RESUMO
Breast cancer is one of the most common and deadly cancers in women worldwide. Current treatments for breast cancer have limitations, such as toxicity, resistance, and side effects. Therefore, there is a need to develop new and effective anti-cancer agents from natural sources. Spinosyn A (SPA) is a natural product derived from soil bacteria. SPA has been reported to have anti-parasitic, insecticidal, and anti-bacterial activities. However, its anti-cancer effects and mechanisms are not well understood. In this study, we investigated the effects of SPA on T47-D, estrogen receptor-positive breast cancer cells. We found that SPA inhibited cell proliferation and migration and induced apoptosis and cell cycle arrest. Flow cytometry and holographic imaging microscopy revealed that SPA activated MAPK and PI3K signaling pathways and altered cellular morphology. Finally, RNA-Seq analysis revealed that SPA treatment altered the expression of 1380 genes in T47-D cells, which were enriched in various biological processes and signaling pathways related to cell proliferation, cholesterol metabolism, growth factor activity, amino acid transport activity, extracellular matrix, PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, and PPAR signaling pathway. Our results suggest that SPA exerts multiple anti-cancer effects on T47-D cells by modulating multiple pathways and cellular processes involved in cell growth, survival, and motility. Our findings provide new insights into the molecular mechanisms of SPA action on breast cancer cells and its potential applications as a novel anti-cancer agent.
Assuntos
Neoplasias da Mama , Macrolídeos , Progesterona , Feminino , Humanos , Progesterona/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Proliferação de Células , Apoptose , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND/AIM: Breast cancer, one of the most prevalent cancers globally, is marked by its cellular heterogeneity. A key aspect of breast cancer research is understanding the distinct morphological features of cancerous and non-cancerous cells, which could serve as potential targets for novel therapeutic interventions. In this light, our study aimed to comprehensively analyze the morphological features of the MCF10A and MCF7 cell lines, representing normal breast and breast cancer cells, respectively. The ultimate objective was to identify the most significant features that differentiate these cell lines. MATERIALS AND METHODS: We utilized advanced imaging techniques such as holographic time-lapse microscopy, which provides real-time, three-dimensional imaging of cells, to conduct our comparative analysis. This allowed us to examine dynamic cellular morphology and behavior with exceptional sensitivity and resolution over time. The primary features assessed in our study included texture clustershade, area (µm2), eccentricity, irregularity, phaseshift sum, optical volume (µm3), shape convexity, and Hull convexity. RESULTS: Our findings highlighted significant differences in the morphological features of MCF10A and MCF7 cells. MCF10A cells showed a higher texture clustershade value, suggesting less symmetry than MCF7 cells. On the other hand, MCF7 cells had smaller cellular area, higher eccentricity, lower irregularity, higher phase shift sum, higher optical volume, higher shape convexity, and higher hull convexity compared to MCF10A cells. These results suggest that MCF7 cells are smaller, more circular, less irregular, exhibit different light properties, and have a closer to perfect 3D shape relative to MCF10A cells. CONCLUSION: The identified morphological differences between MCF10A and MCF7 cells offer valuable insights into the characteristics distinguishing normal breast cells from breast cancer cells. These findings not only contribute to our understanding of the morphological variability in breast cancer but also underscore the potential utility of these differences in future cancer diagnostics and treatment strategies.
Assuntos
Neoplasias da Mama , Microscopia , Humanos , Feminino , Células MCF-7 , Imagem com Lapso de Tempo , Imageamento TridimensionalRESUMO
The H3K4 demethylase KDM5B is overexpressed in multiple cancer types, and elevated expression levels of KDM5B is associated with decreased survival. However, the underlying mechanistic contribution of dysregulated expression of KDM5B and H3K4 demethylation in cancer is poorly understood. Our results show that loss of KDM5B in multiple types of cancer cells leads to increased proliferation and elevated expression of cancer stem cell markers. In addition, we observed enhanced tumor formation following KDM5B depletion in a subset of representative cancer cell lines. Our findings also support a role for KDM5B in regulating epigenetic plasticity, where loss of KDM5B in cancer cells with elevated KDM5B expression leads to alterations in activity of chromatin states, which facilitate activation or repression of alternative transcriptional programs. In addition, we define KDM5B-centric epigenetic and transcriptional patterns that support cancer cell plasticity, where KDM5B depleted cancer cells exhibit altered epigenetic and transcriptional profiles resembling a more primitive cellular state. This study also provides a resource for evaluating associations between alterations in epigenetic patterning upon depletion of KDM5B and gene expression in a diverse set of cancer cells.
Assuntos
Histona Desmetilases com o Domínio Jumonji , Neoplasias , Linhagem Celular Tumoral , Epigênese Genética , Epigenômica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Repressoras/genéticaRESUMO
Epigenetic regulation of chromatin plays a critical role in controlling stem cell function and tumorigenesis. The histone lysine demethylase, KDM5B, which catalyzes the demethylation of histone 3 lysine 4 (H3K4), is important for embryonic stem (ES) cell differentiation, and is a critical regulator of the H3K4-methylome during early mouse embryonic pre-implantation stage development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates expression of oncogenes and tumor suppressors by modulating H3K4 methylation levels. In this review, we examine how KDM5B regulates gene expression and cellular fates of stem cells and cancer cells by temporally and spatially controlling H3K4 methylation levels.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Epigênese Genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Biomarcadores Tumorais , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Metilação , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Células-Tronco/metabolismoRESUMO
PURPOSE: Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. EXPERIMENTAL DESIGN: Small-molecule drug discovery and extensive biological characterization of a lead compound. RESULTS: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. CONCLUSIONS: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos Hormonais/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/uso terapêutico , Animais , Antineoplásicos Hormonais/química , Antineoplásicos Hormonais/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator Inibidor de Leucemia/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Linhagem Celular , Autorrenovação Celular/genética , Células Cultivadas , Epigênese Genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Fator Inibidor de Leucemia/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Transcrição Gênica , TranscriptomaRESUMO
Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Ribonucleoproteínas/metabolismo , Arsenitos/farmacologia , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/química , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/isolamento & purificação , Humanos , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação a Y-Box/química , Proteína 1 de Ligação a Y-Box/isolamento & purificação , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys(17), Trp(20), Tyr(24), or Arg(26) are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.