Human Breast Cell Lines Research Articles

Long-term exposure of MCF-7 breast cancer cells to ethanol stimulates oncogenic features.

Alcohol consumption is a risk factor for breast cancer. Little is known regarding the mechanism, although it is assumed that acetaldehyde or estrogen mediated pathways play a role. We previously showed that long-term exposure to 2.5 mM ethanol (blood alcohol ~0.012%) of MCF-12A, a human normal epithelial breast cell line, induced epithelial mesenchymal transition (EMT) and oncogenic transformation. In this study, we investigated in the human breast cancer cell line MCF-7, whether a similar exposure to ethanol at concentrations ranging up to peak blood levels in heavy drinkers would increase malignant progression. Short-term (1-week) incubation to ethanol at as low as 1–5 mM (corresponding to blood alcohol concentration of ~0.0048–0.024%) upregulated the stem cell related proteins Oct4 and Nanog, but they were reduced after exposure at 25 mM. Long-term (4-week) exposure to 25 mM ethanol upregulated the Oct4 and Nanog proteins, as well as the malignancy marker Ceacam6. DNA microarray analysis in cells exposed for 1 week showed upregulated expression of metallothionein genes, particularly MT1X. Long-term exposure upregulated expression of some malignancy related genes (STEAP4, SERPINA3, SAMD9, GDF15, KRT15, ITGB6, TP63, and PGR, as well as the CEACAM, interferon related, and HLA gene families). Some of these findings were validated by RT-PCR. A similar treatment also modulated numerous microRNAs (miRs) including one regulator of Oct4 as well as miRs involved in oncogenesis and/or malignancy, with only a few estrogen-induced miRs. Long-term 25 mM ethanol also induced a 5.6-fold upregulation of anchorage-independent growth, an indicator of malignant-like features. Exposure to acetaldehyde resulted in little or no effect comparable to that of ethanol. The previously shown alcohol induction of oncogenic transformation of normal breast cells is now complemented by the current results suggesting alcohol's potential involvement in malignant progression of breast cancer.

In Vitro Study on Effect of Germinated Wheat on Human Breast Cancer Cells

This research investigated the possible anticancer effects of germinated wheat flour (GWF) on growth and apoptosis of human breast cancer cells. In a series of in vitro experiments, estrogen‐receptor‐positive (MCF‐7) and ‐negative (MDA‐MB‐231) cells were cultured and treated with GWF that was obtained after a three‐day germination. The GWF significantly reduced the growth of both of MCF‐7 and MDA‐MB‐231 cells. In analysis with a flow cytometer, the GWF significantly up‐regulated apoptosis of both cell lines. These results indicate that GWF induced apoptosis and inhibited growth of the human breast cell lines, thereby demonstrating its potential inhibition of breast cancer cell proliferation.

Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells

Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy.

CO-releasing properties and anticancer activities of manganese complexes with imidazole/benzimidazole ligands

Carbon monoxide (CO) is an important signaling molecule which plays significant roles in the pathogenesis of cancer. CO is produced by enzymatic degradation of heme in mammals. Heme oxygenase 1 (HO-1) catalyzes the breakdown of heme into CO, ferrous iron, and biliverdin. CO induces HO-1 and inhibits cell proliferation. Cancer cells exposed to several stress factors (hypoxia, reactive oxygen species, cis-platin, and oxidative stress), and HO-1 displays cytoprotective role against oxidative stress and inhibits apoptosis, metastases, angiogenesis, and cell proliferation processes. Therefore, metal containing CO-releasing molecules (CORMs) have been designed as an effective cancer treatment strategy. CORMs are responsible for releasing controlled amounts of CO to cells and tissues. Thus, we synthesized [Mn(CO)3(bpy)L]X manganese containing CORMs [bpy = 2,2′-bipyridine, X = hexafluorophosphate (PF6), trifluoromethanesulfonate (OTf), L = imidazole, methylimidazole, benzimidazole, N-benzylbenzimidazole, N-(4-chlorobenzyl)benzimidazole] to release CO in human invasive ductal breast (MCF-7) cell line. In vitro experiments indicated that the compounds inhibited cell proliferation and exhibited cytotoxic effect on breast cancer cells. Moreover, side groups of the compounds enhanced the anticancer effects in MCF-7 cell line. These manganese containing CORMs gave promising results and may be used as a drug template for effective treatment of invasive ductal breast carcinoma.

Integrin α6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy.

Integrin α6 (ITGA6), a transmembrane glycoprotein adhesion receptor protein, is widely upregulated in many types of tumors and promotes migration and invasion in cancer cells. However, the role that the ITGA6-associated signaling network plays in radiosensitivity in breast cancer has not been described. The expression of ITGA6 was examined in human breast cancer and normal breast cell lines using western blot analysis. We also explored the role of ITGA6 in the regulation of radiation sensitivity in breast cancer using the colony formation assays, cell cycle analyses, apoptosis assays and immunofluorescence analyses. The results showed that the protein and mRNA expression levels of ITGA6 was higher in breast cancer cells than in normal cells. ITGA6 protectived responses to radiotherapy in breast cancer cells by altering cell apoptosis, DNA damage repair and cell-cycle regulation. Furthermore, ITGA6 enhanced radiation resistance via PI3K/Akt and MEK/Erk signaling. In addition, overexpressing ITGA6 promoted radiation resistance in cells, and this effect was neutralized by the PI3K inhibitor LY294002 and MEK inhibitor U0126. Taken together, these findings indicate that ITGA6 might be involved in a mechanism that underlies radiation resistance and that ITGA6 could be a potential target for therapies aimed at overcoming radiation resistance in breast cancer.

Macrocyclic [N5] transition metal complexes: synthesis, characterization and biological activities

Novel penta-azamacrocyclic 21-membered [N5] ligand [L] and its transition metal complexes with Co(II), Ni(II), Cu(II), Ru(III) and Pd(II) have been isolated and characterized. The mode of bonding and overall geometry of the complexes have been inferred through IR, MS, UV–Vis, EPR, 1H NMR spectral studies, molar conductivity, magnetic, thermal and microanalyses, On the basis of above studies, an octahedral geometry has been proposed for all complexes except Pd(II) chloride complex which adopt square planar geometry. The in vitro antitumor activity of the synthesized ligand and some selected complexes against human breast and human hepatocarcinoma cell lines (MCF-7) and (HePG2), respectively has been studied. The results show that the tested compounds are potent antitumor agents. Also the ligand and some selected complexes have been tested for their inhibitory effect on the growth of bacteria: Streptococcus pyogenes as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria. The activity data show that most of the tested compounds exhibit remarkable antibacterial activity against these organisms.

Synthesis and anticancer activities of novel (tetrahydrobenzo [4,5] thieno [2,3-d] pyrimidine-4-yl)-pyrolidine-2-carboxylic acid derivatives

Cancer chemotherapy has been one of the major medical advances in the last few decades. The medications involved in treatment have a narrow therapeutic index, and often the responses produced are only just palliative as well as unpredictable. In contrast, targeted therapy such as Tyrosine kinases inhibitors, that has been introduced in recent years is directed against cancer-specific molecules and signaling pathways with some side effects Therefore, we studied these class of molecules to obtain new candidates of remarkable activities and minimal side effects. We herein reported SAR driven synthesis of 16 novel molecules and their anticancer profiles in specifically targeted human Breast cell line MCF-7 and Human Colon Cancer Cell Line HCT-15. Compounds 7e and 8f showed descent antitumor activities when compared to Doxorubicine (GI50 < 0.1) for human Breast cell line MCF-7. The synthesized analogs were ensured by spectral and elemental analysis.

Abstract 3751: Apoptosis inducer gene delivery by polylysine-calcium complexes attenuates mouse lung carcinoma allograft growth after intravenous injection or intratracheal spray

Abstract Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Nanoparticle-based gene delivery technique potentially overcomes these concerns and may be applicable to cancer gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Our previous study demonstrated that an apoptosis inducer, angiotensin II type 2 receptor plasmid DNA (pAT2R) encapsulated in a modified HIV-1 TAT peptide (dTAT-pAT2R), significantly attenuated the growth of Lewis lung carcinoma (LLC) allograft in mouse lungs (Kawabata et al., Cancer Res, 2012). Here, we report a newly synthesized polylysine CPP (K9 peptide)-based gene therapy for lung cancer treatment. The pAT2R and K9 peptide (K9-pAT2R) complexes were condensed using calcium chloride (K9-pAT2R-Ca2+). The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro. This simple non-viral formulation approach showed negligible cytotoxicity in several different human and mouse cell lines (human cervix, breast, kidney, and human and mouse lung cell lines). Additionally, this K9-pDNA-Ca2+ complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray into LLC orthotopic allograft-bearing mice. Average lung weights (mg) of the K9-pAT2R-Ca2+ IT (190.6±48.3) and the K9-pAT2R-Ca2+ IV (201.6±67.0) treated groups were significantly smaller than that of the control PBS group (325.7±69.4, P&amp;lt;0.05). In histological examination of tumors in H&amp;E stained lung, average numbers of tumor nodules in the lungs in the K9-pAT2R-Ca2+ IT (6.0±4.2) and the K9-pAT2R-Ca2+ IV (4.6±3.4) groups were also significantly smaller than that of the control PBS group (17.8±6.0, P&amp;lt;0.01). This tumor attenuating effect was not observed in K9-pLUC-Ca2+ IT and K9-pLUC-Ca2+ IV treatment, thus it is suggested that LLC tumor growth was attenuated by apoptosis inducer gene, pAT2R, delivery. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth offering preclinical proof of concept for a novel non-viral gene delivery method exhibiting effective lung tumor gene therapy via either IV or IT administration. This work is supported in part by Savara Pharmaceuticals (CB and MT), Faculty of Pharmacy of King Abdulaziz University, Jeddah, Saudi Arabia (NAA), University of Kansas Macromolecule and Vaccine Stabilization Center (CB), Kansas State University Johnson Cancer Research Center (MT), NIH grants U43 CA165462 (MT), P20 GM103418 (MT), and Kansas Bioscience Authority Collaborative Cancer Research grant (MT). Citation Format: Susumu Ishiguro, Nabil A. Alhakamy, Deepthi Uppalapati, Cory J. Berkland, Masaaki Tamura. Apoptosis inducer gene delivery by polylysine-calcium complexes attenuates mouse lung carcinoma allograft growth after intravenous injection or intratracheal spray. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3751.

Design and synthesis of pyrimidinyl acyl thioureas as novel Hsp90 inhibitors in invasive ductal breast cancer and its bone metastasis

Invasive ductal carcinoma is the most common breast malignancies tumors and has tendency to bone metastases. Many oncogenic client proteins involved in formation of metastatic pathways. Stabilization, regulation, and maintenance of these oncogenic client proteins are provided with Heat Shock Protein 90 (Hsp90). Hsp90 perform these processes through its ATP binding and subsequent hydrolysis energy. Therefore, designing Hsp90 inhibitors is a novel cancer treatment method. However, many Hsp90 inhibitors have solubility problems and showed adverse effects in clinical trials. Thus, we designed and synthesized novel pyrimidinyl acyl thiourea derivatives to selectively inhibit Hsp90 alpha in human invasive ductal breast (MCF-7) and human bone osteosarcoma (Saos-2) cell lines. In vitro experiments showed that the compounds inhibited cell proliferation, ATP hydrolysis, and exhibited cytotoxic effect on these cancer cell lines. Further, gene expression was analyzed by microarray studies on MCF-7 cell lines. Several genes that play vital roles in breast cancer pathogenesis displayed altered gene expression in the presence of a selected pyrimidinyl acyl thiourea compound. Molecular docking studies were also performed to determine interaction between Hsp90 ATPase domain and pyrimidinyl acyl thiourea derivatives. The results indicated that the compounds are able to interact with Hsp90 ATP binding pocket and inhibit ATPase function. The designed compounds powerfully inhibit Hsp90 by an average of 1 μM inhibition constant. And further, the compounds perturb Hsp90 N terminal domain proper orientation and ATP may not provide required conformational change for Hsp90 function as evidenced by in silico experiments. Therefore, the designed compounds effectively inhibited both invasive ductal breast carcinoma and bone metastasis. Pyrimidinyl acyl thiourea derivatives may provide a drug template for effective treatment of invasive ductal breast carcinoma and its bone metastasis as well as new therapeutic perspective for drug design.

Synthesis and in Vitro Antiproliferative Evaluation of C-13 Epimers of Triazolyl-d-Secoestrone Alcohols: The First Potent 13α-d-Secoestrone Derivative.

The syntheses of C-13 epimeric 3-[(1-benzyl-1,2,3-triazol-4-yl)methoxy]-d-secoestrones are reported. Triazoles were prepared from 3-(prop-2-inyloxy)-d-secoalcohols and p-substituted benzyl azides via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The antiproliferative activities of the products and their precursors were determined in vitro against a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines by means of MTT assays. The orientation of the angular methyl group and the substitution pattern of the benzyl group of the azide greatly influenced the cell growth-inhibitory potential of the compounds. The 13β derivatives generally proved to be more potent than their 13α counterparts. Introduction of a benzyltriazolylmethyl group onto the 3-OH position seemed to be advantageous. One 13α compound containing an unsubstituted benzyltriazolyl function displayed outstanding antiproliferative activities against three cell lines.

Synthesis and Biological Testing of Novel Glucosylated Epigallocatechin Gallate (EGCG) Derivatives.

Epigallocatechin gallate (EGCG) is the most abundant component of green tea catechins and has strong physiological activities. In this study, two novel EGCG glycosides (EGCG-G1 and EGCG-G2) were chemoselectively synthesized by a chemical modification strategy. Each of these EGCG glycosides underwent structure identification, and the structures were assigned as follows: epigallocatechin gallate-4′′-O-β-d-glucopyranoside (EGCG-G1, 2) and epigallocatechin gallate-4′,4′′-O-β-d-gluco-pyranoside (EGCG-G2, 3). The EGCG glycosides were evaluated for their anticancer activity in vitro against two human breast cell lines (MCF-7 and MDA-MB-231) using MTT assays. The inhibition rate of EGCG glycosides (EGCG-G1 and EGCG-G2) is not obvious. The EGCG glycosides are more stable than EGCG in aqueous solutions, but exhibited decreasing antioxidant activity in the DPPH radical-scavenging assay (EGCG > EGCG-G2 > EGCG-G1). Additionally, the EGCG glycosides exhibited increased water solubility: EGCG-G2 and EGCG-G1 were 15 and 31 times as soluble EGCG, respectively. The EGCG glycosides appear to be useful, and further studies regarding their biological activity are in progress.

Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines.

In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.

Abstract B34: A novel role for β-adrenergic receptor in mammary branching morphogenesis and its implication in breast cancer

Abstract The mammary gland develops from embryogenesis to infancy-puberty and adulthood, by the input of circulating hormones. At birth the gland is composed of a rudimentary ductal system that grows allometrically until puberty (4-weeks in mice). Later on, estrogens, growth hormone and insulin like growth factor induce expansive proliferation. Normal breast has three types of lobules, type 1, 2 and 3. Full term pregnancy and lactation results in the development of lobule type 3 into type 4, preparing the gland for lactation. This transformation is known to inhibit carcinogenic initiation through the induction of differentiation. β2-adrenergic receptors (β2-AR) have been well characterized in several human breast cell lines (normal and tumor) and in tissue samples. Recently, we have demonstrated that β2-AR expression and stimulation are associated with a benign breast tumor cell phenotype, reducing proliferation and cell migration, and increasing adhesion, suggesting that this receptor might be an important factor during tumor progression. In order to understand the implications of β2-AR on breast development and cancer, we evaluated β-activation in experimental models of normal and tumor breast, using cell lines, cultures 3D and in vivo approaches. Non tumor breast cell line MCF-10A cultured during 15 days on matrigel, formed gland-like organoids. Tubular structures mimicking mammary ducts were recognized, with type 1 and 2 lobules. No sign of lumen was observed. When treated with the β-agonist Isoproterenol (1 μM ISO) MCF-10A cells showed an increase in type 2 and 3 lobules (p&amp;lt;0.001) and the presence of lumen was observed. Furthermore, breast cancer cells MCF-7 growing in 3D, formed unorganized espheroid structures. When treated with ISO these structures acquire a polarized phenotype and some of them develop lumen. Later, Balb/c female virgin mice, weaning and 2 months old were treated daily with ISO 1 mg/Kg during 15 days. Control groups received saline solution. Mice were sacrificed and the 4th mammary glands were removed for Whole Mount and hematoxylin and eosin staining. Interestingly, ISO induced a statistically significant difference in mammary gland branching in weaning (150±32 vs 57±21 branches/field in controls, p&amp;lt;0.05) as well as in puberal mice (196±16 vs 94±14, p&amp;lt;0.01). It is well established that branching is controlled by progesterone receptor (PR), estrogen receptor alpha (ERα), FGF2, FGF10 and FGFR2, hence we studied their involvement in β-AR effect. First, ISO action was assessed on PR knockout mice in which branches were also significantly increased (p&amp;lt;0.05). After that, we addressed ER related signaling comparing ovariectomized (OVX) or ER depleted (by fulvestrant administration) mice treated or not with ISO. In both cases, despite the administration of ISO, branching was not induced. These results suggest that estrogen receptor and its classical ligand, estrogen, are required for ISO-induced branching. In addition, ISO induced a significant increase of FGF2, FGF10 and FGFR2 expression in both in vitro treated cells and in ISO treated mammary glands (p&amp;lt;0.05). Finally, we tested ISO in a mouse model of mammary ductal carcinoma. Surprisingly, tumors exhibited newly rough lumens and changes in cell polarity towards them, reinforcing the differentiating role of ISO also in a pathological context. In conclusion, β-AR stimulation seems to be involved in normal mammary development, leading to a terminal stage phenotype. In agreement with this, the analysis of a publicly available MicroArray dataset (GSE8191) showed an increase in β-AR expression levels during lactation. This study highlights a possible physiological role of β-AR in the development of mammary gland. Understanding the function of β-AR in normal development is crucial to elucidate its role in breast cancer and its possible use as a therapeutic target. Citation Format: Lucía Gargiulo, María May, Ezequiel Mariano Rivero, Lydon John, Caroline Lamb, Claudia Lanari, Isabel Alicia Luthy, Ariana Bruzzone. A novel role for β-adrenergic receptor in mammary branching morphogenesis and its implication in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B34.

Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

Transcriptional signature induced by a metastasis‐promoting c‐Src mutant in a human breast cell line

Deletions at the C-terminus of the proto-oncogene protein c-Src kinase are found in the viral oncogene protein v-Src as well as in some advanced human colon cancers. They are associated with increased kinase activity and cellular invasiveness. Here, we analyzed the mRNA expression signature of a constitutively active C-terminal mutant of c-Src, c-Src(mt), in comparison with its wild-type protein, c-Src(wt), in the human non-transformed breast epithelial cell line MCF-10A. We demonstrated previously that the mutant altered migratory and metastatic properties. Genome-wide transcriptome analysis revealed that c-Src(mt) de-regulated the expression levels of approximately 430 mRNAs whose gene products are mainly involved in the cellular processes of migration and adhesion, apoptosis and protein synthesis. 82.9% of these genes have previously been linked to cellular migration, while the others play roles in RNA transport and splicing processes, for instance. Consistent with the transcriptome data, cells expressing c-Src(mt), but not those expressing c-Src(wt), showed the capacity to metastasize into the lungs of mice in vivo. The mRNA expression profile of c-Src(mt)-expressing cells shows significant overlap with that of various primary human tumor samples, possibly reflecting elevated Src activity in some cancerous cells. Expression of c-Src(mt) led to elevated migratory potential. We used this model system to analyze the transcriptional changes associated with an invasive cellular phenotype. These genes and pathways de-regulated by c-Src(mt) may provide suitable biomarkers or targets of therapeutic approaches for metastatic cells. This project was submitted to the National Center for Biotechnology Information BioProject under ID PRJNA288540. The Illumina RNA-Seq reads are available in the National Center for Biotechnology Information Sequence Read Archive under study ID SRP060008 with accession numbers SRS977414 for MCF-10A cells, SRS977717 for mock cells, SRS978053 for c-Src(wt) cells and SRS978046 for c-Src(mt) cells.

Differential Response of Two Human Breast Cancer Cell Lines to the Phenolic Extract from Flaxseed Oil.

Many studies have evidenced that the phenolic components from flaxseed (FS) oil have potential health benefits. The effect of the phenolic extract from FS oil has been evaluated on two human breast cancer cell lines, MCF7 and MDA-MB231, and on the human non-cancerous breast cell line, MCF10A, by SRB assay, cellular death, cell cycle, cell signaling, lipid peroxidation and expression of some key genes. We have evidenced that the extract shows anti-proliferative activity on MCF7 cells by inducing cellular apoptosis, increase of the percentage of cells in G0/G1 phase and of lipid peroxidation, activation of the H2AX signaling pathway, and upregulation of a six gene signature. On the other hand, on the MDA-MB2131 cells we verified only an anti-proliferative activity, a weak lipid peroxidation, the activation of the PI3K signaling pathway and an up-regulation of four genes. Overall these data suggest that the extract has both cytotoxic and pro-oxidant effects only on MCF7 cells, and can act as a metabolic probe, inducing differences in the gene expression. For this purpose, we have performed an interactomic analysis, highlighting the existing associations. From this approach, we show that the phenotypic difference between the two cell lines can be explained through their differential response to the phenolic extract.

P53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer.

p125 is one of four subunits of human DNA polymerases – DNA Pol δ as well as one of p53 target protein encoded by POLD1. However, the function and significance of p125 and the role that p53 plays in regulating p125 expression are not fully understood in breast cancer. Tissue sections of human breast cancer obtained from 70 patients whose median age was 47.6 years (range: 38–69 years) with stage II–III breast cancer were studied with normal breast tissue from the same patients and two human breast cell lines (MCF-7 and MCF-10A). p53 expression levels were reduced, while p125 protein expression was increased in human breast cancer tissues and cell line detected by Western blot and quantitative reverse transcriptase-polymerase chain reaction. The methylation level of the POLD1 gene promoter was greater in breast cancer tissues and cells when compared with normal tissues and cells. In MCF-7 cell model, p53 overexpression caused a decrease in the level of p125 protein, while the methylation level of the p125 gene promoter was also inhibited by p53 overexpression. To further investigate the regulating mechanism of p53 on p125 expression, our study focused on DNA methyltransferase 1 (DNMT1) and transcription factor Sp1. Both DNMT1 and Sp1 protein expression were reduced when p53 was overexpressed in MCF-7 cells. The Sp1 binding site appears to be important for DNMT1 gene transcription; Sp1 and p53 can bind together, which means that DNMT1 gene expression may be downregulated by p53 through binding to Sp1. Because DNMT1 methylation level of the p125 gene promoter can affect p125 gene transcription, we propose that p53 may indirectly regulate p125 gene promoter expression through the control of DNMT1 gene transcription. In conclusion, the data from this preliminary study have shown that p53 inhibits the methylation of p125 gene promoter by downregulating the activities of Sp1 and DNMT1 in breast cancer.

A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells.

According to the World Health Organization (WHO), breast cancer is the most common cancer affecting women worldwide. In the USA ~12.3% of all women are expected to be diagnosed with various types of breast cancer, exhibiting varying degrees of therapeutic response rates. Therefore, the identification of novel anti-breast cancer drugs is of paramount importance. The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore was incorporated into a number of cytotoxins. Three of the resulting dienones, 2a, 2b and 2c, were tested for their anti-neoplastic potencies in a variety of human breast cancer-derived cell lines, including the triple negative MDA-MB-231 cell line and its metastatic variant, using a live-cell bio-imaging method. Special emphasis was put on dienone 2c, since its anti-cancer activity and its mode of inflicting cell death have so far not been reported. We found that all three dienones exhibited potent cytotoxicities towards the breast cancer-derived cell lines tested, whereas significantly lower toxicities were observed towards the non-cancerous human breast cell line MCF-10A. The dienones 2b and 2c exhibited the greatest selective cytotoxicity at submicromolar concentration levels. We found that these two dienones induced phosphatidylserine externalization in MDA-MB-231 cells in a concentration-dependent manner, suggesting that their cytotoxic effect might be mediated by apoptosis. This possibility was confirmed by our observation that the dienone 2c can induce mitochondrial depolarization, caspase-3 activation, cell cycle disruption and DNA fragmentation in MDA-MB-231 cells. Our findings indicate that dienone 2c uses the mitochondrial/intrinsic pathway to inflict apoptosis in triple negative MDA-MB-231 breast cancer-derived cells. This observation warrants further assessment of dienone 2c as a potential anti-breast cancer drug.

Abstract P5-04-10: Phosphodiesterase type 5 promotes the invasive potential of breast cancer cells through Rho GTPase activation

Abstract The impairment of cyclic guanosine monophosphate (cGMP) signaling by overexpression of PDE5 isoform has been recently described in multiple human carcinomas. In addition, accumulating evidences indicate that PDE5 inhibitors could have direct anti-cancer activities as well as they may enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs. However, despite these studies, neither the expression of PDE5 in breast cancer subtypes nor the underlying regulatory molecular mechanisms by which PDE5 expression may contribute to breast cancer progression have been deeply studied. We demonstrated that PDE5 was expressed in different subtypes of breast cancer cell lines at higher levels than in non tumorogenic human epithelial breast cell lines. Increased levels were detected in more aggressive endocrine non responsive basal-like breast cancer cells. Interestingly, PDE5 was expressed at very low levels in luminal A-type breast cancer cell lines, which display low ki67 expression, weak invasive behavior and endocrine responsiveness (MCF-7 and T47D cells) compared to luminal B-like cells (such as ZR-75 cells). These results well correlated with data obtained in immunohistochemistry analyses of human breast cancer tissues, showing PDE5 expression in 30 of 35 tumor entities analyzed, with the highest intensity staining in high-grade tumors. Concomitantly, no cytoplasmic PDE5 staining was observed in non neoplastic tissues examined (n=5). In addition, retrospective analyses (n=1959, median follow-up time: 25 years) showed that high PDE5 expression in breast cancer patients was correlated with a statistically significant poorer survival compared to low PDE5-expressing patients. A more relevant discrimination is achieved in lymphnode-negative patients, suggesting a role of PDE5 for identifying early patients at high risk of rapid progression. In order to better ascertain the role of PDE5 in breast tumorogenesis, we selected a breast tumor cell line that express low levels of this enzyme, MCF-7 and engineered stable clones for overexpression studies. Both vector- and PDE5-stable MCF-7 clones demonstrated comparable proliferation rates; whereas, cell motility and invasion were dramatically increased in PDE5-overexpressing cells. RNA sequencing to compare the transcriptomes of vector- and PDE5-overexpressing MCF-7 cells identified differential expression of genes involved in cell migration and invasion. Particularly, based on pathway analysis we found marked changes in the expression of Rho GTPase family members, proteins involved in cell cytoskeleton organization, migration, and metastasis dissemination (Rho A, cdc42 and Rac signaling, activation score= 1.9, 1.342, and 0.302, respectively). Indeed, Rho and cdc42 pull-down assays revealed increased Rho GTPase activity in cells overexpressing PDE5. Moreover, the selective ROCK inhibitor Y-27632 as well as the PDE5 inhibitor sildenafil were able to significantly reduce both migration and invasion of PDE5 clones. Our data reveal that PDE5 expression enhances motility and invasiveness of breast cancer cells through the activation of the Rho family of GTPases, and highlight, for the first time, a novel role for PDE5 as a marker of poor outcome in breast cancer patients. Citation Format: Barone I, Campana A, Giordano C, Tarallo R, Rinaldi A, Bruno G, Gyorffy B, Lanzino M, Bonofiglio D, Catalano S, Ando' S. Phosphodiesterase type 5 promotes the invasive potential of breast cancer cells through Rho GTPase activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-10.

Synthesis, structural characterization, and in vitro anti-cancer activities of new phenylacrylonitrile derivatives

The present study was designed to both synthesize phenylacrylonitrile compounds (1a–k) and their anti-tumor activities on human breast cancer cell line (MCF-7) were determined. The structures of all the compounds were defined by melting point, elemental analysis, FT-IR, 1H, 13C, 13C-APT, and HETCOR-NMR spectroscopy. Anti-tumor activities of these compounds on cell viability were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against MCF-7. The MCF-7 cell lines were treated with 1, 5, 25, 50, and 100 μM concentrations of phenylacrylonitrile compounds for 24 h. At the end of the experiments, 1a, 1b, 1c, 1g, and 1h compounds reduced cell viability (p < 0.01). Additionally, the anti-cancer activities of these compounds on MCF-7 were investigated by comparing IC50 values. In conclusion, while some of the synthesized phenylacrylonitrile compounds (1a, 1b, 1c, 1g, and 1h) have anti-tumor activity, other phenylacrylonitrile compounds (1d, 1e, 1f, 1k, and 1h) have no effect on human breast cell lines.