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Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.
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Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).
Assuntos
Canais de Cálcio Tipo T/metabolismo , Movimento Celular , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Substituição de Aminoácidos , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas Associadas aos Microtúbulos/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição da Família Snail/genéticaRESUMO
BACKGROUND: Autologous tumour lysate dendritic cell vaccine (ADC) has T-cell stimulatory capacity and, therefore, potential antitumour activity. We designed a phase II randomised trial of ADC + best supportive care (BSC) (experimental arm [EA]) compared with BSC (control arm [CA]), in pre-treated metastatic colorectal cancer (mCRC) patients. PATIENTS AND METHODS: Patients with progressive mCRC, at least to two chemotherapy regimens and Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2, were randomised to EA versus CA. Stratification criteria: ECOG PS (0-1 versus 2) and lactate dehydrogenase (
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Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/terapia , Células Dendríticas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/secundário , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Análise de SobrevidaRESUMO
Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP-AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP-AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF-ĸB and MAPK/ERK. However, NVP-AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP-AUY922 and PFT-µ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT-µ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT-µ further enhanced NVP-AUY922-induced cytotoxic effects. These data suggest a potential therapeutic role for NVP-AUY922 used in combination with PFT-µ, in melanoma.
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Antineoplásicos/uso terapêutico , Glutationa/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Resorcinóis/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Isoxazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Camundongos SCID , NF-kappa B/metabolismo , Metástase Neoplásica , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resorcinóis/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Current evidence suggests that the presence of tumor-initiating cells (TICs) in epithelial ovarian cancer (EOC) has a role in chemoresistance and relapse. Surface markers such as CD44(+)/CD24(-), CD117(+), and CD133(+) expression have been reported as potential markers for TICs related to ovarian cancer and tumorigenic cell lines. In this study, we have investigated if spheroid forms are TIC specific or whether they can also be produced by somatic stem cells from healthy tissue in vitro. In addition, we also investigated the specificity of surface markers to identify TICs from papillary serous EOC patients. METHODS: Cells were obtained from fresh tumors from 10 chemotherapy-naive patients with EOC, and cells from ovarian and tubal epithelium were obtained from 5 healthy menopausal women undergoing surgery for benign pathology and cultured in standard and in selective medium. Cells forming nonadherent spheroids were considered TICs, and the adherent cells were considered as non-TIC-like. Percentages of CD24(+), CD44(+), CD117(+), CD133(+), and vascular endothelial growth factor receptor (VEGF-R)(+) cell surface markers were analyzed by flow cytometry. RESULTS: Four of 10 EOC cell tissues were excluded from the study. Tumor cells cultured in selective medium developed spheroid forms after 1 to 7 weeks in 5 of 6 EOC patients. No spheroid forms were observed in cultures of cells from healthy women. Unlike previously published data, low levels of CD24(+), CD44(+), CD117(+), and VEGF-R(+) expression were observed in spheroid cells, whereas expression of CD133(+) was moderate but higher in adherent cells from papillary serous EOC cells in comparison with adherent cells from controls. CONCLUSIONS: Papillary serous EOC contains TICs that form spheroids with low expression of CD44(+), CD24(+), CD117(+) and VEGF-R(+). Further research is required to find specific surface markers to identify papillary serous TICs.
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Cistadenocarcinoma Seroso/patologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Ovário/patologia , Esferoides Celulares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Cistadenocarcinoma Seroso/metabolismo , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Projetos Piloto , Prognóstico , Esferoides Celulares/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Food allergy caused by lipid transfer protein (LTP) from peach (Pru p 3) is frequently associated with sensitization to mugwort LTP (Art v 3). Although in vitro cross-reactivity is already well known, it has yet to be elucidated whether a pollen LTP can induce rhinitis. OBJECTIVE: The aim of this study was to investigate whether mugwort LTP could elicit respiratory symptoms and whether a primary food LTP allergy could lead to a respiratory allergy. METHODS: Patients with confirmed Pru p 3 allergy and control subjects were selected. Immediate responses to nasal allergen provocation tests (NAPTs) with Art v 3, Pru p 3, and mugwort were assessed by using the visual analog scale score, total nasal symptom score, and acoustic rhinometry. Tryptase and cysteinyl leukotriene (cysLT) levels were measured in nasal lavage fluid. Immunoblotting, ELISAs, and ELISA inhibition assays were also performed. RESULTS: Fifteen patients and 9 control subjects were selected. NAPT results with Art v 3 and Pru p 3 showed significant changes in acoustic rhinometry, visual analog scale scores, total nasal symptom scores, and cysLT levels (P < .001). Tryptase levels were only increased in NAPTs with Pru p 3. NAPTs with mugwort were used in those patients who were only sensitized to Art v 3, with similar results (P < .05). No significant changes were detected in control subjects. CONCLUSION: The results demonstrated that a pollen LTP can elicit rhinitis in sensitized patients. Findings also suggest that a primary sensitization to Pru p 3 can lead to a respiratory allergy through cross-reactivity.
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Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/complicações , Hipersensibilidade Alimentar/imunologia , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/complicações , Rinite Alérgica Sazonal/imunologia , Adulto , Estudos de Casos e Controles , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade Alimentar/diagnóstico , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Rinite Alérgica Sazonal/diagnóstico , Testes Cutâneos , Adulto JovemRESUMO
AIM is expressed by macrophages in response to agonists of the nuclear receptors LXR/RXR. In mice, it acts as an atherogenic factor by protecting macrophages from the apoptotic effects of oxidized lipids. In humans, it is detected in atherosclerotic lesions, but no role related to atherosclerosis has been reported. This study aimed to investigate whether the role of hAIM extends beyond inhibiting oxidized lipid-induced apoptosis. To accomplish this goal, functional analysis with human monocytic THP1 cells and macrophages differentiated from peripheral blood monocytes were performed. It was found that hAIM reduced oxLDL-induced macrophage apoptosis and increased macrophage adhesion to endothelial ICAM-1 by enhancing LFA-1 expression. Furthermore, hAIM increased foam cell formation, as shown by Oil Red O and Nile Red staining, as well as quantification of cholesterol content. This was not a result of decreased reverse cholesterol transport, as hAIM did not affect the efflux significantly from [(3)H] Cholesterol-laden macrophages driven by plasma, apoA-I, or HDL2 acceptors. Rather, flow cytometry studies indicated that hAIM increased macrophage endocytosis of fluorescent oxLDL, which correlated with an increase in the expression of the oxLDLR CD36. Moreover, hAIM bound to oxLDL in ELISA and enhanced the capacity of HEK-293 cells expressing CD36 to endocytose oxLDL, as studied using immunofluorescence microscopy, suggesting that hAIM serves to facilitate CD36-mediated uptake of oxLDL. Our data represent the first evidence that hAIM is involved in macrophage survival, adhesion, and foam cell formation and suggest a significant contribution to atherosclerosis-related mechanisms in the macrophage.
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Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD36/metabolismo , Células Espumosas/metabolismo , Lectinas Tipo C/metabolismo , Lipoproteínas LDL/metabolismo , Apoptose/imunologia , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Adesão Celular , Endocitose/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células Espumosas/citologia , Células Espumosas/imunologia , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We have recently reported that human melanoma cells express a variety of voltage-gated calcium (Ca(2+) ) channel types, including low-voltage-activated T-type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T-type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G1 -S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in-depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca(2+) homeostasis, autophagy, and cell death were mimicked by T-type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.
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Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Melanoma/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/enzimologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Neoplasias Cutâneas , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Melanoma Maligno CutâneoRESUMO
Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.
Assuntos
Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Técnicas de Cocultura , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estadiamento de Neoplasias , Transplante de Neoplasias , Neoplasias da Próstata , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Neoplasias da Bexiga Urinária , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: Several studies have demonstrated that different genetic profiles contribute to melanoma development and progression. MATERIALS AND METHODS: To evaluate the existence of different molecular aberration patterns in melanoma associated with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) or 9p21 locus alterations, eleven patient-derived melanoma cell lines were characterized. Multiplex ligation probe amplification (MLPA) was used to detect chromosomal alterations. Single- strand conformation analysis and sequencing were performed to study BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (c-KIT), melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R), cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase 4 (CDK4) genes. RESULTS: BRAFV600E mutation was detected in 54% of cell lines. NRAS was mutated in one cell line also carrying multiple copies of NRAS. All cell lines with MC1R variants harboured BRAFV600E. Concurrent loss of MUTYH (1p33), gains of c-MYC (8q24) and of CDK6 (7q21) were found to be significantly associated in cell lines (45%) that harboured biallelic 9p21 deletions including CDKN2B-CDKN2A-MTAP. CONCLUSION: These data suggest the existence of a specific pattern of somatic alterations in genes that are involved in DNA repair (MUTYH) and in cell cycle regulation (c-MYC, CDK6, CDKN2A and CDKN2B). Interestingly, all MC1R variants were associated with BRAFV600E and all cell lines from visceral metastases harboured BRAFV600E.
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Melanoma/genética , Neoplasias Cutâneas/genética , Sequência de Bases , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/genética , Primers do DNA , Genes ras , Humanos , Melanoma/patologia , Mutação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptor Tipo 1 de Melanocortina/genética , Neoplasias Cutâneas/patologiaRESUMO
Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Indóis/farmacologia , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirazinas/farmacologia , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Humanos , Melanoma/patologia , Morfolinas/farmacologia , RNA Interferente Pequeno/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Sunitinibe , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Despite its high incidence as the second most common tumor in males worldwide, primary prostate cancer has been associated with few recurrent chromosomal gains and deletions that are consistent across various studies. Few studies have explored how chromosomal alterations are coupled to abnormal gene expression. Here, we review the major genomic aberrations associated with prostate cancer and describe how detailed transcriptional and computational analyses allowed us to discover a recurrent chromosomal gain in a small region on chromosome 17. Fluorescent in situ hybridization confirmed the presence of a copy number gain in 17q25.3 in tumor-associated preneoplastic lesions of the prostate, 65% of primary tumors, and metastatic samples. These results suggest the involvement of this gain at all steps of prostate cancer progression.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Mutação , Neoplasias da Próstata/genética , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Neoplasias da Próstata/patologia , Transcrição GênicaRESUMO
Syndecans bind to cell adhesion molecules, growth factors and cytokines, and can act as coreceptors, and in this way modulate leukocyte cell function. Here, expression of the syndecans on primary human CD4 T cells was examined. Cell stimulation dramatically increased the amount of syndecan-4, and in a lower extent that of syndecan-2. Expression of syndecan-2 and -4 show different induction kinetics. Whereas syndecan-4 expression is fast and significant, that of syndecan-2 is more delayed and short-lived decreasing its mRNA expression at day 4. Both CD45RA+ naive and CD45RA- memory CD4 T cells express syndecan-2 and -4 upon activation. When incubated with human peripheral blood lymphocytes in a mixed leukocyte reaction, anti-syndecan-4 but not anti-syndecan-2 antibodies, decreased T cell proliferation. However, cross-linking of cell-bound syndecan-2 or syndecan-4 via immobilized antibodies blocked proliferation and decreased TNF production of T cells in the presence of optimal levels of anti-CD3. These findings suggest that syndecan-2 and -4 act as inhibitors of T cell activation. We also investigated the role that MAPK signalling pathways play in control of syndecan expression in T cells. We show that production of syndecan-2 but not syndecan-4 requires signaling via p38 MAP kinase alpha/beta in T CD4 cells. As mechanisms that confer syndecan-2 expression are unknown, we analyse the chromatin hypersensitivity of syndecan-2 promoter proximal region in Jurkat T cells and endothelial cells. The analysis reveals a chromatin accessible site in the +3.5kb intronic region, concomitant with a region showing high evolutionary conservation. We isolate and analyse 5'-flanking regions of human syndecan-2 gene, by transfection assays. The +3.5kb hypersensitive site in the intronic region demonstrates basal promoter activity in Jurkat. This study provides evidence for the up-regulation of syndecan-2 and -4 in human primary CD4 T cells during in vitro activation and suggest an inhibitory role for these syndecans in CD4 T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Sindecana-2/imunologia , Sindecana-4/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/enzimologia , Células Cultivadas , Cromatina/química , Desoxirribonuclease I/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Memória Imunológica/efeitos dos fármacos , Íntrons/genética , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sindecana-2/genética , Sindecana-4/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
OBJECTIVE: It was the aim of this study to analyze the clinical value of the determination of serum S-100beta protein in high-risk melanoma patients. PATIENTS AND METHODS: Patients were tested for serum S-100beta protein by luminoimmunometric assay after melanoma surgical excision, before starting interferon-alpha2b and every 3 months thereafter, until treatment was completed. RESULTS: Ninety-seven patients were included in the study. Median follow-up was 62.9 months (range 32.7-87.4). High baseline S-100beta levels were associated with positive lymph node status (p = 0.02). High S-100beta levels (during therapy) showed a relation with positive lymph node status (p = 0.014), number of positive lymph nodes (p = 0.01), macroscopic lymph node involvement (p = 0.002) and second melanoma diagnosis at study entry (p = 0.001). By univariate analysis, high baseline S-100beta levels were associated with disease-free survival (p = 0.004) and overall survival (p = 0.0007). Similarly, high S-100beta levels during therapy were associated with disease-free survival (p < 0.0001) and overall survival (p < 0.0001). In the multivariate analysis, high S-100beta levels during therapy (hazard ratio 1.017, 95% CI 1.008-1.026; p < 0.0001) and high baseline S-100beta levels (hazard ratio 3.31, 95% CI 1.10-9.89; p = 0.032) were independent prognostic factors for overall survival when compared with low levels while on therapy and low baseline S-100beta levels, respectively. CONCLUSIONS: These results provide evidence of the clinical usefulness of serum S-100beta level determination in high-risk melanoma patients. S-100beta serum determination should be considered to be included in clinical trials that test adjuvant therapies in melanoma patients.
Assuntos
Melanoma/sangue , Fatores de Crescimento Neural/sangue , Proteínas S100/sangue , Neoplasias Cutâneas/sangue , Adulto , Idoso , Biomarcadores Tumorais/sangue , Terapia Combinada , Intervalo Livre de Doença , Feminino , Humanos , Fatores Imunológicos/uso terapêutico , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Estimativa de Kaplan-Meier , Masculino , Melanoma/tratamento farmacológico , Melanoma/mortalidade , Melanoma/patologia , Melanoma/cirurgia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Proteínas Recombinantes , Risco , Subunidade beta da Proteína Ligante de Cálcio S100 , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/cirurgia , Análise de Sobrevida , Resultado do TratamentoRESUMO
Surgically resected stage III melanoma patients commonly receive adjuvant therapy with interferon (IFN) alpha2b. For those patients with high-risk features of draining node recurrence, radiation therapy can also be considered as a treatment option. The purpose of this retrospective study was to assess the efficacy and radiation-related toxicity of this combined therapy. Eighteen patients receiving adjuvant IFNalpha2b therapy during radiation therapy, or within 1 month of its completion, were reviewed retrospectively and analysed for outcome. Radiation was delivered at 600 cGy dose per fraction, in 16 out of 18 patients, twice a week, and at 200 cGy dose per fraction in two patients five times a week. Total radiation dose and number of fractions were as follows: 30 Gy/5 fr (n=8), 36 Gy/6 fr (n=8) and 50 Gy/25 fr (n=2). The percentage of disease-free patients, with no local recurrence, at 3 years was 88%. In 10 patients, IFNalpha2b was administered concurrently with radiotherapy; in three, within 30 days before or after radiation; and in five, more than 30 days after radiation. All the patients experienced acute skin reactions, grade I on the Radiation Therapy Oncology Group (RTOG) scale. Late radiation-related toxicity was seen in one patient with grade III (RTOG) skin reaction and two with grade IV (RTOG) radiation-induced myelitis. Concurrent use of adjuvant radiotherapy and IFNalpha2b might enhance radiation-induced toxicity, and special care should be taken when the spinal cord is included in the radiation field.
Assuntos
Antineoplásicos/uso terapêutico , Interferon-alfa/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Relação Dose-Resposta a Droga , Feminino , Humanos , Sarda Melanótica de Hutchinson/tratamento farmacológico , Sarda Melanótica de Hutchinson/radioterapia , Sarda Melanótica de Hutchinson/secundário , Interferon alfa-2 , Metástase Linfática , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Dosagem Radioterapêutica , Radioterapia Adjuvante , Proteínas Recombinantes , Estudos Retrospectivos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/radioterapiaRESUMO
BACKGROUND: Surgical therapy plays an important role in the management of selected patients with metastatic melanoma. PURPOSE: A retrospective review of 13 patients who underwent surgical resection of lung metastases from melanoma from 1996 to 2003 was performed. The aim of the study was to analyze the clinical outcome and survival time. MATERIALS AND METHODS: Mean age was 45 years old (range: 31-64). Complete tumour resection was confirmed histologically. Nine patients presented one single pulmonary lesion, two lesions (n = 3) and three lesions (n = 1) but in all cases confined in the same pulmonary lobe. RESULTS: Median survival time (MST) for the entire group was 20 months (95% confidence interval (CI): 16-24 months). The median time to disease progression after lung metastasectomy was 5 months (95% CI: 3-7 months). MST, according to the prognostic groups proposed by the International Registry of Lung Metastases, was 17 months (95% CI: 6-28 months) for group I (n = 6), MST of 20 months (95% CI: 16-24 months) for group II (n = 5) and MST of 4 months for group III (n = 2), without differences statistically significant (log-rank p = 0.423). MST regarding the time of disease free interval from diagnostic of primary tumour and lung metastases (< 36 months [n = 5] vs > 36 months [n = 8]) was 20 months and 17 months respectively, without differences statistically significant (log rank p = 0.222). CONCLUSIONS: Surgical resection when feasible provides survival rates superior to any available nonsurgical therapy. In carefully selected patients, when the resection is performed with curative intent, it may result in improved survival.
Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/cirurgia , Melanoma/secundário , Melanoma/cirurgia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
INTRODUCTION: Whole brain irradiation (WBRT) remains a recommended treatment for patients with brain metastases from malignant melanoma in terms of symptom palliation, especially when extracranial systemic disease is present. Temozolomide (TMZ) has shown efficacy in the treatment of metastatic melanoma. The objective was to evaluate the potential benefit in survival of two different schedules of total dose and fractionation (20 Gy/5 fractions vs 30 Gy/10 fractions) and further TMZ based chemotherapy. MATERIALS AND METHOD: We have conducted a retrospective study in a group of twenty-one patients (RTOG Recursive Partitioning Analysis class II) of the use of WBRT with 20 Gy/5 fractions (n = 11) and 30 Gy/10 fractions (n = 10). All patients received further TMZ based chemotherapy administered as a single chemotherapeutic agent or in combination with chemo-immunotherapy. RESULTS: Prognostic variables such as: age, Karnofsky performance status, extracranial metastases and number of brain metastases, were analyzed in both groups of treatment without statistically significant differences. The median survival time (MST) for WBRT 20 Gy group was 4 months (CI 95%: range 2- 6 months) and for WBRT 30 Gy group was 4 months (CI 95%: range 0-7 months) without statistically significant differences (Log rank p = 0.74). There was one complete response and two partial responses. CONCLUSIONS: The results suggest that MST was not significantly affected by the total dose/fractionation schedule.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/secundário , Irradiação Craniana , Dacarbazina/análogos & derivados , Melanoma/secundário , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Cisplatino/administração & dosagem , Estudos de Coortes , Terapia Combinada , Dacarbazina/administração & dosagem , Dacarbazina/uso terapêutico , Esquema de Medicação , Avaliação de Medicamentos , Feminino , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interleucina-2/administração & dosagem , Tábuas de Vida , Masculino , Melanoma/tratamento farmacológico , Melanoma/radioterapia , Pessoa de Meia-Idade , Seleção de Pacientes , Modelos de Riscos Proporcionais , Proteínas Recombinantes , Estudos Retrospectivos , Análise de Sobrevida , Temozolomida , Resultado do Tratamento , Vimblastina/administração & dosagemRESUMO
Standard antineoplastic treatment for metastatic melanoma is ineffective in the large majority of patients. Therefore, alternative approaches need to be investigated. STI571 is a new antineoplastic compound, which selectively inhibits the tyrosine kinase activity of ABL, c-Kit and platelet-derived growth factor receptor (PDGFR). Melanoma may express all of these proteins. The aim of this study was to investigate whether STI571 inhibits the in-vitro growth of melanoma cells. Nineteen cell lines were obtained from four primary and 15 metastatic melanomas of cutaneous origin. The percentages of positive cells for the putative targets of STI571 were as follows: ABL, 41-100%; c-Kit, 8-97%; PDGFR-alpha, 41-98%; PDGFR-beta, 51-99%. 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium (MTT) and viability assays showed that STI571 clearly inhibits the proliferation of eight of the 19 (42.1%) cell lines. No relationship could be established between the expression of c-Kit, ABL, PDGFR-alpha or PDGFR-beta and the response of cell lines to STI571. Our study shows, for the first time, an antiproliferative effect of STI571 on human melanoma cell lines of cutaneous origin, raising the possibility of the future clinical use of STI571. The identification of the target of STI571 in human cutaneous melanoma cells would allow the selection of patients who could benefit from this treatment.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Melanoma/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Análise Mutacional de DNA , Citometria de Fluxo , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Proteínas Oncogênicas v-abl/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Malignant melanoma (MM) early lymph node (LN) metastasis usually appears first in the sentinel LN (SLN). Breslow thickness is the main factor considered in the selection of patients to be submitted to SLN biopsy. The present study aimed to describe other independent prognostic factors useful in SLN candidate selection. During one year, 94 MM patients (90 primary cutaneous MM with Breslow thickness > or = 0.76 mm, and four cutaneous relapses), were submitted to SLN biopsy in the Melanoma Unit at the Hospital Clinic, Barcelona, Spain. The prognostic factors studied were: Breslow thickness, Clark's level of invasion, mitotic rate, cellular type (small, epithelioid, fusocellular, sarcomatoid), vertical growth phase, regression > 50%, severe vascularization, infiltrate (lymphocytic, plasmocytic), ulceration, neurotropism, intravascular/intraneural invasion, protein p16 expression and recurrence. Nineteen SLN (20.2%) were positive and 75 (79.8%) negative. No positive SLN occurred in MM with Breslow thickness < or = 1.0 mm. Breslow thickness > or = 2 mm (P = 0.005), severe vascularization (P = 0.005), small cell (P = 0.000) and ulceration (P = 0.005) were significant prognostic factors by univariate analysis. Small cell (P = 0.008) and ulceration (P = 0.05) were also significant prognostic factors in a multivariate analysis. The probability of finding a positive SLN for small cell was 56.9% [95% confidence interval (CI), 26.8-82.6%]. The probability of positive SLN for ulceration was 35.5% (95% CI, 14.2-64.7%). For small cell and ulceration together the probability increased to 86.3% (95% CI, 54.3-97.1%). The results of this study corroborated ulceration as a prognostic factor for SLN candidate selection and for the first time we have described small cell melanoma morphology as a significant factor associated with positive SLN.
Assuntos
Metástase Linfática/diagnóstico , Melanoma/patologia , Biópsia de Linfonodo Sentinela , Úlcera/patologia , Humanos , Metástase Linfática/patologia , Estadiamento de Neoplasias , Probabilidade , PrognósticoRESUMO
Unresectable metastatic melanoma has no elective treatment. Neither chemotherapy, intravenous IL-2 nor biochemotherapy clearly improves the overall survival. Recent assays with therapeutic vaccines have been recently yielded promising results. Here, we describe the application, clinical tolerance and antitumoural activity of a heterologous polyvalent melanoma whole cell vaccine in patients with metastatic melanoma. Twenty-eight AJCC stage III/IV melanoma patients with progressive unresectable metastatic disease were treated with our heterologous polyvalent melanoma whole cell vaccine between July 1, 1998 and July 1, 2002. All patients had already been unsuccessfully treated with high doses of IFN-alpha2 and/or polychemotherapy and/or biochemotherapy and/or perfusion of extremities, or could not receive other treatments due to their age or underlying illness. Twenty-three were assessable. The vaccine was constituted by 10 melanoma cell lines, derived from primary, lymph node and metastatic melanomas. Prior to intradermal inoculation, the cells were irradiated and mixed with BCG, and 50% were treated with DNFB. After a median follow-up of 19 months, 26% of patients responded: 3 CR (18, 16+, and 26+ months), 2 PR (8 and 22 months) and 1 MR (36+ months). The median survival of the whole group was 20.2 months. None of the 28 patients initially included in the study presented significant toxicity. This vaccination program had specific antitumoural activity in advanced metastatic melanoma patients and was well tolerated. The clinical responses and the median survival of our group of patients, together with the low toxicity of our polyvalent vaccine, suggest that this approach could be applied to earlier metastatic melanoma patients.