β signaling through ZEB1/δEF1, but is not directly involved in EMT.

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Mitochondrial amplification selectively increases doxorubicin sensitivity in breast cancer cells with acquired antiestrogen resistance

The metabolic phenotype of cancer, characterized by uncoupled mitochondrial respiration and increased mitochondrial oxidative stress, is an attractive pharmacological target for sensitizing cancer cells to therapies that rely on oxidative stress for their tumor specific cytotoxicity. The identification of specific cancer sub-types for which metabolic priming of tumors prior to chemotherapy is beneficial is critical, particularly in heterogeneous diseases such as breast cancer. The effects of the thiazolidinedione drug troglitazone were examined in normal mammary epithelial cells and cancer cell lines representing three clinically relevant breast cancer phenotypes. Endpoints measured were PGC1α mRNA expression, proliferation, cell cycle phase distribution, mitochondrial capacity and superoxide generation, and sensitivity to the chemotherapy drug doxorubicin. Troglitazone increases expression of PGC1α, a key mediator of mitochondrial biogenesis, in normal mammary epithelial cells and in breast cancer cell lines. The induction of PGC1α mRNA is at least partially dependent on PPARγ activation. In estrogen receptor negative cells and cells with acquired antiestrogen resistance, troglitazone treatment increased mitochondrial superoxide production and mitochondrial capacity. At pharmacologically achievable doses, troglitazone pretreatment significantly enhanced the sensitivity of cancer cells to the chemotherapy agent doxorubicin. This effect was most dramatic in estrogen receptor positive cells with acquired antiestrogen resistance, in which troglitazone and doxorubicin combined had superadditive effects compared to treatment with either agent alone. In contrast, troglitazone treatment did not appreciably sensitize non-malignant mammary epithelial cells to doxorubicin induced cytotoxicity, despite increasing PGC1α mRNA. These data suggest that troglitazone or a similarly acting compound could be used to selectively prime tumor cells to the cytotoxic effects of anticancer agents such as doxorubicin and ionizing radiation. This novel treatment strategy may be most effective in women with antiestrogen insensitive tumors, a patient population with historically poor response to traditional therapies.

Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress

Maspin, identified as a 42 kDa unique tumor suppressive serine protease inhibitor (serpin), has multifaceted biological functions by interacting with various target molecules under physiological and pathological conditions including oxidative stress. However, the type of post-translational modification that confers the specific binding affinity of maspin to target molecules, such as glutathione S-transferase (GST), has not been determined. The aim of this study, therefore, is to analyze the molecular heterogeneity of maspin and to identify its modifications in the normal mammary epithelial cell line, MCF-10A, which is known to express the maspin protein abundantly, using electrophoretic analysis. Conventional SDS-PAGE analysis of MCF-10A cell extracts showed that endogenous maspin is composed of both an intact form observed as a 42 kDa band and a smaller form observed as a 36 kDa band. Interestingly, a brief exposure of MCF-10A cells to 10 mM hydrogen peroxide (H2O2) led to the appearance of a novel endogenous maspin form, as demonstrated by non-denaturing PAGE and non-reducing SDS-PAGE. Two-dimensional sequential non-reducing/reducing SDS-PAGE supported that this novel form was generated by intramolecular disulfide-bonded linkage under oxidative stress, and this oxidized maspin form also existed under physiological conditions. Furthermore, a glutathione (GSH) bead pull-down assay revealed that the intramolecular disulfide-bonded maspin lost its binding activity to endogenous GST, indicating that intramolecular disulfide-bonded maspin might have some distinct properties under oxidative stress, although the precise biological significance of this modification remains elusive. In conclusion, we have shown that maspin undertakes different modifications under oxidative stress.

Down‐regulation of Orai3 arrests cell‐cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells

Breast cancer (BC) is the leading cancer in the world in terms of incidence and mortality in women. However, the mechanism by which BC develops remains largely unknown. The increase in cytosolic free Ca(2+) can result in different physiological changes including cell growth and death. Orai isoforms are highly Ca(2+) selective channels. In the present study, we analyzed Orai3 expression in normal and cancerous breast tissue samples, and its role in MCF-7 BC and normal MCF-10A mammary epithelial cell lines. We found that the expression of Orai3 mRNAs was higher in BC tissues and MCF-7 cells than in normal tissues and MCF-10A cells. Down-regulation of Orai3 by siRNA inhibited MCF-7 cell proliferation and arrested cell cycle at G1 phase. This phenomenon is associated with a reduction in CDKs 4/2 (cyclin-dependent kinases) and cyclins E and D1 expression and an accumulation of p21(Waf1/Cip1) (a cyclin-dependent kinase inhibitor) and p53 (a tumor-suppressing protein). Orai3 was also involved in MCF-7 cell survival. Furthermore, Orai3 mediated Ca(2+) entry and contributed to intracellular calcium concentration ([Ca(2+)](i)). In MCF-10A cells, silencing Orai3 failed to modify [Ca(2+)](i), cell proliferation, cell-cycle progression, cyclins (D1, E), CDKs (4, 2), and p21(Waf1/Cip1) expression. Our results provide strong evidence for a significant effect of Orai3 on BC cell growth in vitro and show that this effect is associated with the induction of cell cycle and apoptosis resistance. Our study highlights a possible role of Orai3 as therapeutic target in BC therapy.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

IntroductionNormal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues.MethodsWe used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages.ResultsHuman breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions.ConclusionsAs a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues.

Repression of Lim only protein 4-activated transcription inhibits proliferation and induces apoptosis of normal mammary epithelial cells and breast cancer cells

Lim only protein (LMO) 4 acts as a transcriptional adapter and modulates mammary gland morphogenesis as well as breast oncogenesis in transgenic mice. Yet, the molecular and cellular mechanisms of these effects remain to be fully elucidated. Engrailed LMO4 fusion protein is a powerful dominant repressor of LMO4 activated transcription that was successfully used to discover the role of LMO4 as a transcriptional activator in mammary gland development in our previous studies using mouse models. In this manuscript, we investigated the cellular effects of LMO4 in human normal mammary epithelial cells (HMECs) and breast cancer cell lines using the Engrailed-LMO4 fusion protein. HMEC cell growth was inhibited by the expression of the Engrailed-LMO4 fusion protein. The decrease in cell number was due to both decreased cell proliferation and enhanced apoptosis, suggesting that LMO4 promotes proliferation and survival of normal mammary epithelial cells. The expression of the Engrailed-LMO4 fusion protein also suppressed cell growth, and induced apoptosis in two breast cancer cell lines, MDA-MB-231 and T47D, suggesting that LMO4 contributes to oncogenesis by similar mechanisms of enhanced cell survival and proliferation. Taken together, our data indicate that LMO4 has similar cellular effects in normal mammary epithelial cells and breast cancer cells, and also provide direct evidence for the idea that normal development and carcinogenesis share conserved molecular mechanisms.