Normal MCF-10A Cells Research Articles

Alterations in replication timing of cancer‐related genes in malignant human breast cancer cells

The replication timing of nine genes commonly involved in cancer was investigated in the MCF10 cell lines for human breast cancer progression. Six of these nine genes are part of a constellation of tumor suppressor genes that play a major role in familial human breast cancer (TP53, ATM, PTEN, CHK2, BRCA1, and BRCA2). Three other genes are involved in a large number of human cancers including breast as either tumor suppressors (RB1 and RAD51) or as an oncogene (cMYC). Five of these nine genes (TP53, RAD51, ATM, PTEN, and cMYC) show significant differences (P < 0.05) in replication timing between MCF10A normal human breast cells and the corresponding malignant MCF10CA1a cells. These differences are specific to the malignant state of the MCF10CA1a cells since there were no significant differences in the replication timing of these genes between normal MCF10A cells and the non-malignant cancer MCF10AT1 cells. Microarray analysis further demonstrated that three of these five genes (TP53, RAD51, and cMYC) showed significant changes in gene expression (≥2-fold) between normal and malignant cells. Our findings demonstrate an alteration in the replication timing of a small subset of cancer-related genes in malignant breast cancer cells. These alterations partially correlate with the major transcriptional changes characteristic of the malignant state in these cells.

Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

Effect of Botulinum Toxin A on Proliferation and Apoptosis in the T47D Breast Cancer Cell Line

The present study was performed to assess the activity of the botulinum toxin A on breast cancer cells. The T47D cell line was exposed to diverse concentrations of the botulinum toxin A and cell viability and apoptosis were estimated using MTT and propidium iodine/annexin V methods, respectively. Botulinum toxin A exerted greater cytotoxic activity in T47D cells in comparison with MCF10A normal cells; this appeared to be via apoptotic processes caspase-3 and -7. In conclusion, botulinum toxin A induces caspase-3 and -7 dependent apoptotic processes in the T47D breast cancer cell line.

Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Induction of apoptosis in cancer cells by NiZn ferrite nanoparticles through mitochondrial cytochrome C release

The long-term objective of the present study was to determine the ability of NiZn ferrite nanoparticles to kill cancer cells. NiZn ferrite nanoparticle suspensions were found to have an average hydrodynamic diameter, polydispersity index, and zeta potential of 254.2 ± 29.8 nm, 0.524 ± 0.013, and −60 ± 14 mV, respectively. We showed that NiZn ferrite nanoparticles had selective toxicity towards MCF-7, HepG2, and HT29 cells, with a lesser effect on normal MCF 10A cells. The quantity of Bcl-2, Bax, p53, and cytochrome C in the cell lines mentioned above was determined by colorimetric methods in order to clarify the mechanism of action of NiZn ferrite nanoparticles in the killing of cancer cells. Our results indicate that NiZn ferrite nanoparticles promote apoptosis in cancer cells via caspase-3 and caspase-9, downregulation of Bcl-2, and upregulation of Bax and p53, with cytochrome C translocation. There was a concomitant collapse of the mitochondrial membrane potential in these cancer cells when treated with NiZn ferrite nanoparticles. This study shows that NiZn ferrite nanoparticles induce glutathione depletion in cancer cells, which results in increased production of reactive oxygen species and eventually, death of cancer cells.

Extremely high inhibition activity of photoluminescent carbon nanodots toward cancer cells.

Highly water-soluble, biocompatible, and photoluminescent carbon nanodots (C-dots) having an average diameter 3.4 ± 0.8 nm and a quantum yield 4.3% are obtained from used green tea through grinding, calcination and centrifugation. The as-prepared C-dots are stable in high-ionic-strength media (e.g. 500 mM NaCl) and under light irradiation, allowing images of MCF-10A, MCF-7 and MDA-MB-231 cells to be recorded. The C-dots are mostly localized in the cell membranes and cytoplasms, with evidence of excitation-wavelength cell images. Relative to catechin, the C-dots provide greater inhibition efficiency of the growth of MCF-7 and MDA-MB-231 cancer cells, with lower toxicity for the MCF-10A normal cells. The inhibitory activity of C-dots is associated with the generation of greater amounts of reactive oxygen species. A prothrombin time (PT) assay of plasma samples reveals excellent biocompatibility of the C-dots. To the best of our knowledge, for the first time the C-dots provide inhibition efficiencies up to 80% and 82% for MCF-7 and MDA-MB-231 cancer cells, respectively, showing their high potential as cancer inhibitors.

The DNA binding site specificity and antiproliferative property of ternary Pt(ii) and Zn(ii) complexes of phenanthroline and N,N′-ethylenediaminediacetic acid

The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

Experimental Study of Entropy Production in Cells under Alternating Electric Field

We put forward a new method for measuring the entropy production in the living cell. It involves heating the sample by alternating the electric field and recording the outward heat flow. The entropy production in a normal cell MCF10A and a cancerous cell MDA-MB-231 were measured and compared. The results show that the method is effective for the entropy measurement of a living organism. The scaled electro-induced entropy production rate (SEEP) of MDA-MB-231 monotonically increases with the electric field strength at 5–40 V/cm. While that of MCF10A changes non-monotonically and there exists a peak at 5–30 V/cm. The electro-induced entropy production ratio (EEPR) is smaller than 1 in a large range of field strengths, from 5 to 25 V/cm, which reveals that under 5–25 V/cm electric field exposure, the direction of the entropy flow may be changed from normal tissue to cancerous cells. We present a facile and effective strategy for experimentally investigating the thermodynamic properties of the cell and give a deeper insight into the physical difference between normal and cancerous cells under electric field exposure.

Transmission Near-Field Scanning Optical Microscopy Investigation on Cellular Uptake Behavior of Iron Oxide Nanoparticles

Cellular uptake behavior of iron oxide nanoparticles is investigated using a transmission near-field scanning optical microscopy (NSOM) without the need of fluorescent labeling. By using the transmission NSOM system, we could simultaneously explore the near-field optical analysis of the cell interior and record the topographic information of the cell surface. The cell endocytosis of iron oxide nanoparticles by normal breast MCF10A cells is first studied by this transmission NSOM system, and this dual functional nanoscale-resolution microscopy shows the capability of mapping the spatial localization of nanoparticles in/outside cell surface without the need of fluorescence labeling. Nanoscale optical signature patterns for iron oxide nanoparticle-loaded vesicles inside the cells were observed and analyzed.

PTTG1 Oncogene Promotes Tumor Malignancy via Epithelial to Mesenchymal Transition and Expansion of Cancer Stem Cell Population

The prognosis of breast cancer patients is related to the degree of metastasis. However, the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize at a distant site are not entirely understood. Here, we analyzed expression levels of pituitary tumor-transforming gene-1 (PTTG1), a relatively uncharacterized oncoprotein, in patient-derived breast cancer tissues with corresponding normal breast tissues. We found that PTTG1 is highly expressed in breast cancer patients, compared with normal tissues. Also, PTTG1 expression levels were correlated with the degree of malignancy in breast cancer cell lines; the more migratory and invasive cancer cell lines MDA-MB-231 and BT549 displayed the higher expression levels of PTTG1 than the less migratory and invasive MCF7 and SK-BR3 and normal MCF10A cell lines. By modulating PTTG1 expression levels, we found that PTTG1 enhances the migratory and invasive properties of breast cancer cells by inducing epithelial to mesenchymal transition, as evidenced by altered morphology and epithelial/mesenchymal cell marker expression patterns and up-regulation of the transcription factor Snail. Notably, down-regulation of PTTG1 also suppressed cancer stem cell population in BT549 cells by decreasing self-renewing ability and tumorigenic capacity, accompanying decreasing CD44(high) CD24(low) cells and Sox2 expression. Up-regulation of PTTG1 had the opposite effects, increasing sphere-forming ability and Sox2 expression. Importantly, PTTG1-mediated malignant tumor properties were due, at least in part, to activation of AKT, known to be a key regulator of both EMT and stemness in cancer cells. Collectively, these results suggest that PTTG1 may represent a new therapeutic target for malignant breast cancer.

Hic-5 promotes invadopodia formation and invasion during TGF-β–induced epithelial–mesenchymal transition

Transforming growth factor β (TGF-β)-stimulated epithelial-mesenchymal transition (EMT) is an important developmental process that has also been implicated in increased cell invasion and metastatic potential of cancer cells. Expression of the focal adhesion protein Hic-5 has been shown to be up-regulated in epithelial cells in response to TGF-β. Herein, we demonstrate that TGF-β-induced Hic-5 up-regulation or ectopic expression of Hic-5 in normal MCF10A cells promoted increased extracellular matrix degradation and invasion through the formation of invadopodia. Hic-5 was tyrosine phosphorylated in an Src-dependent manner after TGF-β stimulation, and inhibition of Src activity or overexpression of a Y38/60F nonphosphorylatable mutant of Hic-5 inhibited matrix degradation and invasion. RhoC, but not RhoA, was also required for TGF-β- and Hic-5-induced matrix degradation. Hic-5 also induced matrix degradation, cell migration, and invasion in the absence of TGF-β via Rac1 regulation of p38 MAPK. These data identify Hic-5 as a critical mediator of TGF-β-stimulated invadopodia formation, cell migration, and invasion.

Abstract 5747: Elucidating the mechanisms by which Snail transcription factor promotes resistance to chemotherapy in breast carcinomas

Abstract Snail (Snail1) is a zinc finger transcription factor that can induce EMT and is expressed at high levels in tumors. Maspin is a potential tumor suppressor, whose expression is lost as breast and prostate tumors progress during EMT. Maspin can be transcriptionally repressed by androgen receptor (AR) in prostate cancer cells. We hypothesized that overexpression of Snail will inhibit maspin expression and confer resistance to chemotherapy. Initially, we examined the expression of Snail and maspin in a panel of breast epithelial and breast cancer cells by RT-PCR and Western blot analysis. Preliminary results suggest that breast cancer cells express variable levels of Snail that is higher in breast cancer cells than in the normal MCF10A cells. We then utilized MCF-7 cells transfected stably with an empty Neo vector (MCF-7 Neo) or constitutively active Snail cDNA (MCF-7 Snail) that have been shown previously to represent an EMT model, and examined maspin, estrogen receptor alpha (ER-α) and AR levels by western blot analysis. MCF-7 Snail cells had upregulated levels of Snail and lower levels of maspin, ER-α and AR, as compared to MCF-7 Neo. MCF-7 Neo and MCF-7 Snail cells were also xenografted into nude mice and tissue sections stained immunohistochemically for Snail, maspin, and ER-α. Tumor volumes of MCF-7 Snail tumor xenografts were significantly larger than the MCF-7 Neo xenografts. MCF-7 Snail tissue sections expressed higher levels of Snail and lower levels of maspin and ER-α as compared to MCF-7 Neo sections. MCF-7 Neo and MCF-7 Snail cells were also treated with estradiol (E2, 0.01uM), tamoxifen (TAM, 10uM), and docetaxel (DOX, 1uM) individually; and in combination with E2 plus TAM and TAM plus DOX for periods of 24h and 72h to analyze changes in cell morphology and EMT marker expression. No effect on cell morphology or cell death was observed in MCF-7 Snail cells after TAM treatments, whereas TAM induced cell death in MCF-7 Neo. Expression of Snail was increased with E2 treatments and decreased with TAM, DOX, and combination treatments in MCF-7 Neo and MCF-7 Snail. AR expression decreased further in MCF-7 Snail cells and MCF-7 Neo following treatments with TAM and DOX. Overall, the Snail overexpression appears to promote resistance to TAM and DOX chemotherapeutic agents. The mechanism of Snail-regulated EMT needs to be investigated further to determine changes in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5747. doi:1538-7445.AM2012-5747

Abstract 22: Differential IGF-II response of AA and CA normal breast cell lines to prolactin treatment

Abstract African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22

Abstract 1296: Identification of human nuclear factor 1 (NF-1) as a potential transcription factor which regulates the gene expression of Sulfotransferase isoform 1A1 (SULT1A1) in human breast cancer

Abstract Sulfotransferase isoform 1A1 (SULT1A1) plays a central role in drug metabolism. SULT1A1 expression is tissue specific, with little to no expression in normal breast epithelia but plentiful protein expression in most breast tumors. Revelation of the regulators of SULT1A1 expression in breast tissues is important in predicting breast tissue response to therapeutic agents that are substrates of SULT1A1, particularly tamoxifen which can be used for both breast cancer treatment and chemoprevention. In order to identify the appropriate transcription factors that regulate the gene expression of SULT1A1, we performed transcription factor (TF) Activation Profiling Plate Array assay (Signosis, Sunnyvale, CA) in normal breast MCF10A cells and breast cancer ZR-75 cells. TF activation profile differences in MCF10A cells and ZR-75 cells were quantitatively analyzed and compared. Out of 48 TFs DNA-binding activities allowed per plate, 18 TFs’ activities were at least 1.5 fold higher in ZR75 cells compared to that of in MCF10A cells. Matching of the 18 TFs to the possible TF binding consensus sequences in SULT1A1, which were predicted by software Alibaba 2.0, was performed and we found that nuclear factor 1 (NF-1) and GATA transcription factor 1 (GATA-1) were putative transcription factors involved in the regulation of SULT1A1 expression. To further study the relationship between NF-1 and GATA-1 and SULT1A1, we measured mRNA levels of NF-1, GATA-1, and SULT1A1 using total RNA isolated from 97 human liver samples. The liver samples were obtained from 43 female and 54 male health subjects (age range 26-102 year, mean 59.0 years). The real-time RT-PCR results showed that only NF-1, not GATA-1, mRNA level showed significant correlation with SULT1A1 mRNA level (r=0.64, p&amp;lt;0.0001). Therefore, we have identified that NF-1 as a new strong candidate for SULT1A1 gene expression regulation. The finding has laid foundation for the study on the effect of NF-1 on SULT1A1 gene expression, which could provide a mechanistic explanation for the differential expression of SULT1A1 between normal breast tissue and breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1296. doi:1538-7445.AM2012-1296

Obscurins Invade Breast Cancer Research

Obscurins are giant multidomain proteins, encoded by the single OBSCN gene, which play important roles in the structural organization and contractile activity of striated muscles. Recently, genetic linkage analysis identified OBSCN and TP53 as the only common and most frequently mutated genes in breast and colorectal cancers among 13,023 genes studied. Our laboratory set forth to study the expression profile of the OBSCN gene in breast cancer. Our data revealed that obscurins are readily expressed in normal breast epithelial cells (MCF10A), but their amounts are significantly diminished in breast cancer cells (MCF7 &amp; MDA‐MB‐ 231). Down‐regulation of obscurins in MCF10A cells via siRNA technology resulted in decreased levels of β‐catenin and Ecadherin, both of which are major components of adherens junctions (AJ) and are heavily implicated in the formation and metastasis of breast tumors. Residual β‐catenin showed a preferential nuclear accumulation in obscurin‐null MCF10A cells, and a concomitant loss from cell‐cell junctions. Interestingly, TGF‐β1 treatment of normal MCF10A cells resulted in down‐regulation of obscurins, β‐catenin, and E‐cadherin. In summary, our findings indicate that TGF‐β1 may negatively regulate the expression of obscurins in breast epithelium, loss of which results in diminished levels and altered distribution of major AJ proteins, and thus disruption of cell‐cell contacts.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Role of autophagy in chemoresistance: Regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA–PKcs and PARP-1

Capsaicin treatment was previously reported to reduce the sensitivity of breast cancer cells, but not normal MCF10A cells, to apoptosis. The present study shows that autophagy is involved in cellular resistance to genotoxic stress, through DNA repair. Capsaicin treatment of MCF-7 cells induced S-phase arrest and autophagy through the AMPKα–mTOR signaling pathway and the accumulation of p53 in the nucleus and cytosol, including a change in mitochondrial membrane potential. Capsaicin treatment also activated δ-H2AX, ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA–PKcs), and poly(ADP-ribose) polymerase (PARP)-1. Genetic or pharmacological disruption of autophagy attenuated capsaicin-induced phospho-ATM and phospho-DNA–PKcs and enhanced apoptotic cell death. ATM inhibitors, including Ku55933 and caffeine, and the genetic or pharmacological inhibition of p53 prevented capsaicin-induced DNA–PKcs phosphorylation and stimulated PARP-1 cleavage, but had no effect on microtubule-associated protein light chain 3 (LC3)-II levels. Ly294002, a DNA–PKcs inhibitor, boosted the capsaicin-induced cleavage of PARP-1. In M059K cells, but not M059J cells, capsaicin induced ATM and DNA–PKcs phosphorylation, p53 accumulation, and the stimulation of LC3II production, all of which were attenuated by knockdown of the autophagy-related gene atg5. Ku55933 attenuated capsaicin-induced phospho-DNA–PKcs, but not LC3II, in M059K cells. In human breast tumors, but not in normal tissues, AMPKα, ATM, DNA–PKcs, and PARP-1 were activated and LC3II was induced. The induction of autophagy by genotoxic stress likely contributes to the sustained survival of breast cancer cells through DNA repair regulated by ATM–mediated activation of DNA–PKcs and PARP-1.

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.

Elevated p-CREB-2 (ser 245) expression is potentially associated with carcinogenesis and development of breast carcinoma

CREB-2, also known as ATF-4, belongs to the CREB proteins, a family of transcription factors phosphorylated at serine residues by protein kinase A (PKA). This family is known to stimulate the transcription of genes containing CRE elements. Recently, some studies have demonstrated elevated CREB-2 expression in certain tumor types, including breast carcinoma, compared to their corresponding non-tumor tissues. However, the expression and clinical significance in malignant tumors, including breast carcinoma, of p-CREB-2 (ser 245), a phosphorylated form of the CREB-2 protein at serine 245 site, which is believed to be an active type of this protein, have not been clearly documented. In the present study, we investigated the expression of p-CREB-2 (ser 245) in a group of tumor and non-tumor breast tissues, including normal breast epithelia, hyperplasia, dysplasia, carcinoma in situ and infiltrating carcinoma of the breast using tissue microarray and immunohistochemistry (IHC). p-CREB-2 (ser 245) immunostaining was detected in the nucleus and cytoplasm of these tissues. Compared to normal breast epithelia and breast hyperplasia (total positive rate 13.3%), there was increased expression of p-CREB-2 (ser 245) in dysplasia, carcinoma in situ (total positive rate 35.7%) and infiltrating carcinoma of the breast (total positive rate 60.0%) (p<0.05). The highest expression of p-CREB-2 (ser 245) was found in infiltrating breast carcinoma (total positive rate 60%) compared to normal breast epithelia and all types of non-infiltrating lesions (total positive rate 27.6%) (p<0.05). In addition, increased expression of p-CREB-2 (ser 245) was found to be associated with lymph node metastasis in infiltrating breast carcinoma (p<0.05). Immunofluorescent staining confirmed stronger staining of p-CREB-2 (ser 245) in breast cancer MCF 7 and MDA-MB-231 cells compared with normal breast epithelial MCF 10A cells. Western blotting revealed elevated expression levels of p-CREB-2 (ser 245) in 17 cases of breast carcinoma compared with corresponding normal breast tissues (p<0.05). These results indicate that elevated expression of p-CREB-2 (ser 245) may potentially contribute to carcinogenesis and cancer development of breast carcinoma.

A Novel Prodrug of Epigallocatechin-3-gallate: Differential Epigenetic hTERT Repression in Human Breast Cancer Cells

Epigallocatechin-3-gallate (EGCG), a major component of green tea polyphenols (GTP), has been reported to downregulate telomerase activity in breast cancer cells thereby increasing cellular apoptosis and inhibiting cellular proliferation. However, the major concerns with GTPs are their bioavailability and stability under physiologic conditions. In the present study, we show that treatments with EGCG and a novel prodrug of EGCG (pro-EGCG or pEGCG) dose- and time-dependently inhibited the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells but not normal control MCF10A cells. Furthermore, both EGCG and pro-EGCG inhibited the transcription of hTERT (human telomerase reverse transcriptase), the catalytic subunit of telomerase, through epigenetic mechanisms in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cells. The downregulation of hTERT expression was found to be because of hTERT promoter hypomethylation and histone deacetylations, mediated at least partially through inhibition of DNA methyltransferase and histone acetyltransferase activities, respectively. In addition, we also observed that EGCG and pEGCG can remodel chromatin structures of the hTERT promoter by decreasing the level of acetyl-H3, acetyl-H3K9, and acetyl-H4 to the hTERT promoter. EGCG and pEGCG induced chromatin alterations that facilitated the binding of many hTERT repressors such as MAD1 and E2F-1 to the hTERT regulatory region. Depletion of E2F-1 and MAD1 by using siRNA reversed the pEGCG downregulated hTERT expression and associated cellular apoptosis differently in ER-positive and ER-negative breast cancer cells. Collectively, our data provide new insights into breast cancer prevention through epigenetic modulation of telomerase by using pro-EGCG, a more stable form of EGCG, as a novel chemopreventive compound.