RESUMO
Since sodium stibogluconate (SSG) inhibited phosphatases including SHP-1 and augmented anti-tumor actions of IFN-α2b in vitro and in mice, two Phase I trials of SSG/IFN-α2b combination were undertaken to evaluate safety and target inhibition. Escalating doses of SSG (200-1200 mg/m2) and fixed doses of IFN-α2b (3x106 units/m2) with or without chemotherapy (dacarbazine, vinblastine, cisplatin) were evaluated for side effects and impact on SHP-1 phospho-substrates and IFNα-stimulated-genes (ISGs) in peripheral blood in 40 patients with metastatic melanoma, soft tissue sarcomas, gastrointestinal stromal tumors, and breast or colorectal carcinomas who did not have other established treatment options. Common adverse events were bone marrow suppression, fatigue, gastrointestinal upset, and asymptomatic lipase elevation (n=13); the latter was dose related and mostly after 10d of SSG/IFN-α2b in combination. Levels of SHP-1 substrates (pSTAT1, pSTAT3, pLck and pSlp76) were increased (up to 3x) in peripheral blood cells following SSG with no potentiation by combination with IFN-α2b. Representative ISGs in peripheral blood were induced after IFN-α2b at 4 and 24 hrs with selective modulations by combination. The median time on trials was 2.3 months (10-281d) with no objective regression of disease. Alive at 1y were 17/40 (43%) patients and after 2y were 8/40 (20%) following treatment initiation. These data demonstrate that SSG impacted signal molecules consistent with PTP inhibition and was tolerated in combination with IFN-α2b. Phase II investigations of SSG could safely utilize doses of up to 1200 mg/m2 of SSG for up to 10d alone or in combination with IFN-α2b with or without chemotherapy.
Assuntos
Gluconato de Antimônio e Sódio , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Interferon-alfa/uso terapêutico , Adulto , Idoso , Gluconato de Antimônio e Sódio/administração & dosagem , Gluconato de Antimônio e Sódio/efeitos adversos , Gluconato de Antimônio e Sódio/farmacocinética , Gluconato de Antimônio e Sódio/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Cisplatino/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Dacarbazina/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/sangue , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Sarcoma/tratamento farmacológico , Vimblastina/farmacologiaRESUMO
Drug resistance is a major obstacle in cancer treatments and diminishes the clinical efficacy of biological, cytotoxic, or targeted therapeutics. Being an antiapoptotic mediator of chemoresistance in breast and lung cancer cells, MKP1 phosphatase might be targeted for overcoming chemoresistance and improving therapeutic efficacy. In this work, tyrosine phosphatase inhibitor-3 (TPI-3) was identified as a novel small molecule inhibitor of MKP1 and was capable of sensitizing tumors to bio- and chemotherapeutics in mice as a tolerated oral agent. Effective against recombinant MKP1, TPI-3 selectively increased MKP1 phosphosubstrates in Jurkat cells and induced cell death via apoptosis at nanomolar concentrations. TPI-3 also increased MKP1 phosphosubstrates in WM9 human melanoma cells and synergized with biotherapeutic IFN alpha 2b in the growth inhibition of melanoma cells in vitro (combination index, <1). WM9 xenografts unresponsive to individual agents were significantly inhibited (62%, P = 0.001) in mice by a tolerated combination of oral TPI-3 (10 mg/kg, 5 d/wk) and IFN alpha 2b. MKP1 expression was detected in human melanoma cell lines and tissue samples at levels up to six times higher than those in normal or nonmalignant melanocytes. TPI-3 also interacted positively with chemotherapeutics, 5-fluorouracil/leucovorin, against MC-26 colon cancer cells in vitro and in mice. Altogether, our data show the preclinical activities of TPI-3 in overcoming cancer resistance to bio- and chemotherapeutics, implicate MKP1 as a drug-resistant molecule in melanoma, and support the targeting of MKP1 for improving cancer therapeutic efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Interferon-alfa/uso terapêutico , Melanoma/tratamento farmacológico , Tiazóis/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Proteínas Recombinantes , Especificidade por Substrato/efeitos dos fármacos , Tiazóis/química , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Src homology region 2 domain-containing phosphatase 1 (SHP-1) has been implicated as a potential cancer therapeutic target by its negative regulation of immune cell activation and the activity of the SHP-1 inhibitor sodium stibogluconate that induced IFN-gamma(+) cells for anti-tumor action. To develop more potent SHP-1-targeted anti-cancer agents, inhibitory leads were identified from a library of 34,000 drug-like compounds. Among the leads and active at low nM for recombinant SHP-1, tyrosine phosphatase inhibitor-1 (TPI-1) selectively increased SHP-1 phospho-substrates (pLck-pY394, pZap70, and pSlp76) in Jurkat T cells but had little effects on pERK1/2 or pLck-pY505 regulated by phosphatases SHP-2 or CD45, respectively. TPI-1 induced mouse splenic-IFN-gamma(+) cells in vitro, approximately 58-fold more effective than sodium stibogluconate, and increased mouse splenic-pLck-pY394 and -IFN-gamma(+) cells in vivo. TPI-1 also induced IFN-gamma(+) cells in human peripheral blood in vitro. Significantly, TPI-1 inhibited ( approximately 83%, p < 0.002) the growth of B16 melanoma tumors in mice at a tolerated oral dose in a T cell-dependent manner but had little effects on B16 cell growth in culture. TPI-1 also inhibited B16 tumor growth and prolonged tumor mice survival as a tolerated s.c. agent. TPI-1 analogs were identified with improved activities in IFN-gamma(+) cell induction and in anti-tumor actions. In particular, analog TPI-1a4 as a tolerated oral agent completely inhibited the growth of K1735 melanoma tumors and was more effective than the parental lead against MC-26 colon cancer tumors in mice. These results designate TPI-1 and the analogs as novel SHP-1 inhibitors with anti-tumor activity likely via an immune mechanism, supporting SHP-1 as a novel target for cancer treatment.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Administração Oral , Animais , Western Blotting , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Dual specificity protein tyrosine phosphatase PRL-2 is overexpressed in pediatric acute myeloid leukemia (AML) and is located at human chromosome 1p35, a region often rearranged or amplified in malignant lymphoma and B-cell chronic lymphocytic leukemia (B-CLL). Little is known of the significance of PRL-2 expression in hematopoietic malignancies. Herein we demonstrated that ectopic expression of PRL-2 in murine pre-B-cell line Baf3ER and mouse bone marrow cells induced key features associated with malignant progression and metastasis. PRL-2-transfected Baf3ER cells had augmented growth responses to hematopoietic growth factors Epo or IL-3 with shortened cell cycle, reduced requirement (5x) for Epo in cell survival, increased cell migration (3x), reduced cell adhesion (5x), and conversion to an immature cell morphology in association with increased expression (3x) of stem cell marker Bmi-1. When transduced into mouse bone marrow cells, PRL-2 increased Epo-induced colony formation (4x) and gave rise to larger colonies. These observations provide evidences implicating PRL-2 as a pathogenic molecule in hematopoietic malignancies and suggest its potential as a novel therapeutic target.
Assuntos
Eritropoetina/farmacologia , Interleucina-3/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Proteínas Tirosina Fosfatases/fisiologia , Animais , Células da Medula Óssea/citologia , Adesão Celular/efeitos dos fármacos , Desdiferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular/citologia , Linhagem Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Epoetina alfa , Neoplasias Hematológicas/etiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Linfócitos B/citologia , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes , Fator de Transcrição STAT5/biossíntese , Fator de Transcrição STAT5/genética , Transdução GenéticaRESUMO
Sodium stibogluconate (SSG), an inhibitor of SHP-1 that negatively regulates cytokine signaling and immunity, suppressed growth of murine Renca tumors in combination with interleukin-2 (IL-2) via a T-cell-dependent mechanism. The ability of SSG to interact with IL-2 in activating primary human immune cells was evaluated herein by assessing its induction of interferon (IFN)-gamma(+) TH1 cells in human peripheral blood in vitro. The significance of IFN-gamma(+) cells was also investigated by assessing SSG/IL-2 antitumor activity in wild-type and IFN-gamma(-/-) mice. IFN-gamma(+) cells but not IL-5(+) cells were induced markedly (9.1x) in healthy peripheral blood by SSG/IL-2 in contrast to the modest induction by SSG alone (2.1x) at its clinically achievable dose (20 microg/mL) or by IL-2 (3.1x) at its C(max) of low-dose schedule (30 IU/mL). SSG at a higher dose (100 microg/mL) was less effective alone (1.5x) or in combination with IL-2 (7.8x). Peripheral IFN-gamma(+) cells were induced after 4 or 16 h treatment with SSG/IL-2 within CD4(+) and CD8(+) lymphocytes coincided with heightened CD69 expression (approximately 3-4x). SSG/IL-2 was also more effective than the single agents in inducing IFN-gamma(+) cells in the peripheral blood of melanoma patients, whose basal IFN-gamma(+) cell levels were approximately 5% of healthy controls. Renca tumor growth was inhibited by SSG/IL-2 in wild-type but not IFN-gamma(-/-) mice. These results demonstrate SSG interactions with IL-2 in vitro to activate key antitumor immune cells in peripheral blood of healthy and melanoma donors, providing further evidence for proof of concept clinical trials for effecting augmentation of IL-2 through inhibiting negative regulatory protein tyrosine phosphatases.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Gluconato de Antimônio e Sódio/farmacologia , Antineoplásicos/farmacologia , Inibidores do Crescimento/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-5/metabolismo , Neoplasias Renais/imunologia , Leucócitos Mononucleares/imunologia , Melanoma/imunologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Gluconato de Antimônio e Sódio/administração & dosagem , Antineoplásicos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Inibidores do Crescimento/administração & dosagem , Humanos , Imunofenotipagem , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Lectinas Tipo C , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transplante de Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Transdução de SinaisRESUMO
Protein tyrosine phosphatases (PTPases) are attractive targets for developing novel cancer therapeutics. Activated via gain-of-function point mutations or overexpression, several PTPases have been identified as critical oncogenic molecules in human malignancies that may be targeted with small chemical inhibitors as a therapeutic strategy. Tumor suppressor PTPases have also been discovered as contributing factors in cancer development that may be targeted via intervention of downstream signaling events for therapeutic purposes. In addition, PTPases have been identified as key negative regulators of cytokines or immune cells. Targeting these negative PTPases may improve the efficacy of cytokine therapy and immunotherapy, which currently have modest response rates and limited survival benefit. Inhibitors of selective PTPases have demonstrated significant preclinical antitumor activity, leading to early-phase clinical trials. Further research and development could lead to PTPase-targeted cancer therapeutics in the near future.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Genes Supressores de Tumor , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias/enzimologia , Monoéster Fosfórico Hidrolases/fisiologia , Mutação Puntual , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Resultado do TratamentoRESUMO
BACKGROUND: Pre-clinical activity of SSG against melanoma and renal cancer has been identified recently although the drug's mechanism of action and activity against tumors of additional histological-types remain undefined. METHODS: The effects of SSG and SSG combination with other agents on DU145 human prostate carcinoma xenograft tumors in mice and on DU145 cell subpopulations of differential SSG sensitivities were evaluated. RESULTS: DU145 tumor growth was inhibited by SSG (69%), IFNalpha2 (33%) or the combination (80%) that induced complete regression of WM9 human melanoma tumors. DU145 cells in culture were also partially growth inhibited by SSG at killing doses (200-800 mug/ml) for WM9 cells, indicating a correlation of SSG inhibition of cancer cell growth in vitro and in vivo. DU145 cells formed multiple micro tumors in mice treated with SSG or SSG/IFNalpha2 in contrast to the single large tumors in the control or IFNalpha2-treated mice, suggesting the existence of an SSG-resistant subpopulation in DU145 cells. Indeed, DU145 but not WM9 cells formed colonies (approximately 4% frequency) when cultured in the presence of SSG. Single cell clone (DU145-7) isolated from DU145 cells showed SSG-resistant growth in culture, unassociated with cross-resistance to IFNalpha2 and converted to SSG-responsive cells by BSO that inhibited intracellular glutathione levels. CONCLUSIONS: These results implicate a role for direct growth inhibition in SSG anti-tumor action, provide novel insights into the mechanism of tumor resistance to the drug and suggest a therapeutic potential for SSG and its combinations with IFNalpha2 or BSO for prostate cancer that warrants further investigation.
Assuntos
Gluconato de Antimônio e Sódio/uso terapêutico , Interferon-alfa/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Animais , Gluconato de Antimônio e Sódio/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Interferon-alfa/administração & dosagem , Masculino , Melanoma , Camundongos , Camundongos Nus , Neoplasias da Próstata/patologia , Transplante HeterólogoRESUMO
Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Tirosina Fosfatases/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linhagem Celular , Células Cultivadas , Células Clonais , Inibidores Enzimáticos/farmacologia , Interferon gama/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Proto-Oncogênicas c-vav/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Recombinantes , Tetraciclina/farmacologia , TransfecçãoRESUMO
IL-2 therapy results in 10-20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mphi). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mphi (4-fold), whereas IL-2 alone had little effect. Mphi increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mphi expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-gamma T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-gamma capable of activating tumoricidal Mphi. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-gamma production and expansion/activation of Mphi. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.
Assuntos
Gluconato de Antimônio e Sódio/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Interleucina-2/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Animais , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Feminino , Fatores Imunológicos/administração & dosagem , Interferon gama/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Recombinantes/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania. Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein [J. Biol. Chem. 277 (2002) 50190]. To characterize this apparent Leishmania virulence factor further, the cDNA encoding L. donovani EF-1 alpha was cloned and sequenced. Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L. donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes. Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion. Consistent with these structural differences, the sub-cellular distributions of L. donovani EF-1 alpha and host EF-1 alpha were strikingly different. Interestingly, infection of macrophages with L. donovani caused redistribution of host as well as pathogen EF-1 alpha. Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.
Assuntos
Leishmania donovani/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Fracionamento Celular , Clonagem Molecular , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/metabolismo , Proteína Fosfatase 1 , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
Interferon (IFN)-dependent cellular effects are mediated by transcriptional induction of responsive genes, collectively referred to as IFN-stimulated genes (ISGs). Which ISGs regulate the potent antiviral, antiproliferative, apoptosis-inducing, antiangiogenic, and immunologic effects of IFNs remains largely undetermined. To identify genes that might be useful for predicting or targeting apoptosis induction in response to IFNs, WM9 melanoma cells were assessed. WM9 cells had equivalent antiviral activity in response to IFN-beta and IFN-alpha2 but underwent apoptosis only in response to IFN-beta. RNA samples from WM9 cells and WM35 cells, a second melanoma cell line, treated with IFN-alpha2 or IFN-beta were assessed on oligonucleotide arrays. For 95% of genes assessed, IFN-beta was more potent than IFN-alpha2 in inducing ISG expression. Using a 22,000-gene oligonucleotide array, the largest yet reported for assessing ISG induction, approximately 910 genes were identified as induced by IFN-beta at 500 U/ml, and 260 ISGs were identified as significantly induced by IFN-beta at both 50 and 500 U/ml. Of these 260, 209 were defined as new ISGs based on the array analysis. Confirmation by Northern blot or semiquantitative or quantitative PCR was undertaken for 28, and all were confirmed. Nearly half of the 260 genes were functionally categorized as encoding growth-regulatory proteins. Of the 104 with described growth-regulatory function, 71 were induced more than three times by 500 U/ml and twice by 50 U/ml IFN-beta, and 48 of these were new ISGs. Included in this latter category were tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), XIAP-associated factor 1 (XAF1), galectin 9, a cyclin E binding protein, amphiphysin 1, MyD88, and several ubiquitin pathway genes. The diversity of stimulated genes suggests the full therapeutic potential of IFN regulation of gene expression has yet to be realized.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Melanoma/genética , Melanoma/patologia , Apoptose/genética , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Galectinas/genética , Perfilação da Expressão Gênica , Humanos , Cinética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-alpha synergized to overcome IFN-alpha resistance in various human cancer cell lines in culture and eradicated IFN-alpha-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-alpha-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.
Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antineoplásicos/farmacologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Gluconato de Antimônio e Sódio/uso terapêutico , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Quimioterapia Combinada , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/uso terapêutico , Humanos , Interferon-alfa/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Fator de Transcrição STAT1 , Transativadores/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.
Assuntos
Leishmania donovani/metabolismo , Macrófagos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Glutationa Transferase/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Leishmania donovani/patogenicidade , Macrófagos/parasitologia , Espectrometria de Massas , Camundongos , Microscopia de Fluorescência , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fenótipo , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 1 , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Domínios de Homologia de srcRESUMO
Fcgamma receptor-mediated phagocytosis is a complex process involving the activation of protein tyrosine kinases, events that are potentially down-regulated by protein tyrosine phosphatases. We used the J774A.1 macrophage cell line to examine the roles played by the protein tyrosine phosphatase SHP-1 in the negative regulation of Fcgamma receptor-mediated phagocytosis. Stimulation with sensitized sheep red blood cells (sRBCs) induced tyrosine phosphorylation of CBL and association of CBL with CRKL. These events were completely or partially abrogated by PP1 or the heterologous expression of dominant-negative SYK, respectively. Heterologous expression of wild-type but not catalytically inactive SHP-1 also completely abrogated the phagocytosis of IgG-sensitized sRBCs. Most notably, overexpressed SHP-1 associates with CBL and this association led to CBL dephosphorylation, loss of the CBL-CRKL interaction, and the suppression of Rac activation. These data represent the first direct evidence that SHP-1 is involved in the regulation of Fcgamma receptor-mediated phagocytosis and suggest that activating signals mediated by SRC family kinases SYK, CBL, phosphatidyl inositol-3 (PI-3) kinase, and Rac are directly opposed by inhibitory signals through SHP-1.
Assuntos
Fagocitose/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de IgG/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/fisiologia , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Transdução de Sinais , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Domínios de Homologia de src/fisiologiaRESUMO
SHP-1 protein tyrosine phosphatase is a critical regulator of signaling in hematopoietic cells as illustrated by the lethal hematopoietic disorders in SHP-1-deficient mice. We and others have shown in previous studies that SHP-1 regulates membrane receptor signaling: it binds via its N-terminal region SH2 domains to tyrosine phosphorylated membrane receptors to dephosphorylate key substrates in the receptor complexes. Here we demonstrate that the SHP-1 C-terminal region contains a bipartite NLS that mediates SHP-1 nuclear localization in response to cytokines. This NLS was located within amino acids 576-595 of the PTPase and, when fused by itself to EGFP, targeted the fluorescent protein into the nuclei of transiently transfected NIH3T3 fibroblasts and Bac1.2f5 macrophage cells. When positioned within SHP-1, the activity of the NSL was under tight regulation as indicated by the predominant cytoplasmic distribution of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and the exclusive cytoplasmic localization of the endogenous SHP-1 in hematopoietic cell line PBLC-1. Activation of the NLS in SHP-1 by IL-4 was demonstrated by increased nuclear localization of the EGFP/SHP-1 fusion protein in NIH3T3 transfectants and of the endogenous SHP-1 protein in PBCL-1 cells at 4, 6 and 8 h post-IL-4 stimulation. SHP-1 nuclear localization in PBCL-1 cells was also induced by IL-7 in a similar manner, suggesting it as a common event in cytokine signaling. In comparison to that of the wild-type phosphatase, an SHP-1 mutant lacking the NLS showed only approximately half of the activity in inhibiting proliferation of NIH3T3 transfectants. These results provide evidence of cytokine-regulated SHP-1 nuclear localization mediated by a bipartite NLS and suggest that SHP-1 regulates nuclear signaling in cell growth control.
Assuntos
Transporte Ativo do Núcleo Celular , Citocinas/farmacologia , Sinais de Localização Nuclear/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Células 3T3 , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Interleucina-4/farmacologia , Interleucina-7/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia de Fluorescência , Sinais de Localização Nuclear/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Mice homozygous for the viable motheaten (Hcph(me-v)) mutation are deficient in SHP-1 protein-tyrosine phosphatase, resulting in severe systemic autoimmunity and immune dysfunction. A high percentage of B-cells in viable motheaten mice express the cell surface glycoprotein CD5, in contrast to wild type mice that express CD5 on only a small percentage of B-cells. CD5(+) B-cells have been associated with autoantibody production. To determine the role of CD5 in the development of the inflammatory disease in me(v)/ me(v) mice, we created a stock of CD5(null)me(v)/ me(v) mice. The longevity of CD5(null)me(v)/ me(v) mice was increased 69% in comparison to me(v)/ me(v) mice on a similar (B6;129) background. The increased lifespan was associated with a marked reduction in pulmonary inflammation. Flow cytometry analysis of spleen cells from CD5(null)me(v)/ me(v) mice at 9-12 weeks of age revealed significant decreases in percentages of IgM/B220 double positive B-cells, Mac-1/Gr-1 double positive cells and CD4(+) T-cells compared with me(v)/ me(v) mice. CD5(null)me(v)/ me(v) mice also had significantly lower serum IgM levels in comparison to me(v)/ me(v) mice. Study of CD5(null)me(v)/ me(v) mice may provide further insight into the role of CD5 in cell signaling and may help explain the observed association of CD5(+) B-cells with autoimmune disease.
Assuntos
Antígenos CD5/metabolismo , Pneumonia/etiologia , Pneumonia/imunologia , Proteínas Tirosina Fosfatases/deficiência , Animais , Autoanticorpos/sangue , Contagem de Células Sanguíneas , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Antígenos CD5/genética , Feminino , Imunoglobulinas/sangue , Peptídeos e Proteínas de Sinalização Intracelular , Longevidade , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Pneumonia/enzimologia , Pneumonia/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Baço/imunologia , Baço/patologiaRESUMO
The PRL family oncogenic phosphatases are attractive targets for developing inhibitors as anticancer therapeutics given their potentially pathogenic role in human malignancies. Herein we demonstrate that pentamidine, an anti-protozoa drug with an unknown mechanism of action, is an inhibitor of PRLs with anticancer potential. Pentamidine at its therapeutic doses inhibited recombinant PRL phosphatases in vitro and inactivated ectopically expressed PRLs in NIH3T3 transfectants with an effective duration more than 24 h after a pulse cell treatment. The drug had in vitro growth-inhibitory activity against human cancer cell lines that express the endogenous PRLs. Pentamidine at a tolerable dose markedly inhibited the growth of WM9 human melanoma tumors in nude mice coincident with the induction of tumor cell necrosis and is capable of inactivating ectopically expressed PRL-2 in the cancer cells. These observations suggest the potential of pentamidine in anticancer therapies and may provide a basis for developing novel PTPase-targeted therapeutics.