RESUMO
Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Colágeno , Combinação de Medicamentos , Células Epiteliais , Hidrogéis , Laminina , Peptídeos , Proteoglicanas , Laminina/farmacologia , Laminina/química , Hidrogéis/química , Hidrogéis/farmacologia , Proteoglicanas/farmacologia , Proteoglicanas/química , Colágeno/química , Colágeno/farmacologia , Peptídeos/farmacologia , Peptídeos/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/citologia , Humanos , Feminino , Técnicas de Cultura de Células em Três Dimensões/métodos , Sobrevivência Celular/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Glândulas Mamárias Humanas/citologia , Organoides/efeitos dos fármacos , Organoides/citologia , Técnicas de Cultura de Células/métodosRESUMO
As a major apurinic/apyrimidinic endonuclease and a redox signaling protein in human cells, APE1 plays a crucial role in cellular function and survival. The relationship between alterations of APE1 expression and subcellular localization and the initiation, development and treatment of various cancers has received extensive attention. However, comparing the in-vivo activity of APE1 in normal and cancerous breast live cells remains challenging due to the low efficiency of commonly used liposome transfection methods in delivering DNA substrate probes into human normal breast epithelial cells (MCF-10A). In this work, we develop a DNA/RNA hybrid-based small magnetic fluorescent nanoprobe (25 ± 3 nm) that can be taken up by various live cells under magnetic transfection. The D0/R-nanoprobe demonstrates an outstanding specificity toward APE1 and strong resistance to the cellular background interference. Using this nanoprobe, we are not only able to visualize the intracellular activity of APE1 in breast ductal carcinoma (MCF-7) live cells, but also demonstrate the APE1 activity in MCF-10A live cells for the first time. The method is then extended to observe the changes in APE1 levels in highly metabolically active neuroendocrine cells under normal conditions and severe attacks by reactive oxygen species in real-time. The fluorescent nanoprobe provides a useful tool for studying the dynamic changes of intracellular APE1 in normal or cancerous live cells. It also displays the potential for visible and controllable release of miRNA drugs within live cells for therapeutic purposes.
Assuntos
Neoplasias da Mama , MicroRNAs , Humanos , Feminino , Neoplasias da Mama/patologia , DNA , Neurônios/metabolismo , Endonucleases , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismoRESUMO
BACKGROUND: Aerotaxis, the chemotactism to oxygen, is well documented in prokaryotes. We previously reported for the first time that non-tumorigenic breast epithelial cells also display unequivocal directional migration towards oxygen. This process is independent of the hypoxia-inducible factor (HIF)/prolyl hydroxylase domain (PHD) pathway but controlled by the redox regulation of epidermal growth factor receptor (EGFR), with a reactive oxygen species (ROS) gradient overlapping the oxygen gradient at low oxygen concentration. Since hypoxia is an acknowledged hallmark of cancers, we addressed the putative contribution of aerotaxis to cancer metastasis by studying the directed migration of cancer cells from an hypoxic environment towards nearby oxygen sources, modelling the in vivo migration of cancer cells towards blood capillaries. METHODS: We subjected to the aerotactic test described in our previous papers cells isolated from fresh breast tumours analysed by the Pathology Department of the Saint-Etienne University Hospital (France) over a year. The main selection criterion, aside from patient consent, was the size of the tumour, which had to be large enough to perform the aerotactic tests without compromising routine diagnostic tests. Finally, we compared the aerotactic properties of these primary cells with those of commonly available breast cancer cell lines. RESULTS: We show that cells freshly isolated from sixteen human breast tumour biopsies, representative of various histological characteristics and grades, are endowed with strong aerotactic properties similar to normal mammary epithelial cell lines. Strikingly, aerotaxis of these primary cancerous cells is also strongly dependent on both EGFR activation and ROS. In addition, we demonstrate that aerotaxis can trigger directional invasion of tumour cells within the extracellular matrix contrary to normal mammary epithelial cells. This contrasts with results obtained with breast cancer cell lines, in which aerotactic properties were either retained or impaired, and in some cases, even lost during the establishment of these cell lines. CONCLUSIONS: Altogether, our results support that aerotaxis may play an important role in breast tumour metastasis. In view of these findings, we discuss the prospects for combating metastatic spread. TRIAL REGISTRATION: IRBN1462021/CHUSTE.
Assuntos
Neoplasias da Mama , Receptores ErbB , Humanos , Feminino , Espécies Reativas de Oxigênio , Receptores ErbB/metabolismo , Neoplasias da Mama/genética , Oxigênio/metabolismo , HipóxiaRESUMO
The effects of electric fields (EFs) on various cell types have been thoroughly studied, and exhibit a well-known regulatory effect on cell processes, implicating their usage in several medical applications. While the specific effect exerted on cells is highly parameter-dependent, the majority of past research has focused primarily on low-frequency alternating fields (<1 kHz) and high-frequency fields (in the order of MHz). However, in recent years, low-intensity (1-3 V/cm) alternating EFs with intermediate frequencies (100-500 kHz) have been of topical interest as clinical treatments for cancerous tumours through their disruption of cell division and the mitotic spindle, which can lead to cell death. These aptly named tumour-treating fields (TTFields) have been approved by the FDA as a treatment modality for several cancers, such as malignant pleural mesothelioma and glioblastoma multiforme, demonstrating remarkable efficacy and a high safety profile. In this work, we report the results of in vitro experiments with HeLa and MCF-10A cells exposed to TTFields for 18 h, imaged in real time using live-cell imaging. Both studied cell lines were exposed to 100 kHz TTFields with a 1-1 duty cycle, which resulted in significant mitotic and cytokinetic arrest. In the experiments with HeLa cells, the effects of the TTFields' frequency (100 kHz vs. 200 kHz) and duty cycle (1-1 vs. 1-0) were also investigated. Notably, the anti-mitotic effect was stronger in the HeLa cells treated with 100 kHz TTFields. Additionally, it was found that single and two-directional TTFields (oriented orthogonally) exhibit a similar inhibitory effect on HeLa cell division. These results provide real-time evidence of the profound ability of TTFields to hinder the process of cell division by significantly delaying both the mitosis and cytokinesis phases of the cell cycle.
Assuntos
Glioblastoma , Mesotelioma Maligno , Glioblastoma/terapia , Células HeLa , Humanos , Mitose , Fuso AcromáticoRESUMO
Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.
Assuntos
Neoplasias da Mama , Nanoestruturas , Humanos , Feminino , Fosfolipídeos , Sistemas de Liberação de Medicamentos , Nanomedicina , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/uso terapêuticoRESUMO
BACKGROUND: Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. METHODS: To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. RESULTS: We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. CONCLUSIONS: Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity.
Assuntos
Mama/metabolismo , Proteínas de Membrana/metabolismo , Biomarcadores/metabolismo , Mama/citologia , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Humanos , Células-Tronco/metabolismo , Células Estromais/metabolismoRESUMO
Background The impact of hormones on the development of breast cancer is despite extensive studies, incompletely understood. Combined estrogen-progestogen treatment augments the risk for breast cancer beyond that of estrogen alone, according to numerous studies. The role of breast cell proliferation as a promoter in the development and growth of breast cancer is well recognized. Materials and methods Seventy-nine patients from three randomised trials were subject to a re-analysis of breast cell proliferation: (1) 22 women received continuous combined treatment with oral estradiol (E2) 2 mg/norethisterone acetate (NETA) 1 mg once daily for 3 months. (2) Thirty-seven women received 2 months of sequential treatment with oral conjugated equine estrogens (CEE) 0.625 mg daily combined with medroxyprogesterone acetate (MPA) 5 mg for 14/28 days of each cycle. (3) Twenty women received oral estradiol-valerate (E2V) 2 mg daily combined with levonorgestrel (LNG) intrauterine system (IUS), 20 µg/24 h for 2 months. Fine needle aspiration (FNA) (studies 1 and 3) and core needle biopsy (CNB) (study 2) were used for the assessment of breast cell proliferation. Results There were no baseline proliferation differences, but at the end of treatment there was a highly significant between-group difference for E2V/LNG IUS versus the other two groups (p = 0.0025). E2/NETA and CEE treatments gave a 4-7-old increase in proliferation during treatment (p = 0.04) and (p = 0.007), respectively, which was absent in the E2V/LNG group, showing a significant correlation with insulin-like growth factor binding protein-3 (IGFBP-3) serum levels. Conclusion E2V in combination with very low serum concentrations of LNG in the IUS gives no increase in proliferation in the normal breast.
Assuntos
Neoplasias da Mama/etiologia , Terapia de Reposição de Estrogênios/efeitos adversos , Levanogestrel/efeitos adversos , Glândulas Mamárias Humanas/efeitos dos fármacos , Progestinas/efeitos adversos , Administração Oral , Idoso , Proliferação de Células , Feminino , Humanos , Levanogestrel/administração & dosagem , Levanogestrel/uso terapêutico , Glândulas Mamárias Humanas/patologia , Acetato de Medroxiprogesterona/administração & dosagem , Acetato de Medroxiprogesterona/efeitos adversos , Acetato de Medroxiprogesterona/uso terapêutico , Pessoa de Meia-Idade , Acetato de Noretindrona/administração & dosagem , Acetato de Noretindrona/efeitos adversos , Acetato de Noretindrona/uso terapêutico , Progestinas/administração & dosagem , Progestinas/uso terapêutico , ÚteroRESUMO
We have characterized two highly tumorigenic and metastatic basal B TNBC cell lines, XtMCF and LmMCF, with the additional values of having the normal and early-stage counterparts of them. This model allows the study of the evolution of TNBC, and investigates molecular pathways at different stages of transformation and progression in a relatively constant genetic background. This constitutes an ideal model for developing targeted therapy in two important fields in cancer biology which are the epithelial mesenchymal transition (EMT) and cancer stem cells (CSC).
Assuntos
Linhagem Celular Tumoral , Modelos Biológicos , Neoplasias de Mama Triplo Negativas , Transição Epitelial-Mesenquimal , Humanos , Técnicas In Vitro , Células-Tronco NeoplásicasRESUMO
BACKGROUND: BRCA1-associated breast cancer originates from luminal progenitor cells. BRCA1 functions in multiple biological processes, including double-strand break repair, replication stress suppression, transcriptional regulation, and chromatin reorganization. While non-malignant cells carrying cancer-predisposing BRCA1 mutations exhibit increased genomic instability, it remains unclear whether BRCA1 haploinsufficiency affects transcription and chromatin dynamics in breast epithelial cells. METHODS: H3K27ac-associated super-enhancers were compared in primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Non-tumorigenic MCF10A breast epithelial cells with engineered BRCA1 haploinsufficiency were used to confirm the H3K27ac changes. The impact of BRCA1 mutations on enhancer function and enhancer-promoter looping was assessed in MCF10A cells. RESULTS: Here, we show that primary mammary epithelial cells from women with BRCA1 mutations display significant loss of H3K27ac-associated super-enhancers. These BRCA1-dependent super-enhancers are enriched with binding motifs for the GATA family. Non-tumorigenic BRCA1mut/+ MCF10A cells recapitulate the H3K27ac loss. Attenuated histone mark and enhancer activity in these BRCA1mut/+ MCF10A cells can be partially restored with wild-type BRCA1. Furthermore, chromatin conformation analysis demonstrates impaired enhancer-promoter looping in BRCA1mut/+ MCF10A cells. CONCLUSIONS: H3K27ac-associated super-enhancer loss is a previously unappreciated functional deficiency in ostensibly normal BRCA1 mutation-carrying breast epithelium. Our findings offer new mechanistic insights into BRCA1 mutation-associated transcriptional and epigenetic abnormality in breast epithelial cells and tissue/cell lineage-specific tumorigenesis.
Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Células Epiteliais/metabolismo , Genes BRCA1 , Haploinsuficiência , Glândulas Mamárias Humanas/metabolismo , Mutação , Sítios de Ligação , Biomarcadores Tumorais , Linhagem Celular , Transformação Celular Neoplásica/genética , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas , Humanos , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-α was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-α was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-α, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.
Assuntos
Quimiocina CCL2/metabolismo , Células Epiteliais/metabolismo , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Macrófagos/citologia , Fenótipo , Regulação para Cima/efeitos dos fármacosRESUMO
Iodine deficiency (ID), which affects almost two billion people worldwide, is associated with breast pathologies such as fibrosis in human and induces breast atypia in animal models. Because ID induces vascular activation in the thyroid, another iodide-uptaking organ, and as breast is also sensitive to ID, we aimed to characterize ID-induced effects on the breast microvasculature in vivo and in two different breast cell lines in vitro. Virgin and lactating NMRI mice received an iodide-deficient diet and a Na+/I- symporter inhibitor for 1 to 20 days. Some virgin mice were treated with vascular endothelial growth factor A (VEGF) or VEGF receptor inhibitors. In vitro, ID was induced in MCF7 and MCF12A cells by replacing the iodide-containing medium by an iodide-deficient medium. In vivo, VEGF expression was increased following ID in mammary tissues. Consequently, ID induced a transient increase in mammary gland blood flow, measured after anesthesia, in virgin and lactating mice, which was repressed by VEGF or VEGF receptor inhibitors. In MCF7 cells, ID induced a transient increase in reactive oxygen species, followed by an increase in hypoxia-inducible factor-1α (HIF-1α) protein and VEGF mRNA expression. Antioxidant N-acetylcysteine and mammalian target of rapamycin (mTOR) inhibitor blocked ID-induced HIF-1α protein increase and VEGF transcription. However, mTOR activity was not inhibited by N-acetylcysteine. Similar responses were observed in MCF12A cells. These data indicate that ID activates the canonical VEGF pathway and mTOR in breast tissues, which provides new insights to better understand the correlation between ID, vascular activation, and breast pathologies.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Iodo/deficiência , Glândulas Mamárias Humanas/metabolismo , Microvasos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acetilcisteína/metabolismo , Animais , Antioxidantes/metabolismo , Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Lactação/metabolismo , Células MCF-7 , Glândulas Mamárias Animais/metabolismo , Camundongos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Telomere dysfunction drives chromosome instability through endless breakage-fusion-bridge (BFB) cycles that promote the formation of highly rearranged genomes. However, reactivation of telomerase or ALT-pathway is required for genome stabilisation and full malignant transformation. To allow the unrestricted proliferation of cells at risk of transformation, we have established a conditional system of telomere deprotection in p16INK4a-deficient MCF-10A cells with modified checkpoints. After sustained expression of a dominant negative form of the shelterin protein TRF2 (TRF2ΔBΔM), cells with telomere fusion did progress to anaphase but no signs of ongoing BFB cycles were observed, thus anticipating proliferation defects. Indeed, 96 h TRF2ΔBΔM expression resulted in noticeable growth proliferation defects in the absence of cell cycle disturbances. Further transient periods of 96 h telomere uncapping did not result in cell cycle disturbances either. And reduction of the telomere damage to short acute deprotection periods did not in any case engender cells with a reorganised karyotype. Strikingly, the growth arrest imposed in cells showing dysfunctional telomeres was not accompanied by an activation of the DNA damage response at cellular level, or by the presence of visible markers of senescence or apoptosis. We propose that the deprotection of many telomeres simultaneously, even for a short time, results in a local activation of the cellular stress response which consequently triggers gradual cell withdrawal from cell cycle, restraining the onset of genomic instability.
RESUMO
Non-malignant breast epithelial cells cultured in three-dimensional laminin-rich extracellular matrix (lrECM) form well organized, growth-arrested acini, whereas malignant cells form continuously growing disorganized structures. While the mechanical properties of the microenvironment have been shown to contribute to formation of tissue-specific architecture, how transient external force influences this behavior remains largely unexplored. Here, we show that brief transient compression applied to single malignant breast cells in lrECM stimulated them to form acinar-like structures, a phenomenon we term 'mechanical reversion.' This is analogous to previously described phenotypic 'reversion' using biochemical inhibitors of oncogenic pathways. Compression stimulated nitric oxide production by malignant cells. Inhibition of nitric oxide production blocked mechanical reversion. Compression also restored coherent rotation in malignant cells, a behavior that is essential for acinus formation. We propose that external forces applied to single malignant cells restore cell-lrECM engagement and signaling lost in malignancy, allowing them to reestablish normal-like tissue architecture.
Assuntos
Mama/metabolismo , Células Epiteliais/metabolismo , Óxido Nítrico/metabolismo , Estresse Mecânico , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Mama/citologia , Mama/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Laminina/metabolismo , Laminina/farmacologia , Microscopia Confocal , Transdução de Sinais/efeitos dos fármacos , Imagem com Lapso de Tempo/métodosRESUMO
OBJECTIVES: Breast cancer is a leading cause of death among women in both developed and Third World countries. Fucoidan is a natural plant metabolite produced by brown seaweeds with proven anticancer potential. This study determined the cytotoxic, apoptotic and cell cycle effects of fucoidan alone and in combination with first-line anticancer drugs (cisplatin, doxorubicin and taxol) in MCF-7 breast cancer cells and non-malignant MCF-12A breast epithelial cells as control. METHODS: Cytotoxicity was evaluated using the MTT reduction assay. Cell cycle distribution and apoptosis were assessed by flow cytometry using Annexin VFITC/PI and Hoechst 33342 staining, and caspases-3, -7 and -9 activation. RESULTS: Fucoidan alone was significantly more cytotoxic to MCF-7 breast cancer cells compared to the MCF-12A non-cancerous breast epithelial cell line. In MCF-7 cells, the presence of fucoidan caused cell cycle arrest at G1 with accumulation of cells in the sub-G1 phase with the activation of caspases-3,-7 and -9. Furthermore, combination of fucoidan with the standard chemotherapeutic agents-cisplatin, doxorubicin and taxol-significantly enhanced the cytotoxicity of these drugs and accumulation of cells in the G2/M and sub-G1 phases, and induction of apoptosis. No significant differences were observed between fucoidan-treated and untreated MCF-12A cells with respect to cytotoxicity and cell cycle distribution profiles. By contrast, in non-cancerous MCF-12A cells, fucoidan attenuated the toxicity of doxorubicin and cisplatin in combination by increasing their IC50 values. This effect was not demonstrated with the taxol combination. CONCLUSIONS: Fucoidan is an effective antitumor agent, either alone or in combination with cisplatin, doxorubicin and taxol in MCF-7 breast cancer cells. Drug combinations that discriminate between cancerous and non-cancerous cells afford a plausible and viable strategy of attaining therapeutic efficacy and avoiding possible toxicity and side effects. These findings suggest that fucoidan is a promising candidate for cancer combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Polissacarídeos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Polissacarídeos/administração & dosagemRESUMO
ATBF1, a large transcription factor, was normally localized in nuclei, and its mislocalization to cytoplasm was reported in multiple cancers. However, localization of ATBF1 in breast epithelial cells and its potential functions were unknown. Here, we investigated ATBF1 localization via immunofluorescence staining in different kinds of breast epithelial cells. In MCF10A cells and normal mice mammary gland tissues, ATBF1 was mainly localized in nuclei. Knockdown of ATBF1 expression in MCF10A cells by siRNA promoted cell proliferation. Moreover, ATBF1 was co-localized with chromosome during mitosis, indicating its potential function in mitosis. In an estrogen receptor (ER)-positive breast cancer cell line (MCF7), estrogen induced ATBF1 translocation from cytoplasm to nuclei in an ER dependent pathway. In ER-negative cells (Hs578T and MDA-MB-231), ATBF1 was co-localized with GM130 in cytoplasm, indicating ATBF1 localization was associated with protein modification in golgi body. The results were beneficial for intensive investigation of ATBF1's function with different cellular localization in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Células Epiteliais/patologia , Proteínas de Homeodomínio/análise , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Transporte ProteicoRESUMO
Activation of free radical oxidation in different cell types, including breast epithelial cells, may result in damage to macromolecules, in particular, proteins taking part in regulation of cell proliferation and apoptosis. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. Due to this fact, modulation of cellular redox status under the effect of an SH group inhibitor and an SH group protector may be used as a model for studying the role of redox proteins and glutathione in regulating cell proliferation in different pathological processes. In this study we have evaluated the state of the thioredoxin, glutaredoxin and glutathione systems as well as their role in regulating proliferation of HBL-100 breast epithelial cells under redox status modulation with N-ethylmaleimide (NEM) and 1,4-dithioerythriol (DTE). Modulating the redox status of breast epithelial cells under the effect of NEM and DTE influences the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase through changes in the GSH and GSSG concentrations. In HBL-100 cells under redox-status modulation, we have found an increase in the number of cells in the S-phase of the cell cycle and a decrease in the number of cells in the G0/G1 and G2/Ð phases, as opposed to the values in the intact culture. The proposed model of proliferative activity of cells under redox status modulation may be used for development of new therapeutic approaches for treatment of diseases accompanied by oxidative stress generation.
Assuntos
Ditioeritritol/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Etilmaleimida/farmacologia , Substâncias Protetoras/farmacologia , Catalase/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Etilmaleimida/antagonistas & inibidores , Citometria de Fluxo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
BACKGROUND: Adipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes. METHODS: We used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post-hoc tests were performed within each individual treatment. RESULTS: hATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes. CONCLUSIONS: We conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Mama/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteína ADAMTS1/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Mama/citologia , Mama/patologia , Neoplasias da Mama/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Microambiente Celular , Progressão da Doença , Células Epiteliais/citologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Receptores de Adiponectina/metabolismo , Versicanas/metabolismoRESUMO
Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. ß-adrenergic receptors (ß-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in ß2-AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of ß2-AR. To study regulation of the breast cell phenotype by ß2-AR, we over-expressed ß2-AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down ß2-AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the ß2-AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that ß2-AR expression level is a key player in cell behavior. ß-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of ß-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through ß-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context.
Assuntos
Neoplasias da Mama/metabolismo , Morfogênese/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Catecolaminas/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/metabolismo , Feminino , Fulvestranto , Humanos , Isoproterenol/farmacologia , Células MCF-7 , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Morfogênese/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologiaRESUMO
Inhibitor kappa-B kinase-beta (IKK-ß) controls the activation of nuclear transcription factor kappa-B and has been linked to inflammation and cancer. Therefore, inhibitors of this kinase should have potent anti-inflammatory and anticancer properties. Accordingly, we explored the pharmacophoric space of 218 IKK-ß inhibitors to identify high-quality binding models. Subsequently, genetic algorithm-based quantitative structure activity relationship (QSAR) analysis was employed to select the best possible combination of pharmacophoric models and physicochemical descriptors that explain bioactivity variation among training compounds. Three successful pharmacophores emerged in 2 optimal QSAR equations (r12175 = 0.733, r12LOO = 0.52, F1 = 65.62, r12PRESS against 43 test inhibitors = 0.63 and r22175 = 0.683, r22LOO = 0.52, F2 = 72.66, r22PRESS against 43 test inhibitors = 0.65). Two pharmacophores were merged in a single binding model. Receiver operating characteristic curve validation proved the excellent qualities of this model. The merged pharmacophore and the associated QSAR equations were applied to screen the National Cancer Institute list of compounds. Ten hits were found to exhibit potent anti-IKK-ß bioactivity, out of which, one illustrates IC50 of 11.0nM.
Assuntos
Quinase I-kappa B/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Algoritmos , Linhagem Celular Tumoral , Simulação por Computador , Descoberta de Drogas , Feminino , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Relação Quantitativa Estrutura-Atividade , Curva ROCRESUMO
The effects of the SH-group blocker N-ethylmaleimide (NEM) and thiol group protector 1,4-dithioerythritol (DTE) on the redox status of cells HBL-100 cells, oxidative modification of their proteins and the state of glutathione and thioredoxin systems have been investigated. Breast epithelial cells cultivated in the presence of NEM were characterized by decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of protein and amino acids. Cultivation of HBL-100 cells in the presence of DTE resulted in a shift of the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).