RESUMO
Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.
Assuntos
Âmnio/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Biomarcadores/metabolismo , Forma Celular , Células Cultivadas , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Teratoma/patologia , Transcrição GênicaRESUMO
Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods - flow cytometry, confocal imaging, teratoma formation and transcriptional profiling - is also described. The newly generated lines express markers of embryonic stem cells - Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 - while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.
Assuntos
Líquido Amniótico/citologia , Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular/fisiologia , Humanos , Camundongos , Camundongos SCIDRESUMO
With the recent successes in immuno-oncology, renewed interest in the role of immune checkpoint modulators, such as the B7 family proteins, has escalated. The immune checkpoint proteins play a crucial role in the regulation of cellular immunity; however, their contribution to other aspects of cancer biology remains unclear. Accumulating evidence indicate that immune checkpoint proteins can regulate metabolic energetics of the tumor, the tumor microenvironment, and the tumor-specific immune response, leading to metabolic reprogramming of both malignant cells and immune cells involved in mounting and sustaining this response. Immune cell metabolism impacts the activation status of immune cells and ultimately the immune response in cancer. Tumor cells may deplete nutrients that immune cells require for optimal generation, expansion, and function. They may also generate toxic metabolites in the microenvironment or induce conserved inhibitory pathways that impair immune function and thus inhibit antitumor responses. In this review, we will discuss how cancer cells with altered expression of immune checkpoint proteins can potently inhibit immune function through the alteration of cellular and microenvironmental metabolism, providing a new perspective on the interplay between these pathways and offering a potential therapeutic intervention strategy in the treatment of malignant disease. Cancer Res; 77(6); 1245-9. ©2017 AACR.
Assuntos
Pontos de Checagem do Ciclo Celular/imunologia , Imunidade Celular/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Microambiente Tumoral/imunologia , Animais , Humanos , Neoplasias/patologiaRESUMO
Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or ß-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Plasmídeos/química , Transfecção/métodos , Líquido Amniótico/citologia , Líquido Amniótico/efeitos dos fármacos , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Meios de Cultura/farmacologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos CD15/genética , Antígenos CD15/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Plasmídeos/metabolismo , Análise Serial de Proteínas , Proteoglicanas/genética , Proteoglicanas/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Embrionários Estágio-Específicos/metabolismoRESUMO
Cancer stem cells (CSC) are a distinct subpopulation within a tumor shown to drive tumor progression, metastasis, and recurrence. A review of the literature reveals poor consensus, with the use of a wide variety of surface markers and functional assays to identify and isolate cancer stem cells. Utilizing a novel technology that enables live-cell mRNA quantitation, we have demonstrated the ability to identify and sort viable CSC based on markers associated with stemness in pluripotent cells. Fresh tumor samples from a variety of cancer types were examined by flow cytometry for Nanog expression. Levels of CSC detected ranged from 6% to 19%. This method of CSC detection was cross-validated with other commonly used surface markers with good correlation. Matrigel invasion assays confirmed that CSC isolated using this method are both highly motile and invasive. This approach simplifies the process of identifying viable CSC from fresh tumor tissue, providing a level of accuracy not previously available. This method may also provide a valuable tool for screening and validating new CSC biomarkers.
Assuntos
Citometria de Fluxo , Proteínas de Homeodomínio/genética , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/análise , Biomarcadores Tumorais/análise , Expressão Gênica , Proteínas de Homeodomínio/análise , Humanos , Proteína Homeobox NanogRESUMO
Allyl isothiocyanate (AITC), a constituent of many cruciferous vegetables exhibits significant anticancer activities in many cancer models. Our studies provide novel insights into AITC-induced anticancer mechanisms in human A549 and H1299 non-small cell lung cancer (NSCLC) cells. AITC exposure induced replication stress in NSCLC cells as evidenced by γH2AX and FANCD2 foci, ATM/ATR-mediated checkpoint responses and S and G2/M cell cycle arrest. Furthermore, AITC-induced FANCD2 foci displayed co-localization with BrdU foci, indicating stalled or collapsed replication forks in these cells. Although PITC (phenyl isothiocyanate) exhibited concentration-dependent cytotoxic effects, treatment was less effective compared to AITC. Previously, agents that induce cell cycle arrest in S and G2/M phases were shown to sensitize tumor cells to radiation. Similar to these observations, combination therapy involving AITC followed by radiation treatment exhibited increased DDR and cell killing in NSCLC cells compared to single agent treatment. Combination index (CI) analysis revealed synergistic effects at multiple doses of AITC and radiation, resulting in CI values of less than 0.7 at Fa of 0.5 (50% reduction in survival). Collectively, these studies identify an important anticancer mechanism displayed by AITC, and suggest that the combination of AITC and radiation could be an effective therapy for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Isotiocianatos/farmacologia , Radiação Ionizante , Radiossensibilizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Ciclo Celular/efeitos da radiação , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Citometria de Fluxo , Imunofluorescência , Conservantes de Alimentos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Células Tumorais CultivadasRESUMO
Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Quinases/metabolismo , Fase S , Inibidores da Topoisomerase I/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/química , Sítios de Ligação , Camptotecina/química , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Quinase 1 do Ponto de Checagem , Reparo do DNA , Regulação para Baixo , Resistência a Medicamentos , Fibroblastos/metabolismo , Humanos , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína de Replicação A/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/química , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines. METHODS: We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. RESULTS: Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. CONCLUSION: Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , NF-kappa B/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Proteína Forkhead Box M1 , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Regulação para Cima/efeitos dos fármacosRESUMO
Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.
Assuntos
Desoxicitidina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores CXCR4/genética , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Desoxicitidina/efeitos adversos , Desoxicitidina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/genética , GencitabinaRESUMO
It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.
Assuntos
Mitocôndrias/fisiologia , Receptor ErbB-2/fisiologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Respiração Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transporte de Elétrons/fisiologia , Feminino , Glicólise/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Transporte Proteico , Receptor ErbB-2/metabolismo , TrastuzumabRESUMO
Recent evidence suggests a major role of tumor-stromal interactions in pancreatic cancer pathobiology. The chemokine CXCL12 (stromal cell-derived factor 1 (SDF-1)), abundantly produced by stromal cells, promotes progression, metastasis, and chemoresistance of pancreatic cancer cells. On the other hand, pancreatic tumor cell-derived sonic hedgehog (SHH) acts predominantly on stromal cells to induce desmoplasia and, thus, has a paracrine effect on tumorigenesis and therapeutic outcome. In this study, we examined the association between these two proteins of pathological significance in pancreatic cancer. Our data demonstrate that CXCL12 leads to a dose- and time-dependent up-regulation of SHH in pancreatic cancer cells. CXCL12-induced SHH up-regulation is specifically mediated through the receptor CXCR4 and is dependent on the activation of downstream Akt and ERK signaling pathways. Both Akt and ERK cooperatively promote nuclear accumulation of NF-κB by inducing the phosphorylation and destabilization of its inhibitory protein, IκB-α. Using dominant negative IκB-α, a SHH promoter (deletion mutant) reporter, and chromatin immunoprecipitation assays, we demonstrate that CXCL12 exposure enhances direct binding of NF-κB to the SHH promoter and that suppression of NF-κB activation abrogates CXCL12-induced SHH expression. Finally, our data demonstrate a strong correlative expression of CXCR4 and SHH in human pancreatic cancer tissues, whereas their expression is not observed in the normal pancreas. Altogether, our data reveal a novel mechanism underlying aberrant SHH expression in pancreatic cancer and identify a molecular link facilitating bidirectional tumor-stromal interactions.
Assuntos
Quimiocina CXCL12/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quimiocinas/metabolismo , Citoplasma/metabolismo , Genes Dominantes , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
BACKGROUND: A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). METHODOLOGY/PRINCIPAL FINDINGS: The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. CONCLUSIONS: Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Microesferas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transformação Celular Neoplásica , Humanos , Oligonucleotídeos/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. RESULTS: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. CONCLUSION: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glicólise/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Paclitaxel/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/metabolismo , Lactato Desidrogenase 5RESUMO
The incidence of melanoma is rising at an alarming rate and we are still awaiting an effective treatment for this malignancy. In its early stage, melanoma can be cured by surgical removal, but once metastasis has occurred there is no effective treatment. Recent findings have suggested multiple functional implications of CXCL8 and its cognate receptors, CXCR1 and CXCR2, in melanoma pathogenesis, thus underscoring their importance as targets for cancer therapy. This review provides an update on the roles of CXCL8 and its receptors in melanoma progression and metastasis.
Assuntos
Interleucina-8/metabolismo , Melanoma/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Progressão da Doença , Humanos , Melanoma/patologia , Metástase Neoplásica/patologia , Neoplasias Cutâneas/patologiaRESUMO
Epidermal gamma delta T cells (gammadeltaT) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the 2 major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV-irradiation. Under control conditions, both PRLs reduced the number of gammadeltaT, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in gammadeltaT number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC:gammadeltaT ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis.
Assuntos
Epiderme/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Células de Langerhans/efeitos dos fármacos , Prolactina/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/efeitos dos fármacos , Raios Ultravioleta , Animais , Epiderme/metabolismo , Epiderme/efeitos da radiação , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/metabolismo , Bombas de Infusão Implantáveis , Células de Langerhans/metabolismo , Células de Langerhans/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fosforilação , Prolactina/administração & dosagem , Prolactina/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Linfócitos T/metabolismo , Linfócitos T/efeitos da radiaçãoRESUMO
It is well established that females mount stronger immune responses than males, but only very little is understood about the underlying mechanisms. We have analyzed local cytokine differences among intact females, those that had been ovariectomized (OVX), those receiving estrogen replacement after OVX, and males, both before and after production of delayed-type hypersensitivity (DTH) responses. We report confirmation of a much larger DTH response in females versus males. However, OVX resulted in an even larger response, while estrogen replacement resulted in a smaller response when compared with intact females. In animals exposed for the first time to an antigen (without a DTH response), OVX increased interleukin-6 (IL-6) and estrogen replacement after OVX suppressed IL-6. Of the cytokines that differed between males and females exposed for the first time to an antigen, only IL-6 was higher in females versus males when exposure to antigen occurred for the second time (when the DTH response occurs). Analysis of cytokines with OVX and estrogen replacement after a second exposure to antigen showed that IL-6 did not significantly change. Levels of IL-4; Regulated upon Activation, Normal T-cell Expressed; and Secreted; and thrombopoietin, however, correlated with the DTH response, suggesting direct or indirect positive regulation by estrogen. These results suggest an important role for both IL-6 and IL-4 in determining the degree of DTH response, with IL-6 (which appears negatively regulated by estrogen) increasing and IL-4 (which appears positively regulated by estrogen) decreasing the response. The results further suggest that IL-6 may play a role in predisposing to a larger DTH response, while IL-4 levels seem more important during an active response.
Assuntos
Citocinas/sangue , Terapia de Reposição de Estrogênios , Identidade de Gênero , Hipersensibilidade Tardia/imunologia , Animais , Antígenos/farmacologia , Quimiocina CCL5/sangue , Feminino , Interleucina-4/sangue , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Trombopoetina/sangue , Fatores de TempoRESUMO
CD95 is the quintessential death receptor and, when it is bound by ligand, cells undergo apoptosis. Recent evidence suggests, however, that CD95 mediates not only apoptosis but also diverse nonapoptotic functions depending on the tissue and the conditions.
Assuntos
Apoptose , Receptor fas/metabolismo , Animais , Proteína Ligante Fas/metabolismo , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Progression of mycosis fungoides (MF) to Sézary syndrome (SS) is accompanied by a shift from a T(H)1 to a T(H)2 cytokine profile. Interleukin (IL)-23 is a novel cytokine that shares a common p40 subunit with the T(H)1 inducer, IL-12. IL-23 induces a third profile, T(H)IL-17, that is dominant in inflammation and autoimmunity. Although IL-23 induces an eczematous-like skin reaction in mice, and is expressed in T(H)1-mediated skin disorders such as psoriasis, it has not been evaluated in MF/SS. To study the role of IL-23 in MF/SS development, 40 MF/SS lesions of all stages were immunohistochemically analyzed with a novel anti-human IL-23 antibody raised against full-length human IL-23. IL-23 was detected with the catalyzed signal amplification system. The intensity and frequency of IL-23 staining were semi-quantitatively graded in both the dermal infiltrate and the epidermis. Increased expression of IL-23 was observed throughout the epidermal keratinocytes and in dermal lymphocytes compared to normal skin. IL-23 intensity did not differ significantly among the stages of MF/SS; however, in stage IVB patients, we observed lower frequency of IL-23 expression in dermal lymphocytes than in other stage patients [P = 0.13, analysis of variance (ANOVA)]. Interestingly, clusters of atypical lymphocytes, especially the epidermotropic tumor cells, demonstrated weak or absent IL-23 staining in 18 of 40 (45%) lesions. This finding was present in 4 of 5 (80%) of the stage IVB lesions and 7 of 11 (64%) of the lesions from Sézary patients. These findings indicate that abnormal IL-23 expression may play a role in the pathogenesis and progression of MF/SS.
Assuntos
Interleucina-23/metabolismo , Micose Fungoide/metabolismo , Síndrome de Sézary/metabolismo , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Interleucina-23/genética , Micose Fungoide/genética , Micose Fungoide/patologia , Síndrome de Sézary/genética , Síndrome de Sézary/patologia , Pele/metabolismo , Pele/patologia , Células Th1/patologia , Células Th2/patologiaRESUMO
Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.
Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Melanócitos/fisiologia , Receptor EphB4/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Citometria de Fluxo , Imuno-Histoquímica , Melanócitos/metabolismo , Melanoma/patologia , Camundongos , Microscopia de Vídeo , Reação em Cadeia da Polimerase , Testes de Precipitina , Receptor EphB4/genética , Proteína rhoA de Ligação ao GTP/análiseRESUMO
The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4alpha1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4alpha gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4alpha protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4alpha P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4alpha P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6alpha. p53 bound HNF6alpha in vivo and in vitro and prevented HNF6alpha from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6alpha in vivo. However, since the R249S p53 mutant also bound HNF6alpha, binding HNF6alpha is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4alpha expression in response to cellular stress are discussed.