RESUMO
Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.
Assuntos
Glicólise/fisiologia , Linfócitos T/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Xenopus laevisRESUMO
Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation; however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Glutaratos/farmacologia , Interleucina-12/biossíntese , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida , Células Dendríticas/citologia , Humanos , Lipopolissacarídeos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Espectrometria de Massas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/citologia , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4-0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy.
RESUMO
The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.
RESUMO
Inflammatory signaling pathways orchestrate the cellular response to infection and injury. These pathways are known to be modulated by compounds that alkylate cysteinyl thiols. One class of phytochemicals with strong thiol alkylating activity is the chalcones. In this study we tested fourteen chalcone derivatives, α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH), for their ability to modulate inflammatory responses, as monitored by their influence on heme oxygenase-1 (HO-1) activity, inducible nitric oxide synthase (iNOS) activity, and cytokine expression levels. We confirmed that the transcriptional activity of Nrf2 was activated by α-X-TMCs while for NF-κB it was inhibited. For most α-X-TMCs, anti-inflammatory activity was positively correlated with thiol alkylating activity, i.e. stronger electrophiles (X = CF3, Br and Cl) being more potent. Notably, this correlation did not hold true for the strongest electrophiles (X = CN and NO2) which were found to be ineffective as anti-inflammatory compounds. These results emphasize the idea that chemical fine-tuning of electrophilicity is needed to achieve and optimize desired therapeutic effects.
Assuntos
Anti-Inflamatórios/química , Chalconas/química , Inflamação/metabolismo , Animais , Cisteamina/química , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Glutationa/química , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/química , Macrófagos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Processos Fotoquímicos , Compostos de Sulfidrila/química , Transcrição GênicaRESUMO
Recent years have seen resurging interest in cancer cell metabolism and the role of secreted cancer metabolites in modulating the tumor stroma. Using a combination of nontargeted and targeted LC and GC-MS methods, the exometabolomes of three leukemia, two melanoma, three renal cell carcinoma, two colorectal adenocarcinoma, four hepatocellular carcinoma, three breast cancer, two bladder carcinoma, and one glioblastoma cell line, as well as five primary cultures of human melanocytes, hepatocytes, monocytes, CD4 and CD8 lymphocytes, that had been all cultivated under identical conditions, were investigated. Unsupervised affinity propagation clustering of the metabolic footprints yielded five distinct clusters that grouped the investigated cell cultures mainly according to the tissue of origin. A common expected feature of all neoplastic cells was high lactate production. Extracellular arginine and nicotinamide were major discriminants between normal and neoplastic hepatocytes. Further, significant differences in the assimilation of di- and tripeptides were observed. This finding appears to underscore the importance of peptides for meeting the increased bioenergetic and biosynthetic demands of many cancers.
Assuntos
Metaboloma , Metabolômica/métodos , Neoplasias/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Espectrometria de Massas/métodos , Células Tumorais CultivadasRESUMO
Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diclofenaco/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Transporte Biológico/efeitos dos fármacos , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Ácido Láctico/metabolismo , Leucemia/genética , Leucemia/metabolismo , Melanoma/genética , Melanoma/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Lactate formation in highly proliferative tumors such as malignant gliomas is associated with poor survival and contributes to the suppression of local immunity. Here, we report that diclofenac used at nontoxic concentrations significantly decreased lactate production in murine glioma cells and inhibited the expression of lactate dehydrogenase-A in vitro. Lactate reduction was accompanied by a dose-dependent inhibition of cell growth and a cell cycle arrest at the G2/M checkpoint. In the presence of diclofenac, murine bone marrow-derived dendritic cells (DCs) showed enhanced IL-12, but decreased IL-10 secretion on Toll-like receptor stimulation with R848 that correlated with reduced lactate levels in the glioma cell coculture and a blockade of signal transducers and activators of transcription 3 phosphorylation. In vivo, diclofenac treatment diminished intratumoral lactate levels and resulted in a significant delay of glioma growth. Ex vivo analyses revealed that tumor-infiltrating DCs regained their capacity to produce IL-12 on R848 stimulation. Moreover, diclofenac reduced the number of tumor-infiltrating regulatory T cells and impaired the upregulation of the Treg activation marker CD25. Nevertheless, a single intratumoral injection of R848 combined with diclofenac failed to induce an additional survival advantage in glioma-bearing mice. Further analyses illustrated that the presence of diclofenac during T-cell activation compromised INF-γ production and T-cell proliferation, indicating that immunotherapeutic approaches have to be carefully timed when combined with diclofenac. In summary, diclofenac appears as an attractive agent for targeting lactate production and counteracting local immune suppression in malignant gliomas.
Assuntos
Células Dendríticas/imunologia , Diclofenaco/farmacologia , Glioma/imunologia , Glioma/metabolismo , Ácido Láctico/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células da Medula Óssea/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Glioma/tratamento farmacológico , Imidazóis/farmacologia , Tolerância Imunológica , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Isoenzimas/biossíntese , Isoenzimas/metabolismo , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
About a century ago Otto Warburg observed that tumor cells exhibited increased glycolysis despite the presence of oxygen and stated this metabolic shift to glycolysis as the origin of cancer cell. In the meantime it has become clear, that the altered glucose metabolism is only one piece of the tumor metabolome puzzle. In addition, amino acid, lipid and adenosine metabolism are adapted to fulfill the tumors needs for energy and generation of building blocks such as lipids and nucleotides for new cell structures. The altered tumor metabolism leads to accumulation of specific metabolites in the tumor environment and creates a favorable milieu for tumor growth, progression and metastasis. These tumor-derived metabolites are important players in immune escape mechanisms beside other known factors such as cytokines, chemokines and growth factors. A variety of metabolites re-educate immune cells and prevent an effective immune response against tumor cells. Furthermore, tumor infiltrating immune cells support tumor growth by the secretion of cytokines, growth factors and other metabolic determinants. Hence, a complex interplay of tumor metabolites, cytokines and stromal factors is active in tumors and facilitates their establishment and growth. Pharmacological blockade of tumor metabolites could overcome some limitations of cancer treatment and rescue the endogenous immune response against tumor cells.
Assuntos
Imunomodulação , Neoplasias/metabolismo , Acidose , Aminoácidos/metabolismo , Animais , Transporte Biológico , Progressão da Doença , Glicólise , Humanos , Ácido Láctico/metabolismo , Mitocôndrias/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Via de Pentose Fosfato , Prostaglandina-Endoperóxido Sintases/metabolismo , Nucleosídeos de Purina/metabolismoRESUMO
Depletion of host Langerhans cells (LCs) prevents cutaneous graft-versus-host disease (GvHD) in mice. We analyzed whether UVB irradiation is tolerated during the course of human allogeneic hematopoietic cell transplantation and whether depletion of LCs by broadband UVB could improve GvHD outcome. A total of 17 patients received six whole-body UVB irradiations with 75% of the individually determined minimal erythemal dose after conditioning with a reduced intensity protocol. LCs, dermal dendritic cells (DCs), and macrophages were analyzed before and after UVB irradiation by immunohistochemical analysis. Circulating blood cells and serum factors were analyzed in parallel. In striking contrast to previous data, our irradiation protocol was well tolerated in all patients. UVB treatment decreased the number of LCs and also affected dermal DCs. UVB-treated patients also had significantly higher 25-hydroxyvitamin D3 serum levels and higher numbers of circulating CD4+ FoxP3+ regulatory T cells. Strikingly, nine out of nine patients with complete LC depletion (<1 LC per field) developed only grade I GvHD or no GvHD up to day 100. Our results strongly suggest that prophylactic UVB irradiation post transplant is safe and should be further explored as a clinical strategy to prevent acute (skin) GvHD.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Irradiação Corporal Total/métodos , Doença Aguda , Adulto , Animais , Doença Crônica , Terapia Combinada , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Células de Langerhans/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Doses de Radiação , Especificidade da Espécie , Condicionamento Pré-Transplante/métodos , Transplante HomólogoRESUMO
Lactic acidosis is common to most solid tumors and has been found to affect infiltrating immune cells. Here we document effector phase inhibition of cytotoxic T cells (CTLs) involving complete blockage of cytokine production and partial impairment of lytic granule exocytosis. Lactic acidosis impaired TCR-triggered phosphorylation of JNK, c-Jun and p38, while not affecting MEK1 and ERK. The select targeting of signaling proteins involved in IFNγ production (JNK/c-Jun, p38) without affecting those jointly used in cytokine regulation and granule exocytosis (MEK1/ERK) explains the observed split effect of lactic acidosis on the CTL responses. CTL inhibition by lactic acidosis showed fast dynamics with immediate onset and reversion. Functional recovery by neutralizing the extracellular pH despite continuous presence of lactate holds promise that CTL activity can be improved in the milieu of solid tumors with appropriate anti-acidosis treatment, thereby increasing the efficacy of adoptive T cell therapy.
Assuntos
Acidose Láctica/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ácido Láctico/metabolismo , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Concentração de Íons de Hidrogênio , Interferon gama/biossíntese , Sistema de Sinalização das MAP Quinases , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.1-50 and 0.5-100 nM, respectively. Fully (13)C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%, 1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell culture supernatants from IFN-γ-treated monocytes and immature or mature dendritic cells.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Triptofano/sangue , Triptofano/urina , Linhagem Celular , Meios de Cultura/química , Células Dendríticas/química , Células Dendríticas/metabolismo , Humanos , Monócitos/química , Monócitos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Triptofano/metabolismoRESUMO
Tumor cells have developed multiple mechanisms to escape T-cell-mediated immune recognition. Recent work has revealed that the altered tumor metabolism depletes essential nutrients or leads to the accumulation of immunosuppressive metabolites in the tumor microenvironment. In this review, we discuss the suppressive activity of some metabolic key players, which are upregulated in human tumor cells, including indolamine-2,3-dioxygenase (IDO), arginase, inducible nitric oxide synthetase (iNOS), and lactate dehydrogenase (LDH)-A, on the adaptive immune system. A better understanding of the impact of metabolic alterations of tumor cells on effector T-cell functions could lead to new therapeutic strategies to improve the efficacy of cancer immunotherapy.
Assuntos
Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/imunologia , Humanos , Ácido Láctico/farmacologia , Transdução de Sinais , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Many tumor cells are characterized by a dysregulated glucose metabolism associated with increased glycolysis in the presence of oxygen ("Warburg Effect"). Here, we analyzed for the first time a possible link between glucose metabolism and immune cell infiltration in renal cell carcinoma (RCC). RCC specimens revealed a highly significant increase in the expression of lactate dehydrogenase A (LDHA) and glucose-transporter 1 (GLUT-1) compared to the corresponding normal kidney tissue on mRNA level. Accordingly, tumor cell lines of different origin such as RCC, melanoma and hepatocellular carcinoma strongly expressed LDHA and GLUT-1 compared to their nonmalignant counterparts. In line with this finding, tumor cells secreted high amounts of lactate. High expression of GLUT-1 and LDH5, a tetramer of 4 LDHA subunits, was confirmed by tissue microarray analysis of 249 RCC specimens. Overall, 55/79 (69.6%) and 46/71 (64.7%) cases of clear cell carcinoma showed a constitutive, but heterogeneous expression of GLUT-1 and LDH5, respectively. The number of CD3(+), CD8(+) and FOXP3(+) T cells was significantly elevated in RCC lesions compared to normal kidney epithelium, but effector molecules such as granzyme B and perforin were decreased in tumor infiltrating T cells. Of interest, further analysis revealed an inverse correlation between GLUT-1 expression and the number of CD8(+) T cells in RCC lesions. Together, our data suggest that an accelerated glucose metabolism in RCC tissue is associated with a low infiltration of CD8(+) effector T cells. Targeting the glucose metabolism may represent an interesting tool to improve the efficacy of specific immunotherapeutic approaches in RCC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Neoplasias Renais/imunologia , Neoplasias Renais/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fenótipo , RNA Mensageiro/análise , Análise Serial de Tecidos , Adulto JovemRESUMO
The anti-diabetic thiazolidinedione compound pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, and selective cyclooxygenase-2 inhibitors are clinically used in patients with advanced malignancies. Several previously published in vivo and in vitro studies showed growth inhibitory effects on different cancer cell lines. However, the underlying mechanisms are fairly unclear. Here, we analyzed the effects of pioglitazone in combination with other drugs in a three-dimensional multicellular tumor spheroid culture system (MCTS) generated from the two prostate carcinoma cell lines PC3 and LNCaP. As expected, pioglitazone also inhibited tumor cell proliferation in the MCTS system. Further studies revealed that pioglitazone lowered the pH of the culture medium, decreased oxygen consumption and increased lactate secretion in both tumor cell lines. Other glitazones, troglitazone and ciglitazone, had similar effects. The combination of pioglitazone with 2-deoxyglucose, a potent inhibitor of glycolysis, had an additive effect on the inhibition of cell proliferation and led to MCTS disintegration. Our data propose a new mechanism of growth inhibition by pioglitazone through modulation of the tumor cell metabolism.
Assuntos
Hipoglicemiantes/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Tiazolidinedionas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromanos/farmacologia , Desoxiglucose/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Masculino , PPAR gama/agonistas , Pioglitazona , Neoplasias da Próstata/metabolismo , Esferoides Celulares/patologia , TroglitazonaRESUMO
A considerable fraction of tumor-associated macrophages (TAM) is located in the fibroblast-rich stromal compartment of desmoplastic breast carcinoma. We analyzed the migratory activity of blood monocytes (MO), the precursor cells of TAM, into 3-D cultures of carcinoma cells and fibroblasts from breast tumor origin. MO migration into breast tumor spheroids was highly variable: Hs578T spheroids showed high MO infiltration rates, T47D cultures were intermediate, whereas BT549, BT474 and MCF-7 spheroids were poorly infiltrated. MO infiltration was also high in tumor-derived fibroblast spheroids; however, no MO subpopulation with specific infiltrative potential was identified by CD14/CD16 expression profile. The infiltration of MO could be inhibited by pre-exposure to pertussis and cholera toxins, but only pertussis toxin, which blocks G(i) protein function, entirely inhibited MO migration. The G(i) coupled CCL2 receptor CCR2A/2B was expressed on roughly all MO. Furthermore, highly infiltrated tumor-derived fibroblast and Hs578T spheroids secreted considerable amounts of CCL2. In line with this, the infiltration of MO into fibroblast spheroids was suppressed by either addition of recombinant CCL2 to disturb the CCL2 gradient or by pre-incubation of MO with a CCR2A/2B blocking antibody. MO infiltration of Hs578T spheroids, however, could not be inhibited by CCL2 receptor blockade. Our study clearly shows that the CCL2-CCR2A/2B pathway is crucial for the recruitment of blood MO into tumor fibroblastic areas, whereas additional factors may be relevant for the migration of MO into tumor cell sites.
Assuntos
Neoplasias da Mama/imunologia , Quimiocina CCL2/metabolismo , Fibroblastos/metabolismo , Monócitos/metabolismo , Receptores CCR2/imunologia , Anticorpos Bloqueadores/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Quimiocina CCL2/imunologia , Toxina da Cólera/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Monócitos/efeitos dos fármacos , Monócitos/patologia , Toxina Pertussis/farmacologia , Transdução de Sinais/imunologia , Esferoides Celulares/patologiaRESUMO
High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (< or = 10 mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying the suppression. Uptake of [3-(13)C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-(13)C(2)]glucose to the culture medium and measured glucose uptake and conversion into [2,3-(13)C(2)]lactate. Activation of monocytes increased the glycolytic flux and the secretion of lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-(13)C(2)]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased intracellular [2,3-(13)C(2)]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the tumor environment.
Assuntos
Acidose Láctica/metabolismo , Ácido Láctico/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Acidose Láctica/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Metabolismo Energético/imunologia , Glicólise/imunologia , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Humanos , Imunossupressores/metabolismo , Imunossupressores/toxicidade , Ácido Láctico/toxicidade , Melanoma/imunologia , Melanoma/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Krüppel-like factor 5 (KLF5) is a transcription factor involved in cell transformation, proliferation, and carcinogenesis that can be up-regulated by RAS mutations. However, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Because KRAS is frequently mutated in pancreatic cancer, we investigated the regulation of KLF5 in this cancer entity. Our results show that KLF5 is overexpressed in pancreatic cancer cells and exceeds KLF5 expression of KRAS-mutated colon cancer cells. Surprisingly, inhibition of B-Raf/C-Raf or MAPK/Erk did not reduce KLF5 levels, suggesting that KLF5 expression is not promoted by KRAS-Raf-MEK-Erk signaling in pancreatic cancer. This finding is in striking contrast to reports on MEK-Erk-mediated KLF5 induction in colon cancer cells. Moreover, KLF5 expression levels neither correlated with the mutational status of KRAS nor with MEK phosphorylation in pancreatic cancer cells. Importantly, KLF5 was significantly up-regulated by interleukin (IL)-1beta or hypoxia. The IL-1 beta-mediated induction of KLF5 was diminished by blocking the p38 pathway. In addition, blocking IL-1R reduced the constitutive KLF5 expression, suggesting an autocrine activation loop. Moreover, KLF5 coimmunoprecipitated with hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-1alpha(siRNA) reduced constitutive KLF5. Similarly, KLF5(siRNA) reduced the expression of the HIF-1alpha target gene GLUT-1. Furthermore, KLF5 expression was significantly elevated by high cell density, by anchorage-independent cell growth, and in tumor spheroids. Down-regulation of KLF5 by RNAi reduced the expression of the target genes, survivin, and platelet-derived growth factor-A. In conclusion, overexpression of KLF5 in human pancreatic cancer cells is not mediated by KRAS/Raf/MAPK/Erk signaling, but involves the IL-1beta/IL-1R system, p38, and the transcription factor HIF-1alpha.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-1beta/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pancreáticas/genética , Transdução de Sinais , Regulação para Cima/genética , Contagem de Células , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Interleucina-1beta/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Dendritic cells (DC) are specialized antigen presenting cells that acquire, process, and present tumor-associated antigens to T cells for the induction of antigen-specific tumor immune responses. DC have been shown to infiltrate many tumors but both, circulating and tumor-infiltrating DC from cancer patients, appear to be phenotypically and functionally defective. Several tumor-derived factors such as VEGF, IL-6, IL-10, M-CSF, and STAT-3 have been shown to be responsible for systemic and local DC defects. Furthermore, tumor metabolites such as lactic acid may also critically contribute to DC dysfunction and tumor immune escape. The correction of abnormal DC function might be a requirement for successful vaccine approaches against cancer.
Assuntos
Células Dendríticas/fisiologia , Neoplasias/fisiopatologia , Animais , Diferenciação Celular , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Hipóxia/fisiopatologia , Ácido Láctico/metabolismo , Linfócitos do Interstício Tumoral/fisiologia , Neoplasias/patologia , Prostaglandinas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
A characteristic feature of tumors is high production of lactic acid due to enhanced glycolysis. Here, we show a positive correlation between lactate serum levels and tumor burden in cancer patients and examine the influence of lactic acid on immune functions in vitro. Lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity. A 24-hour recovery period in lactic acid-free medium restored CTL function. CTLs infiltrating lactic acid-producing multicellular tumor spheroids showed a reduced cytokine production. Pretreatment of tumor spheroids with an inhibitor of lactic acid production prevented this effect. Activated T cells themselves use glycolysis and rely on the efficient secretion of lactic acid, as its intracellular accumulation disturbs their metabolism. Export by monocarboxylate transporter-1 (MCT-1) depends on a gradient between cytoplasmic and extracellular lactic acid concentrations and consequently, blockade of MCT-1 resulted in impaired CTL function. We conclude that high lactic acid concentrations in the tumor environment block lactic acid export in T cells, thereby disturbing their metabolism and function. These findings suggest that targeting this metabolic pathway in tumors is a promising strategy to enhance tumor immunogenicity.