Cell Line MCF-10F Research Articles

Breast carcinogenesis induced by organophosphorous pesticides.

Breast cancer is a major health threat to women worldwide and the leading cause of cancer-related death. The use of organophosphorous pesticides has increased in agricultural environments and urban settings, and there is evidence that estrogen may increase breast cancer risk in women. The mammary gland is an excellent model for examining its susceptibility to different carcinogenic agents due to its high cell proliferation capabilities associated with the topography of the mammary parenchyma and specific stages of gland development. Several experimental cellular models are presented here, in which the animals were exposed to chemical compounds such as pesticides, and endogenous substances such as estrogens that exert a significant effect on normal breast cell processes at different levels. Such models were developed by the effect of malathion, parathion, and eserine, influenced by estrogen demonstrating features of cancer initiation in vivo as tumor formation in rodents; and in vitro in the immortalized normal breast cell line MCF-10F, that when transformed showed signs of carcinogenesis such as increased cell proliferation, anchorage independence, invasive capabilities, modulation of receptors and genomic instability. The role of acetylcholine was also demonstrated in the MCF-10F, suggesting a role not only as a neurotransmitter but also with other functions, such as induction of cell proliferation, playing an important role in cancer. Of note, this is a unique experimental approach that identifies mechanistic signs that link organophosphorous pesticides with breast carcinogenesis.

Ionizing Radiation and Estrogen Affecting Growth Factor Genes in an Experimental Breast Cancer Model.

Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/μm) and subsequent growth in the presence or absence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in breast cancer than in normal tissues in breast cancer patients. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expression levels were in stages 3 and 4 of breast cancer patients. It can be concluded that, by using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in breast cancer will make possible the improvement of clinical chemotherapy.

Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell–cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

Apoptotic effect of noscapine in breast cancer cell lines

Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression and also by increase of IκBα gene expression induced by this drug.

Development and characterization of two human triple-negative breast cancer cell lines with highly tumorigenic and metastatic capabilities.

Triple‐negative breast cancer (TNBC) is a group of cancer with high diversity, limited therapies, and poor prognosis. TNBC cell lines and animal models provide effective tools for studies and drug discovery. Here, we report the development of two TNBC cell lines (XtMCF and LmMCF) based on our existing cell model that consists of normal breast epithelial cell line MCF10F, estradiol‐transformed cells trMCF, and Boyden chamber‐selected tumorigenic cells bsMCF. The XtMCF and LmMCF cell line were derived from xenograft and lung metastasis of bsMCF cells, respectively. The bsMCF, XtMCF, and LmMCF cells have undergone epithelial–mesenchymal transition (EMT), exhibiting a mesenchymal‐like feature. In vivo studies showed XtMCF and LmMCF cells were highly tumorigenic and metastatic. The injection of 5 × 104 cells to CB17/SCID mice mammary fat pad produced xenografts in 9/9 mice and tumors reached 10 millimeters in diameter in 5 weeks. The injection of 1 × 106 XtMCF or 8 × 104 LmMCF cells into the mice tail vein was sufficient to form extensive lung metastases in 4 weeks. The two new cell lines exhibited CD44+/CD49f+ and CD44+/EpCAM + cancer stem cell (CSC) characteristics, and the EGF‐like domain of EpCAM was cleaved off. Together with the normal and early transformed counterparts, herein we provide a complete cancer model for the study of initiation, evolution, and identification of new therapeutics for TNBC. The finding that EGF‐like domain of EpCAM was cleaved off in cells which have undergone EMT suggests this cleavage may be involved in the EMT process and the cancer stem cell properties of these cells.

Abstract 2109: All trans-retinoic acid (ATRA) induces redifferentiation of early transformed breast epithelial cells

Abstract Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic, and anti-oxidant properties. The effects of retinoids are mediated by retinoic acid receptors (RARβ;,RARα,RARγ) and the retinoid X receptors (RXR) that bind the DNA as heterodimers. The objective of this project was to study the potential of retinoic acid to revert the neoplastic process using an in vitro model of breast cancer progression. This model was previously developed by treating the human normal breast epithelial MCF-10F cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: (a) the spontaneously immortalized cell line MCF-10F, which does not show any characteristic of invasiveness or tumor formation and therefore is considered to be a normal-like breast epithelial cell line; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the caMCF tumor cells. The bsMCF cells induced tumors when they were injected in the mammary fat pad of SCID mice. We previously showed that RARβ was unmethylated in MCF-10F and trMCF cells, but became hypermethylated in bsMCF and caMCF. We studied the effect of retinoic acid at the different stages in this model by cultivating the cells in 3D-cultures (collagen matrix). In collagen, the MCF-10F cells form tubules resembling the normal mammary gland. After treatment with estradiol, cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with different concentrations of ATRA (10-5M to 10-8M). We found that trMCF clone 11 cells showed a reduction of solid masses and increased number of tubules in collagen after being treated with 10-6M ATRA. A total of 43% of the structures were tubules in trMCF clone 11 cells treated with 10-6M ATRA. Gene expression studies showed that 207 genes up-regulated in transformed trMCF clone 11 cells were down-regulated after ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were down-regulated in trMCF clone 11 were up-regulated after ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 10-6M ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process to a normal stage and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after treatment with ATRA alone or in combination with 5-aza-dC. These results suggest that ATRA could be used as chemopreventive agent to inhibit the progression of premalignant lesions of the breast. This work was supported by The Pennsylvania Breast Cancer Coalition and Friends for an Earlier Breast Cancer Test. Citation Format: Maria F. Arisi, Rebecca A. Starker, Sankar Addya, Yong Huang, Sandra V. Fernandez. All trans-retinoic acid (ATRA) induces redifferentiation of early transformed breast epithelial 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 2109. doi:10.1158/1538-7445.AM2014-2109

Differential expression of cell adhesion molecules in an ionizing radiation-induced breast cancer model system

Cell-cell adhesion is mediated by members of the cadherin-catenin system and among them E-cadherin and β-catenin are important adhesion molecules for epithelial cell function and preservation of tissue integrity. To investigate the importance of cell adhesion molecules in breast carcinogenesis, we developed an in vitro breast cancer model system wherein immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer (LET) α particle radiation (150 keV/µm) and subsequent growth in the presence or absence of 17β-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) parental cell line MCF-10F; ii) MCF-l0F continuously grown with estradiol at 10(-8) (Estrogen); iii) a non-malignant cell line (Alpha3); and iv) a malignant and tumorigenic cell line (Alpha5) and the Tumor2 cell line derived from the nude mouse xenograft of the Alpha5 cell line. Expression levels of important cell adhesion molecules such as α-catenin, β-catenin, γ-catenin, E-cadherin and integrin were found to be higher at the protein level in the Alpha5 and Tumor2 cell lines relative to these levels in the non-tumorigenic MCF-10F, Estrogen and Alpha3 cell lines. In corroboration, cDNA expression analysis revealed elevated levels of genes involved in the cell adhesion function [E-cadherin, integrin β6 and desmocollin3 (DSc3)] in the Alpha5 and Tumor2 cell lines relative to the levels in the MCF-10F, Estrogen and Alpha3 cell lines. Collectively, our results suggest that cell adhesion molecules are expressed at higher levels in malignantly transformed breast epithelial cells relative to levels in non-malignant cells. However, reduced levels of adhesion molecules observed in the mouse xenograft-derived Tumor 2 cell line compared to the pre-tumorigenic Alpha5 cell line suggests that the altered expression levels of adhesion molecules depend on the tumor tissue microenvironment.

Effect of curcumin on irradiated and estrogen-transformed human breast cell lines

Curcumin (diferuloyl methane) is a well known antioxidant that exerts antiproliferative and apoptotic effects. Curcumin effect was evaluated in a breast cancer model that was developed using the immortalized breast epithelial cell line MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17β-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) MCF-10F; ii) Estrogen cell line; iii) a malignant Alpha3 cell line; iv) a malignant and tumorigenic, Alpha5 cell line; and v) a cell line derived from Alpha5 injected into the nude mice that gave rise to Tumor2 cell line. Curcumin decreased anchorage-independent growh in transformed breast cancer cell lines in comparison to their counterparts and increased the percentage of cells from G₀/G₁ with a concomitant increase in G₂/M phases, as well as a decrease in PCNA and Rho-A protein expression. Among the oncogenes, c-Ha-Ras and Ras homologous A (Rho-A) are important cell signaling factors for malignant transformation and to reach their active GTP bound state, Ras proteins must first release bound GDP mediated by a guanine nucleotide releasing factor (GRF). Then curcumin decrease RasGRF1 protein expression in malignant cell lines. Further, differential expression levels of cleaved (ADP) ribose polymerase 1 (PARP-1) and phosphorylated histone H2AX (γ-H2AX) were observed after curcumin treatment. It seems that PARP-1 similar to H2AX, confers cellular protection against radiation and estrogen-induced DNA damage mediated by curcumin. Therefore, targeting either PARP-1 or H2AX may provide an effective way of maximizing the therapeutic value of antioxidants for cancer prevention.

Protective role of curcumin in oxidative stress of breast cells

Curcumin (diferuloylmethane) is a well known antioxidant that exerts anti-proliferative and apoptotic effects. The effects of curcumin were evaluated in a breast cancer model that was developed with the immortalized breast epithelial cell line, MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150keV/µm) of radiation, and subsequently cultured in the presence of 17β-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) a control cell line, MCF-10F, ii) an estrogen-treated cell line, named Estrogen, iii) a malignant cell line, named Alpha3 and iv) a malignant and tumorigenic, cell line named Alpha5. Curcumin decreased the formation of hydrogen peroxide in the control MCF-10F, Estrogen and Alpha5 cell lines in comparison to their counterparts. Curcumin had little effect on NFκB (50kDa) but decreased the protein expression in the Estrogen cell line in comparison to their counterparts. Curcumin enhanced manganese superoxide dismutase (MnSOD) protein expression in the MCF-10F and Alpha3 cell lines. Results indicated that catalase protein expression increased in curcumin treated-Alpha3 and Alpha5 cell lines. Curcumin slightly decreased lipid peroxidation in the MCF-10F cell lines, but significantly (P<0.05) decreased it in the Alpha5 cell line treated with curcumin in comparison to their counterparts as demonstrated by the 8-iso-prostaglandin F2α (8-iso-PGF2α) levels. It can be concluded that curcumin acted upon oxidative stress in human breast epithelial cells transformed by the effect of radiation in the presence of estrogen.

Abstract 75: NRG1 and RARβ hypermethylation in breast cancer progression

Abstract We have developed an in vitro model of breast cancer progression that consists of four derived ER-negative PR-negative cell lines: (a) the normal-like breast epithelial cell line MCF-10F; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the tumorigenic caMCFs cells which shown all characteristics of a fully malignant breast cancer cells [1,2]. Briefly, the normal human breast epithelial MCF-10F cells were treated with high concentration of 17β-estradiol (70 nM) giving origin to trMCF cells; trMCF cells were seeded in Boyden chambers and the cells that crossed the membrane were selected and expanded giving origin to bsMCF. The bsMCF cells were injected in the mammary fat pad of SCID mice and they induced tumors that were poorly differentiated adenocarcinomas that were ESR, progesterone receptor (PR) and ERBB2 negatives. Cells were isolated from 4 different tumors growth in four animals and they gave origin to the tumorigenic caMCFs cells (caMCF1, caMCF2, caMCF3 and caMCF4) [1,2]. This in vitro model of breast cancer progression was used to study DNA-methylation changes at the different stages using sensitive methylation specific PCR (MSP) and, we found that NRG1 and RARβ showed progressive changes in their methylation pattern. NRG1 and RARβ were unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. The clinical relevance of these findings were pursued by studying NRG1 and RARβ methylation in 17 cases of invasive ductal carcinomas (IDC) and 11 normal breast tissue samples (controls). NRG1 was hypermethylated in 14 out of 17 IDC (82.4%) vs. 2 out of 10 (20%) controls. All the ER-negative PR-negative tumors (10/10) showed NRG1 hypermethylated. From the seven ER-positive IDC, only 4 (57%) showed NRG1 hypermethylated. RARβ was methylated in 9 out of 16 IDC (56.3%) vs. 2 out of 11 (18.2%) controls. Most of the ER-negative PR-negative invasive ductal tumors showed RARβ hypermethylated (9/10). None of the ER-positive IDC shown RARβ hypermethylated. Altogether our data shown that the majority (90%) of the ER-negative PR-negative invasive ductal carcinomas shown hypermethylation of both NRG1 and RARβ. None of the ER-positive invasive ductal carcinomas showed RARβ hypermethylation and, only 57% showed hypermethylation of NRG1. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES [1] S.V. Fernandez, K.E. Snider, Y.Z. Wu, I.H. Russo, C. Plass and J. Russo DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. [2] Y. Huang, S.V. Fernandez, S. Goodwin, P.A. Russo, I.H. Russo, T.R. Sutter and J. Russo Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol, Cancer Res 67 (2007) 11147-11157. 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 75. doi:10.1158/1538-7445.AM2011-75

Abstract 4551: Retinoic acid in the reversion of the neoplastic transformation

Abstract Retinoids, usually formed from dietary vitamin A, play a key role during embryonic development, cellular proliferation and differentiation, and are potent inhibitors of cell growth. Retinoids have been recognized as promising anticancer compounds. The effects of retinoids are mediated by retinoic acid receptors (RARβ, RARα, RARγ) and the retinoid X receptors (RXR) that binds the DNA as heterodimers. The objective of this project is to study the potential of retinoic acid to revert the neoplastic process using an in vitro model of breast cancer progression developed in our laboratory. The MCF-10F progression model consists of four derived cell lines: (a) the spontaneously immortalized cell line MCF-10F, which does not show any characteristic of invasiveness or tumor formation and therefore is considered to be a normal-like breast epithelial cell line; (b) the transformed trMCF cells; (c) the invasive bsMCF cell line and; (d) the tumor cell lines, caMCFs, which shown all characteristics of a fully malignant breast cancer cell types. The bsMCF cells induced tumors when they were injected in the mammary fat pad of SCID mice; these tumors were poorly differentiated adenocarcinomas that were estrogen receptor (ER), progesterone receptor (PR) and ERBB2 negatives [1]. Using this model, DNA-methylation studies were performed and we have found that RARβ (retinoic acid receptor β) was unmethylated in MCF-10F and trMCF cells becoming hypermethylated at the invasive (bsMCF) and tumorigenic (caMCF) stages. We studied the effect of retinoic acid at the different stages of the in vitro model of breast cancer progression using 3D-cultures (collagen matrix). Cultivation of normal breast epithelial cells within collagen enables the cells to polarize and to spontaneously differentiate tubular structures. In collagen, the MCF-10F cells form tubules resembling the normal mammary gland. The transformed cells trMCF form tubules and spherical masses; spherical masses are an indication of cell transformation. One solid mass was isolated and the cells expanded, giving origin to trMCF clone 11, which only form solid masses in collagen. The trMCF clone 11 cells were treated with different concentrations of retinoic acid during 15 days and, after the treatments the cells were plated in collagen. The trMCF clone 11 treated with 10-6M and 10-7M retinoic acid shown a reduction of solid masses and increased of tubules in collagen. Although bsMCF and caMCF were treated with 5-aza-dC to reactivate the expression of RARβ in combination with retinoic acid, a change in the phenotype of these cells in collagen was not observed. Our results indicated that retinoic acid was able to re-differentiate transformed cells at early stages of the neoplastic process. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES. [1] S.V. Fernandez et al. DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. 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 4551. doi:10.1158/1538-7445.AM2011-4551

Deleterious MnSOD signals lead to abnormal breast cell proliferation by radiation and estrogen exposure

Manganese superoxide dismutase (MnSOD) seems to have a pivotal role in maintaining the normal phenotype by suppressing cell growth through blocking the entrance of quiescent cells into the cell cycle. MnSOD protein expression has been shown to be dysregulated in malignant cells. A well-established experimental breast epithelial cell cancer model was used to observe the relationship in the presence or absence of such protein and the phenotype of the cells. This model was derived from the spontaneously immortalized breast epithelial cell line MCF-10F, which was transformed with estrogen and radiation. The results of this study showed that deleterious expression of MnSOD enhanced the malignant phenotype demonstrated by cell cycle protein expression changes. Thus, the malignant cell line, called Alpha5, which had high levels of MnSOD protein expression, maintained a similar phenotype to the normal cell line MCF-10F. The cell cycle arrest observed in G1 phase of the Alpha5 cell line was induced by p16 protein expression which has been shown to inhibit the Cyclin D1/CdK4 complex explaining such arrest. It can be concluded from these studies that SOD expression, played a critical role in free radical detoxification and it is directly correlated with the cell cycle, defining one of the most important characteristics of tumor cells, namely cell growth and proliferation. These findings are in agreement with the hypothesis that MnSOD plays a role as a possible tumor suppressor gene. Furthermore, this work is a contribution to understanding the possible changes that occur in α-particle irradiated cells, sensitized with estrogen, due to the presence of superoxide dismutase scavenger that could have significant implications in the design of clinical radiotherapeutic protocols.

Resveratrol and N-acetylcysteine block the cancer-initiating step in MCF-10F cells

Substantial evidence suggests that catechol estrogen-3,4-quinones react with DNA to form predominantly the depurinating adducts 4-hydroxyestrone (estradiol)-1-N3Ade [4-OHE 1(E 2)-1-N3Ade] and 4-OHE 1(E 2)-1-N7Gua. Apurinic sites resulting from these adducts generate critical mutations that can initiate cancer. The paradigm of cancer initiation is based on an imbalance in estrogen metabolism between activating pathways that lead to estrogen–DNA adducts and deactivating pathways that lead to estrogen metabolites and conjugates. This imbalance can be improved to minimize formation of adducts by using antioxidants, such as resveratrol (Resv) and N-acetylcysteine (NAcCys). To compare the ability of Resv and NAcCys to block formation of estrogen–DNA adducts, we used the human breast epithelial cell line MCF-10F treated with 4-OHE 2. Resv and NAcCys directed the metabolism of 4-OHE 2 toward protective pathways. NAcCys reacted with the quinones and reduced the semiquinones to catechols. This pathway was also carried out by Resv. In addition, Resv induced the protective enzyme quinone reductase, which reduces E 1(E 2)-3,4-quinones to 4-OHE 1(E 2). Resv was more effective at increasing the amount of 4-OCH 3E 1(E 2) than NAcCys. Inhibition of estrogen–DNA adduct formation was similar at lower doses, but at higher doses Resv was about 50% more effective than NAcCys. Their combined effects were additive. Therefore, these two antioxidants provide an excellent combination to protect catechol estrogens from oxidation to catechol quinones.

Stage-Specific Expressions of GLUT-1, GLUT-3 and GLUT-12 in Estrogen-Induced Breast Cancer.

Abstract Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. The glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12 have been shown in breast cancer cells in which estrogen play a critical role on its regulation and also associated with poor prognosis. However. the stage-specific GLUT regulation during the process of mammary carcinogenesis is unclear. In the present study we are aiming to study the GLUT expression pattern in an in vitro-in vivo model of estrogen induced carcinogenesis in which the human breast epithelial cell line MCF-10F is considered the normal counterpart E2 the transformed, C5 the invasive and T4 the tumorigenic stage. The gene expression of GLUTs was correlated with the ductulogenic pattern in a collagen matrix and the invasive properties using the Boyden chamber The RT-PCR data showed that the GLUT1 expression was downregulated in MCF10F after the treatment with 17β-estradiol (E2) and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasion capacity. Whereas, GLUT-12 is downregulated in invasive and tumorigenic cells. We conclude that the estrogen-induced malignant transformation is associated with remarkably GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation for transformed MCF-10F cells. (This work was supported by grant R21 ES015894from the NIEHS) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5152.

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

Estrogen metabolism is suggested to play an important role in estrogen-induced breast carcinogenesis. Epidemiologic studies suggest that diets rich in phytoestrogens are associated with a reduced risk of breast cancer. Phytoestrogens are biologically active plant compounds that structurally mimic 17β-estradiol (E 2). We hypothesize that phytoestrogens, may provide protection against breast carcinogenesis by altering the expression of estrogen-metabolizing enzymes cytochrome P450 1A1 (Cyp1A1) and 1B1 (Cyp1B1). Cyp1A1 and Cyp1B1 are responsible for the metabolism of E 2 to generate 2-hydroxyestradiol (2-OHE 2) and 4-hydroxyestradiol (4-OHE 2), respectively. Studies suggest that 2-OHE 2 and 2-methoxyestradiol may protect against breast carcinogenesis, while 4-OHE 2 is carcinogenic in rodent models. Thus, agents that increase the metabolism of E 2 by Cyp1A1 to produce 2-OHE 2 may have chemoprotective properties. The human immortalized non-neoplastic breast cell line MCF10F was treated with quercetin at 10 and 50 μM concentrations for time points ranging from 3 to 48 h. Total RNA and protein were isolated. Real-time PCR was used to measure the expression of Cyp1A1 and Cyp1B1 mRNA. Quercetin treatment produced differential regulation of Cyp1A1 and Cyp1B1 mRNA expression in a time- and dose-dependent manner. Treatment with 10 and 50 μM doses of quercetin produced 6- and 11-times greater inductions of Cyp1A1 mRNA over Cyp1B1 mRNA, respectively. Furthermore, quercetin dramatically increased Cyp1A1 protein levels and only slightly increased Cyp1B1 protein levels in MCF10F cells. Thus, our data suggest that phytoestrogens may provide protection against breast cancer by modulating expression of estrogen-metabolizing genes such that production of the highly carcinogenic estrogen metabolite 4-OHE 2 by Cyp1B1 is reduced and the production of the less genotoxic 2-OHE 2 by Cyp1A1 is increased.

Cancer genes induced by malathion and parathion in the presence of estrogen in breast cells

The identification of genes involved in the process of neoplastic transformation is essential for analyzing the progression of breast cancer when induced by endogenous and exogenous agents, among which are the estrogens and the organophosphorous pesticides, respectively. It is important to consider the impact of such substances when they are present in combination. In vitro experimental models are needed in order to understand breast carcinogenesis. The aim of this work was to examine the effect of 17beta estradiol (estrogen) combined with either malathion or parathion on the transformation of human breast epithelial cells in vitro. Results showed that estrogen combined with either malathion or parathion altered cell proliferation and induced cell transformation as well as exhibited significant invasive capabilities as compared to the control MCF-10F cell line. Several genes were up-regulated by the effect of all of the treatments, such as the cyclins, cyclin D1 and cyclin-dependent kinase 4, IGFBP3 and IGFBP5, and keratin 18. The c-Ha-ras oncogene was up-regulated by the effect of malathion alone and with the combination of estrogen and either malathion or parathion. The DVL1 gene was up-regulated only with malathion alone and the combination of parathion with estrogen. Expression of the HSP 27, MCM2 and TP53 inducible protein 3 genes was up-regulated with malathion alone and with the combination of estrogen and either malathion or parathion while TP53 (Li-Fraumeni syndrome) was up-regulated by estrogen alone and malathion alone. Thus, we suggest that pesticides and estrogens affect human breast cells inducing molecular changes indicative of transformation.

Formation of depurinating N3Adenine and N7Guanine adducts by MCF‐10F cells cultured in the presence of 4‐hydroxyestradiol

Metabolic conversion of endogenous estrogens, estradiol (E2) and estrone (E1), to the catechol estrogens 4-hydroxyE1(E2) [4-OHE1(E2)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4-OHE1(E2) are enzymatically oxidized to E1(E2)-3,4-quinones [E1(E2)-3,4-Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF-10F has been transformed by treatment with E2 or 4-OHE2. We have used MCF-10F cells to study the presence of adducts and conjugates after treatment with 4-OHE2. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF-10F cells were treated with 1 microM 4-OHE2 for a 24-h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4-OHE2 showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4-OHE1(E2)-1-N3Ade. The amount of 4-OHE1(E2)-1-N3Ade adducts was higher after the third treatment; smaller amounts of the 4-OHE1(E2)-1-N7Gua adducts were detected after the second and third treatments. These results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. This DNA damage can play an important role in the 4-OHE2-induced mutations and transformation of MCF-10F cells to malignant cells.

DNA content and chromatin texture of human breast epithelial cells transformed with 17-β-estradiol and the estrogen antagonist ICI 182,780 as assessed by image analysis

The immortalized human breast epithelial MCF-10F cell line, although estrogen receptor α negative, develops cell proliferating activities and invasiveness indicative of neoplastic transformation, after treatment with 17-β-estradiol (E-2). These effects are similar to those produced by benzo[ a]pyrene (BP). Since we have previously reported changes in the nuclear parameters accompanying BP-induced tumorigenesis in MCF-10F cells, we have examined whether similar alterations occur in E-2-treated cells. We therefore studied DNA amounts and other nuclear parameters in Feulgen-stained MCF-10F cells after treatment with various concentrations of E-2, BP, the estrogen antagonist ICI 182,780, and E-2 in the presence of ICI 182,780. E-2 caused a certain loss of DNA and changes in the nuclear size and chromatin supraorganization of MCF-10F cells. Many of these changes were similar to those produced by BP and were indicative of neoplastic transformation. More intense chromatin remodelling was seen with 70 nM E-2. Since these changes were not abrogated totally or partially by ICI 182,780, the neoplastic transformation of MCF-10F cells stimulated by E-2 involved a process that was independent of estrogen α-receptors. The changes produced by ICI 182,780 alone were attributed to effects other than its well-known anti-estrogenic activity.

Protein Kinase C β Enhances Growth and Expression of Cyclin D1 in Human Breast Cancer Cells

Although alterations in the expressions of protein kinase C (PKC) have been implicated in breast carcinogenesis, the roles of specific isoforms in this process remain elusive. In the present study, we examined the specific roles of PKCbeta1 and beta2 in growth control in human breast cancer cell lines. The PKCbeta-specific inhibitor LY379196 significantly inhibited growth of the breast cancer cell lines MCF-7, MDA-MB-231, and BT474, but not the normal mammary epithelial cell line MCF-10F. Treatment of MCF-7 cells with LY379196 caused an increase in the fraction of cells in the G(1) phase of the cell cycle. To explore the roles of PKCbeta1 and beta2, we used cDNA expression vectors that encode wild-type and constitutively activated or dominant negative mutants of these two proteins. When compared with vector controls, derivatives of MCF-7 cells that stably overexpress wild-type PKCbeta1 or PKCbeta2 displayed a slight increase in growth rate; derivatives that stably express the constitutively active mutants of PKCbeta1 or PKCbeta2 displayed a marked increase in growth rate; and derivatives that stably express a dominant negative mutant of PKCbeta1 or beta2 displayed inhibition of growth. The derivatives of MCF-7 cells that stably express the constitutively activated mutants of PKCbeta1 or beta2 were more resistant to growth inhibition by LY379196 than the vector control MCF-7 cells. Immunoblot analysis indicated that MCF-7 cells that stably overexpress wild-type or constitutively activated mutants of PKCbeta1 or beta2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that express a dominant negative mutant had decreased levels of cyclin D1. The derivatives that stably express the constitutively activated mutants of PKCbeta1 or beta2 also displayed increased cyclin D1 promoter activity in transient transfection luciferase reporter assays, and this induction of activity requires activator protein 1. Constitutively activated PKCbeta1 and beta2 also enhanced the transcription of c-fos in transient transfection luciferase reporter assays. Thus, PKCbeta1 and beta2 may play important positive roles in the growth of at least a subset of human breast cancers. Therefore, inhibitors of these isoforms may be useful in breast cancer chemoprevention or therapy.

17‐Beta‐Estradiol induces transformation and tumorigenesis in human breast epithelial cells

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17beta-estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER-alpha) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage-independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11-13. Only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor-negative adenocarcinomas that expressed keratins, EMA, and E-cadherin. Tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of MCF-10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.