Breast Epithelial Cell Line MCF-10A Research Articles

Abstract 4648: Differential regulation of estrogen metabolizing CYP1A1 and CYP1B1 enzymes by novel resveratrol analogs in breast cancer cells

Abstract Evidence from epidemiological and experimental studies suggest an association between diets rich in phytoestrogens and reduced risk of breast cancer. Resveratrol, a bioflavonoid, found naturally in grapes, fruits and nuts, has been shown to be a potent chemopreventive agent in vitro. However, poor bioavailability, rapid metabolism and poor specificity limit its use as the preferred anticancer agent. In an attempt to improve the anticancer properties of resveratrol, we synthesized several analogs of resveratrol with appropriate chemical modifications. Of the compounds that were screened for anticancer activity towards breast cancer cells, two analogs, namely, HPIMBD and TIMBD showed better potency and selectivity in inhibiting the growth of breast cancer cell lines while they did not significantly affect the growth of non-neoplastic breast epithelial cell lines. In this study, we investigated the ability of resveratrol and resveratrol analogs in the regulation of estrogen metabolizing genes, cytochrome P450 (Cyp) 1A1, 1B1 and their upstream regulator AhR in breast cancer and non-neoplastic breast epithelial cells. Non-neoplastic breast epithelial cell line MCF-10A and breast cancer cell lines T47D and MDA-MB-231 were treated with resveratrol, HPIMBD or TIMBD. From cells in culture, mRNA and protein was isolated and expression analyses of CYP1A1, CYP1B1 and AhR carried out by PCR and western blot analyses. Unlike resveratrol, HPIMBD and TIMBD significantly induced the mRNA and protein expression levels of CYP1A1 and AhR while they inhibited that of CYP1B1. Also, HPIMBD and TIMBD induced the expression of CYP1A1 in an AhR-dependent fashion while they inhibited CYP1B1 expression of CYP1B1 by a proteosomal degradation pathway. Cyp1A1 preferentially metabolizes estradiol to 2-hydroxyestradiol and Cyp1B1 to 4-hydroxyestradiol. While, 2-hydroxyestradiol and its methylated product are suggested to be chemo-protective, 4-hydroxyestradiol is regarded as a carcinogenic metabolite of estradiol. Taken together, our findings indicate that novel resveratrol analogs HPIMBD and TIMBD differentially regulate the expressions of CYP1A1 and CYP1B1 in breast cancer cells that may have implications in chemoprevention of breast cancer by these analogs. Note: This abstract was not presented at the meeting. Citation Format: Amruta Mukund Ronghe, Anwesha Chatterjee, Subhash Padhye, Hari K. Bhat. Differential regulation of estrogen metabolizing CYP1A1 and CYP1B1 enzymes by novel resveratrol analogs in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2015-4648

Abstract 1084: An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations

Abstract A recent study of the mutation landscape of >3000 cancers across 12 major cancer types from the Cancer Genome Atlas (TCGA) program revealed PIK3CA as the second most commonly mutated gene, occurring at >10% frequency across most cancer types, with mutations occurring mostly in the PIK3CA kinase (39% overall, 52% for breast cancer) and helical (45% overall, 39% for breast cancer) domains and their most frequent hotspot mutations being H1047R and E545K, respectively. Limited preclinical evidence has suggested that mutations affecting the kinase and helical domains can produce different phenotypic consequences; and our recent analysis of the TCGA data indicates that helical domain mutations, relative to kinase domain mutations, are preferentially associated with pathway/PARADIGM activity features enriched for cell motility mechanisms (e.g. RHO GTPases) and loss of gap junctions, suggesting that helical domain mutations might serve to enhance cell invasiveness and metastatic motility. To validate the functional differences predicted by these two classes of PIK3CA mutations, we turned to the non-malignant breast epithelial cell line MCF10A bearing targeted pathogenic knock-ins of H1047R or E545K at the PIK3CA locus; and we compared RNA-seq expression profiles from the wildtype (wt) and knockin isogenic clones. RSEM was used to estimate gene and isoform abundance; and EBseq was used to perform differential expression analysis. In a three-group comparison, over 2500 differentially expressed genes were identified as differentiating wt cells and the PIK3CA mutated MCF10A clones, with the greatest transcriptional changes (2043 genes) observed between either kinase or helical domain mutations and wt cells. Functional enrichment analysis (DAVID) revealed enrichment of 17 pathway categories among these 2043 genes, representing 3 major functional groups, the most prominent being protein translation. Only 409 genes were differentially expressed across all three groups, suggesting the transcriptome effect differentiating E545K from H2047R bearing MCF10A clones was less pronounced. In a direct comparison of the kinase and helical domain clones, >900 differentially expressed genes were identified that showed enrichment in 64 pathways representing altered cell morphology, motility, apoptosis and phosphorylation, but without enrichment in protein translation function. In particular, MCF10A E545K clones showed significant loss of expression in 19 tight junction-associated genes linked to oncogenic EMT (e.g. OCLN, CLDN-7, TJP1, MYL9). Thus, consistent with our pathway conclusions drawn from the TCGA clinical dataset, helical domain mutations appear to confer an EMT-like expression phenotype beyond the effects of both helical and kinases domain mutations on protein translation. Citation Format: Christina Yau, Joe Garay, Stephen Benz, Joe Gray, Ben Park, Christopher C. Benz. An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2015-1084

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines.

Although the concept of aerobic glycolysis in cancer was already reported in the 1930s by Otto Warburg, the understanding of metabolic pathways remains challenging especially due to the heterogeneity of cancer. In consideration of four different time points (1, 2, 4, and 7 days of incubation), GC-MS profiling of metabolites was performed on cell extracts and supernatants of breast cancer cell lines (MDA-MB-231, -453, BT-474) with different sub classification and the breast epithelial cell line MCF-10A. To the exclusion of trypsinization, direct methanolic extraction, cell scraping and cell disruption was executed to obtain central metabolites. Major differences in biochemical pathways have been observed in the breast cancer cell lines compared to the breast epithelial cell line, as well as between the breast cancer cell lines themselves. Characteristics of breast cancer subtypes could be correlated to their individual metabolic profiles. PLS-DA revealed the discrimination of breast cancer cell lines from MCF-10A based on elevated amino acid levels. The observed metabolic signatures have great potential as biomarker for breast cancer as well as an improved understanding of subtype specific phenomenons of breast cancer.

MiR-27a modulates radiosensitivity of triple-negative breast cancer (TNBC) cells by targeting CDC27.

BackgroundMiR-27a is significantly overexpressed in triple-negative breast cancer (TNBC). However, the exact biological function of MiR-27a in TNBC is not fully understood. In this study, we verified miR-27a expression in TNBC cells and explored how its overexpression modulates radiosensitivity of the cells.Material/MethodqRT-PCR analysis was performed to study miR-27a expression in TNBC lines MDA-MB-435 and MDA-MB-231 and in normal human breast epithelial cell line MCF10A. Dual luciferase assay was performed to verify a putative downstream target of miR-27a, CDC27. CCK-8 assay was used to assess the influence of miR-27a-CDC27 axis on cell proliferation under irradiation (IR) treatment.ResultsWe confirmed significantly higher miR-27a expression in 2 TNBC cell lines – MDA-MB-435 and MDA-MB-231 – than in human breast epithelial cell line MCF10A. miR-27a could modulate proliferation and radiosensitivity of TNBC cells. CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.ConclusionsThe miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells. Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.

C-MYC is a radiosensitive locus in human breast cells.

Ionising radiation is a potent human carcinogen. Epidemiological studies have shown that adolescent and young women are at increased risk of developing breast cancer following exposure to ionising radiation compared with older women, and that risk is dose-dependent. Although it is well understood which individuals are at risk of radiation-induced breast carcinogenesis, the molecular genetic mechanisms that underlie cell transformation are less clear. To identify genetic alterations potentially responsible for driving radiogenic breast transformation, we exposed the human breast epithelial cell line MCF-10A to fractionated doses of X-rays and examined the copy number and cytogenetic alterations. We identified numerous alterations of c-MYC that included high-level focal amplification associated with increased protein expression. c-MYC amplification was also observed in primary human mammary epithelial cells following exposure to radiation. We also demonstrate that the frequency and magnitude of c-MYC amplification and c-MYC protein expression is significantly higher in breast cancer with antecedent radiation exposure compared with breast cancer without a radiation aetiology. Our data also demonstrate extensive intratumor heterogeneity with respect to c-MYC copy number in radiogenic breast cancer, suggesting continuous evolution at this locus during disease development and progression. Taken together, these data identify c-MYC as a radiosensitive locus, implicating this oncogenic transcription factor in the aetiology of radiogenic breast cancer.

Abstract 240: Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC-bZip transcription factors in estrogen-induced breast carcinogenesis

Abstract Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C (VC) or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the role of CNC b-zip transcription factors in antioxidant-mediated prevention of breast cancer. Human non-neoplastic breast epithelial cell line MCF-10A was treated with E2, VC, VC + E2, Resveratrol (Res) and Res + E2 for up to 72 hrs. mRNA and protein expression levels of phase-II detoxifying enzymes SOD1, SOD2, SOD3, GPx, NQO1 and transcription factors from CNC b-zip family: Nrf1, Nrf2 and Nrf3 were quantified after treatment with E2 and compared with that of cells treated with antioxidants either alone or in combination with E2. The mRNA as well as protein expression levels of SOD3 and NQO1 were suppressed in MCF-10A cells treated with E2. There was no change in mRNA and protein expression levels of SOD1, SOD2 and GPX in MCF-10A cells treated with E2. Nrf2 mRNA and protein expression levels were also significantly suppressed in E2-treated MCF-10A cells. Vitamin C or Res treatment prevented E2-mediated decrease in SOD3, NQO1 and Nrf2 mRNA and protein expression levels. A significant increase in Nrf1 mRNA and protein expression levels in MCF-10A cells treated with E2 was noted. Further, treatment with antioxidants Res or VC also increased mRNA and protein expression levels of Nrf1. Nrf3 mRNA and protein expression levels were significantly increased in MCF-10A cells treated with E2. This induction was overcome by co-treatment with E2 and VC or Res. Our studies demonstrate that antioxidant-mediated protection against estrogen-induced breast cancer could be through up regulation of phase-II detoxifying enzymes via an Nrf-dependent pathway involving a complex interplay among Nrfs 1, 2 and 3. Note: This abstract was not presented at the meeting. Citation Format: Anwesha Chatterjee, Amruta Ronghe, Fatma Abdalla, Hari K. Bhat. Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC-bZip transcription factors in estrogen-induced breast carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 240. doi:10.1158/1538-7445.AM2014-240

TGF-β-induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops.

The process of epithelial-to-mesenchymal transition (EMT) is an essential type of cellular plasticity associated with a change from epithelial cells that function as a barrier consisting of a sheet of tightly connected cells to cells with properties of mesenchyme that are not attached to their neighbors and are highly motile. This phenotypic change occurs during development and also contributes to pathological processes, such as cancer progression. The molecular mechanisms controlling the switch between the fully epithelial and fully mesenchymal phenotypes and cells that have characteristics of both (partial EMT) are controversial, and multiple theoretical models have been proposed. To test these theoretical models, we systematically measured the changes in the abundance of proteins, mRNAs, and microRNAs (miRNAs) that represent the core regulators of EMT induced by transforming growth factor-β1 (TGF-β1) in the human breast epithelial cell line MCF10A at the population and single-cell levels. We provide experimental confirmation for a model of cascading switches in phenotypes associated with TGF-β1-induced EMT of MCF10A cells that involves two double-negative feedback loops: one between the transcription factor SNAIL1 and the miR-34 family and another between the transcription factor ZEB1 and the miR-200 family. Furthermore, our data showed that whereas the transition from epithelial to partial EMT was reversible for MCF10A cells, the transition from partial EMT to mesenchymal was mostly irreversible at high concentrations of TGF-β1.

Abstract 3589: Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy

Abstract Autologous adipose stem cell transplantation is increasingly being used for soft tissue reconstruction, particularly after mastectomy. However, a recent study demonstrated recurrence of invasive ductal carcinoma in the breast within four months of autologous fat grafting. Previous studies have documented how cancer cells commandeer adipose stem cells (ASC) to support their survival by altering extracellular matrix composition and stiffness. This study focused on delineating the effects of ASCs on the self-renewal capacity of breast epithelial cells. The immortalized breast epithelial cell line MCF-10A, MCF-10A overexpressing Slug, and MCF-10A overexpressing SRC were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profile. MCF-10A-Slug cells provided a unique model system to study breast epithelial cell dedifferentiation as Slug functions downstream of the breast cancer susceptibility gene BRCA1 and is a major suppressor of progenitor cell lineage commitment and differentiation. Interestingly, results with these established cell lines were reproduced in primary breast epithelial cells isolated from a high-risk patient. We show that secreted factors from ASCs promote self-renewal of all cell types examined. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells in 2D culture and mammosphere assays. ASCs secrete Interferon gamma (IFNγ), CD40 ligand, IL-23 and SERPINE1. Among these factors, only IFNγ treatment altered stem cell hierarchy; while MCF-10A-Slug cells displayed only luminal progenitor phenotype, cells treated with IFNγ contained a distinct stem-like cell population. These results suggest that paracrine activity of adipose stem cell-derived factors can either enhance dedifferentiation and promote proliferation of dormant tumor cells or predispose normal cells to transformation by blocking differentiation. Financial support: Susan G. Komen For the Cure SAC110025 and Zeta Tau Sorority (HN) Citation Format: Manjushree Anjanappa, Riesa M. Burnett, Stephanie Merfeld-Clauss, William A. Wooden, Keith L. March, Sunil Tholpady, Harikrishna Nakshatri. Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2014-3589

Abstract 1234: Tamoxifen synergizes cytotoxic activity of 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD), a novel resveratrol analogue against breast cancer cells

Abstract Tamoxifen is currently the first line of therapy against estrogen receptor (ER)-positive breast cancers. Prolonged treatment with tamoxifen has been associated with the development of resistant tumor phenotypes. Moreover, this treatment strategy is ineffective against ER-negative breast cancers. In this study, we have investigated the role of 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD), a novel analog of resveratrol, either alone or in combination with tamoxifen on the growth inhibition of breast cancer cells. Breast cancer cell lines MCF-7, T47D, MDA-MB-231 and MDA-MB-468 as well as non-neoplastic breast epithelial cell lines MCF-10A, MCF-10F and HMEC were treated with different doses of HPIMBD or a combination of tamoxifen (2µM) and HPIMBD (at its IC50 dose). Cell proliferation was determined by MTT assays. No effect on cell proliferation was observed upon treatment of non-neoplastic breast cells with HPIMBD. A dose dependent growth inhibition was demonstrated upon treatment of breast cancer cells with HPIMBD. IC50 values of HPIMBD were determined. Tamoxifen at a dose of 2µM showed no effect on the growth of breast cancer or non-neoplastic breast epithelial cell lines. However, a significant increase in inhibition of cellular proliferation of breast cancer cells was demonstrated when tamoxifen (2µM) was used in combination with HPIMBD at its IC50 dose. To investigate the mechanism of inhibition, autophagy markers, beclin-1 and LC3B were quantified by Western blot analyses. The combination treatment significantly increased the expression of autophagy marker beclin-1 and the cleavage of LC3BI to LC3BII. Cell cycle analysis showed an arrest in the S-phase by HPIMBD and G0/G1-phase by tamoxifen. Our studies suggest that a combination of tamoxifen and HPIMBD synergistically inhibit the proliferation of breast cancer cells. Autophagy and cell cycle arrest appear to be the primary mechanisms of inhibition by the combination treatment. Citation Format: Amruta Mukund Ronghe, Anwesha Chatterjee, Fatma Abdalla, Hari K. Bhat. Tamoxifen synergizes cytotoxic activity of 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD), a novel resveratrol analogue against breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2014-1234

TIMP-1 via TWIST1 induces EMT phenotypes in human breast epithelial cells.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates intracellular signaling networks for inhibition of apoptosis. Tetraspanin (CD63), a cell surface binding partner for TIMP-1, was previously shown to regulate integrin-mediated survival pathways in the human breast epithelial cell line MCF10A. In the current study, we show that TIMP-1 expression induces phenotypic changes in cell morphology, cell adhesion, cytoskeletal remodeling, and motility, indicative of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecule E-cadherin with an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Signaling through TIMP-1, but not TIMP-2, induces the expression of TWIST1, an important EMT transcription factor known to suppress E-cadherin transcription, in a CD63-dependent manner. RNAi-mediated knockdown of TWIST1 rescued E-cadherin expression in TIMP-1-overexpressing cells, demonstrating a functional significance of TWIST1 in TIMP-1-mediated EMT. Furthermore, analysis of TIMP-1 structural mutants reveals that TIMP-1 interactions with CD63 that activate cell survival signaling and EMT do not require the matrix metalloproteinase (MMP)-inhibitory domain of TIMP-1. Taken together, these data demonstrate that TIMP-1 binding to CD63 activates intracellular signal transduction pathways, resulting in EMT-like changes in breast epithelial cells, independent of its MMP-inhibitory function. TIMP-1's function as an endogenous inhibitor of MMP or as a "cytokine-like" signaling molecule may be a critical determinant for tumor cell behavior.

Natural antioxidants exhibit chemopreventive characteristics through the regulation of CNC b-Zip transcription factors in estrogen-induced breast carcinogenesis.

quinone oxidoreductase 1 (NQO1) were quantified. The treatment with E2 suppressed, whereas VC and Res prevented E2-mediated decrease in the expression levels of SOD3, NQO1, Nrf2 mRNA, and protein in MCF-10A cells. The treatment with E2, Res, or VC significantly increased mRNA and protein expression levels of Nrf1. 17β-Estradiol treatment significantly increased but VC or Res decreased Nrf3 mRNA and protein expression levels. Our studies demonstrate that estrogen-induced breast cancer might be prevented through upregulation of antioxidant enzymes via Nrf-dependent pathways.

Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCɛ signaling shown in a xenograft breast cancer model.

MiR-200c inhibits autophagy and enhances radiosensitivity in breast cancer cells by targeting UBQLN1.

Radioresistance is a major challenge during the treatment of breast cancer. A further understanding of the mechanisms of radioresistance could provide strategies to address this challenge. In our study, we compared the expression of miR-200c in four distinct breast cancer cell lines: two representative basal cancer cells (MDA-MB-231 and BT549) vs. two representative luminal cancer cells (MCF-7 and BT474). The results revealed practically lower expression of miR-200c in the two basal cancer cell lines and higher expression of miR-200c in luminal cancer cells compared to the normal breast epithelial cell line MCF-10A. Ectopic expression of miR-200c in MDA-MB-231 cells inhibited irradiation-induced autophagy and sensitized the breast cancer cells to irradiation. We also identified UBQLN1 as a direct functional target of miR-200c involved in irradiation-induced autophagy and radioresistance. In 35 human breast cancer tissue samples, we detected an inverse correlation between the expression of miR-200c vs. UBQLN1 and LC3. These results indicate that the identified miR-200c/UBQLN1-mediated autophagy pathway may help to elucidate radioresistance in human breast cancer and might represent a therapeutic strategy.

Suppressive role of miR-502-5p in breast cancer via downregulation of TRAF2

TRAF2 promotes cancer cell survival, proliferation and metastasis through the NF-κB pathway by directly interacting with various TNF recepors. However, the molecular mechanism of TRAF2 dysregulation in breast cancer remains to be elucidated. In the present study, miR-502-5p was predicted as a potential regulator of TRAF2. miR-502-5p was significantly downregulated in breast cancer tissues when compared to the level in paired normal breast tissues. The breast cancer cell lines including MCF-7 and MDA-MB-231 expressed a lower level of miR-502-5p when compared to the level in the non-malignant breast epithelial cell line MCF-10A. In vitro, miR-502-5p enhanced early apoptosis and inhibited proliferation of breast cancer cells. Luciferase reporter assay results showed that miR-502-5p could bind to the 3'-untranslated region of the TRAF2 gene, thus, exerting an inhibitory effect on TRAF2. Furthermore, silencing of TRAF2 exhibited effects similar to those of exogenous miR‑502-5p, while overexpression of TRAF2 partially abrogated miR-502-5p-mediated suppression in breast cancer cells. In conclusion, miR-502-5p may act as a tumor-suppressor gene by targeting oncogenic TRAF2 in breast cancer and, therefore, may be a potential diagnostic and anticancer therapeutic marker for breast cancer.

Histone Deacetylase Inhibitors Improve the Replication of Oncolytic Herpes Simplex Virus in Breast Cancer Cells

New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV) is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC) inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (<, = , and >LD50) for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI) following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.

Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells

The goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser. In the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated. Both MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a γ-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells. Our results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells.

A novel cytotoxic terpenoid from the flowers of Kaunia lasiophthalma Griseb

A phytochemical study of the flowers of Kaunia lasiophthalma G. (Asteraceae) yielded a novel triterpene (1) together with several known sesquiterpenoids. The structure of the new compound was elucidated by analysis of the spectroscopic data. The biosynthetic origin of 1 is proposed to be a dimerization of an oxidized derivative (3) of the germacrane sesquiterpene costunolide (2), also present in the flowers. The anticancer activity of 1 in the five breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7 and SK-BR-3 was compared with the cytotoxicity in the normal-like breast epithelial cell line MCF-10A. 1 exhibited high cytotoxicity in all investigated cancer cell lines with IC50 values ranging from 0.67 to 7.0μM, although it is lacking selectivity as the MCF-10A cells were almost as sensitive.

Traditional Chinese medicinal formula Si-Wu-Tang prevents oxidative damage by activating Nrf2-mediated detoxifying/antioxidant genes

BackgroundInduction of Nrf2-mediated detoxifying/antioxidant genes has been recognized as an effective strategy for cancer chemoprevention. Si-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. The purpose of this study is to determine the effects of SWT on Nrf2 pathway in vitro and in vivo and to identify the active component(s).ResultsCell viability and apoptosis were analyzed in the non-cancerous breast epithelial cell line MCF-10A after H2O2 treatment in the presence or absence of SWT using the Sulphorhodamine B assay, Annexin-V/Propidium iodide staining and flow cytometry. SWT strongly reduced H2O2 -induced cytotoxicity and apoptosis in MCF-10A cells. Expression of Nrf2 and Nrf2-regulated genes HMOX1 (heme oxygenase 1) and SLC7A11 (xCT) was evaluated by quantitative RT-PCR, Western Blot and immunocytochemistry. SWT strongly induced Nrf2-regulated genes at mRNA and protein levels and increased the nuclear translocation of Nrf2 in MCF-10A cells. The in vivo pharmacodynamic effect of SWT was evaluated in healthy female Sprague–Dawley rats. Short-term oral administration of SWT (1,000 mg/kg per day for six consecutive days) to rats resulted in an increased expression of Nrf2-regulated genes Hmox1 and Slc7A11 in the liver detected by quantitative RT-PCR. Among nine compounds that have been identified previously in the SWT products, z-liguistilide was discovered as the main component responsible for the effect of Nrf2 activation using the antioxidant response element-luciferase reporter gene assay. Z-liguistilide was confirmed with a high potency to induce Nrf2-regulated genes and Nrf2 nuclear translocation.ConclusionsOur results demonstrated that SWT and its component z-liguistilide are able to activate the Nrf2 pathway in non-cancerous cells and organs in vitro and in vivo, suggesting that SWT might be an orally effective and nontoxic agent for cancer chemoprevention.

Cytotoxic Sesquiterpene Lactones from Kauna lasiophthalma Griseb

Two new eudesmane derivatives (3 and 8) were isolated from the ethanol extract of the aerial parts of Kaunia lasiophthalma Griseb, together with 14 known eudesmane, germacrane, and guaiane sesquiterpenes, and four flavones. The structures and relative configurations of all the compounds were established by NMR spectroscopy and high-resolution mass spectrometry. The anticancer activity of sesquiterpenes 1, 3, 6–9, 11, 12, 14, and 16 was evaluated in vitro with the breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7, and SK-BR-3, and compared with the cytotoxicity in the non-cancerous breast epithelial cell line MCF-10A. All compounds were found to possess anticancer activity, and compound 1 was the most potent in all of the investigated cancer cell lines with IC50 values ranging between 2.0 and 6.2 μM. In order to demonstrate the importance of the α-methylene-γ-lactone/ester moiety present in all compounds for the effects on the cells, the methyl cysteine adduct 21 was prepared from 9 and found to be inactive or considerably less potent.

Methylated DNA Binding Domain Protein 2 (MBD2) Coordinately Silences Gene Expression through Activation of the MicroRNA hsa-mir-496 Promoter in Breast Cancer Cell Line

Methylated DNA binding protein 2 (MBD2) binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA) antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets.