RESUMO
BACKGROUND: Inhibition of kinases is the ever-expanding therapeutic approach to various types of cancer. Typically, assessment of the treatment response is accomplished by standard, volumetric imaging procedures, performed weeks to months after the onset of treatment, given the predominantly cytostatic nature of the kinase inhibitors, at least when used as single agents. Therefore, there is a great clinical need to develop new monitoring approaches to detect the response to kinase inhibition much more promptly. Noninvasive 1H magnetic resonance spectroscopy (MRS) can measure in vitro and in vivo concentration of key metabolites which may potentially serve as biomarkers of response to kinase inhibition. METHODS: We employed mantle cell lymphoma (MCL) cell lines demonstrating markedly diverse sensitivity of inhibition of Bruton's tyrosine kinase (BTK) regarding their growth and studied in-depth effects of the inhibition on various aspects of cell metabolism including metabolite synthesis using metabolomics, glucose and oxidative metabolism by Seahorse XF technology, and concentration of index metabolites lactate, alanine, total choline and taurine by 1H MRS. RESULTS: Effective BTK inhibition profoundly suppressed key cell metabolic pathways, foremost pyrimidine and purine synthesis, the citrate (TCA) cycle, glycolysis, and pyruvate and glutamine/alanine metabolism. It also inhibited glycolysis and amino acid-related oxidative metabolism. Finally, it profoundly and quickly decreased concentration of lactate (a product of mainly glycolysis) and alanine (an indicator of amino acid metabolism) and, less universally total choline both in vitro and in vivo, in the MCL xenotransplant model. The decrease correlated directly with the degree of inhibition of lymphoma cell expansion and tumor growth. CONCLUSIONS: Our results indicate that BTK inhibition exerts a broad and profound suppressive effect on cell metabolism and that the affected index metabolites such as lactate, alanine may serve as early, sensitive, and reliable biomarkers of inhibition in lymphoma patients detectable by noninvasive MRS-based imaging method. This kind of imaging-based detection may also be applicable to other kinase inhibitors, as well as diverse lymphoid and non-lymphoid malignancies.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Linfoma de Célula do Manto , Inibidores de Proteínas Quinases , Humanos , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Animais , Tirosina Quinase da Agamaglobulinemia/metabolismo , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Biomarcadores/metabolismoRESUMO
Bonded cumomers are sets of isotopomers of 13 C-labeled metabolites containing a particular sequence of contiguously or singly labeled carbon atoms. Only these isotopomers contribute to multiplet structure in the 13 C NMR spectrum. We discuss the application of this technique to the study of quantitative tumor metabolism, bioenergetics, and the Warburg effect. The advantages and sensitivity of bonded cumomer analysis over positional enrichment analysis are discussed. When sensitivity requirements are met, bonded cumomer analysis enables the extraction of fluxes through specific metabolic pathways with higher precision. In conjunction with isotopomer control analysis, we evaluate the sensitivity of experimentally measurable metabolite multiplets to determine the robustness of flux analysis in 13 C spectra of tumors. This review examines the role of glycolytic and tricarboxylic acid cycle metabolism with special emphasis on flux through the pentose phosphate pathway (PPP). The impact of reversibility of the nonoxidative branch of the PPP with various 13 C glucose tracers on fine-structure multiplets is analyzed.
Assuntos
Modelos Biológicos , Neoplasias , Humanos , Espectroscopia de Ressonância Magnética/métodos , Metabolismo Energético , Ciclo do Ácido Cítrico , Glucose/metabolismo , Isótopos de Carbono/metabolismoRESUMO
The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.
Assuntos
Antineoplásicos/farmacologia , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Indazóis/farmacologia , Mitocôndrias/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Diacetil/análogos & derivados , Diacetil/farmacologia , Complexo II de Transporte de Elétrons/metabolismo , Fumaratos/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , Humanos , Malatos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Análise do Fluxo Metabólico , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , NADP/metabolismo , Naftalenos/farmacologia , Oxirredução/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/metabolismoRESUMO
A network model for the determination of tumor metabolic fluxes from (13)C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was â¼ 50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was â¼ 6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.
Assuntos
Melanoma/metabolismo , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Modelos Teóricos , Mutação de Sentido Incorreto , Oxigênio/metabolismo , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, and photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells. It has been reported to alter the bioenergetics of tumor cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggested that it also inhibited l-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Recent studies have demonstrated that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5µM) and cooperatively inhibits l-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill coefficient values of 36-40µM and 1.65-1.85, respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~7µM) than other substrates including glutamate (IC50~20µM). LND inhibits the succinate-ubiquinone reductase activity of respiratory Complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through Complex II and has been reported to promote cell death by suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated l-lactic acid efflux, Complex II and glutamine/glutamate oxidation.
Assuntos
Antineoplásicos/farmacologia , Indazóis/farmacologia , Animais , Hexoquinase/antagonistas & inibidores , Humanos , Concentração de Íons de Hidrogênio , Indazóis/toxicidade , Proteínas de Membrana Transportadoras/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki2.5 µM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevisoocytes with K0.5 and Hill coefficient values of 36-40 µM and 1.65-1.85 respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~ 7 µM) than other substrates including glutamate (IC50~ 20 µM). In isolated DB-1 melanoma cells 1-10 µM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 µM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND onL-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC).
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Membrana Celular/metabolismo , Indazóis/farmacologia , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Ácido Láctico/metabolismo , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/metabolismo , Ratos , Ratos Wistar , Proteínas Carreadoras de Solutos , Simportadores/genética , Simportadores/metabolismoRESUMO
RATIONALE: Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role. METHODS: An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity. RESULTS: HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells. CONCLUSIONS: A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.
Assuntos
Ácidos Carboxílicos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Etilaminas/química , Fosfatos/metabolismo , Propanóis/química , Ácidos Carboxílicos/análise , Linhagem Celular Tumoral , Técnicas Citológicas , Humanos , Redes e Vias Metabólicas , Fosfatos/análiseRESUMO
We seek to exploit the natural tendency of melanomas and other tumors to convert glucose to lactate as a method for the selective intracellular acidification of cancer cells and for the potentiation of the activity of nitrogen-mustard antineoplastic agents. We performed this study to evaluate whether the induction of hyperglycemia (26 mM) could enhance the effects of lonidamine (LND, 100 mg/kg; intraperitoneally) on the induction of intracellular acidification, bioenergetic decline and potentiation of the activity of melphalan (LPAM) against DB-1 melanoma xenografts in mice. Intracellular pH (pHi ), extracellular pH (pHe ) and bioenergetics (ß-nucleoside triphosphate to inorganic phosphate ratio, ß-NTP/Pi) were reduced by 0.7 units (p < 0.001), 0.3 units (p > 0.05) and 51.4% (p < 0.05), respectively. The therapeutic response to LPAM (7.5 mg/kg; intravenously) + LND (100 mg/kg; intraperitoneally) was reduced by about a factor of three under hyperglycemic conditions relative to normoglycemia, producing a growth delay of 7.76 days (tumor doubling time, 5.31 days; cell kill, 64%) compared with LND alone of 1.70 days and LPAM alone of 0.29 days. Under normoglycemic conditions, LND plus LPAM produced a growth delay of 17.75 days, corresponding to a cell kill of 90% at the same dose for each of these agents. The decrease in tumor cell kill under hyperglycemic conditions correlates with an increase in tumor ATP levels resulting from increased glycolytic activity. However, hyperglycemia substantially increases lactic acid production in tumors by a factor of approximately six (p < 0.05), but hyperglycemia did not increase the effects of LND on acidification of the tumor, most probably because of the strong buffering action of carbon dioxide (the pKa of carbonic acid is 6.4). Therefore, this study demonstrates that the addition of glucose during treatment with LND diminishes the activity of this agent.
Assuntos
Ácidos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Hiperglicemia/complicações , Indazóis/farmacologia , Melanoma/metabolismo , Melfalan/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Melanoma/complicações , Melanoma/patologia , Camundongos Nus , Especificidade de ÓrgãosRESUMO
We demonstrate that the effects of lonidamine (LND, 100 mg/kg, i.p.) are similar for a number of xenograft models of human cancer including DB-1 melanoma and HCC1806 breast, BT-474 breast, LNCaP prostate and A2870 ovarian carcinomas. Following treatment with LND, each of these tumors exhibits a rapid decrease in intracellular pH, a small decrease in extracellular pH, a concomitant monotonic decrease in nucleoside triphosphate and an increase in inorganic phosphate over a 2-3 h period. We have previously demonstrated that selective intracellular tumor acidification potentiates response of this melanoma model to melphalan (7.5 mg/kg, i.v.), producing an estimated 89% cell kill based on tumor growth delay analysis. We now show that, in both DB-1 melanoma and HCC1806 breast carcinoma, LND potentiates response to doxorubicin, producing 95% cell kill in DB-1 melanoma at 7.5 mg/kg, i.v. doxorubicin and 98% cell kill at 10.0 mg/kg doxorubicin, and producing a 95% cell kill in HCC1806 breast carcinoma at 12.0 mg/kg doxorubicin. Potentiation of doxorubicin may result from cation trapping of the weakly basic anthracycline. Recent experience with the clinical treatment of melanoma and other forms of human cancer suggests that these diseases will probably not be cured by a single therapeutic procedure other than surgery. A multimodality therapeutic approach will be required. As a potent modulator of tumor response to N-mustards and anthracyclines as well as tumor thermo- and radiosensitivity, LND promises to play an important clinical role in the management and possible complete local control of a number of prevalent forms of human cancer.
Assuntos
Trifosfato de Adenosina/deficiência , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Indazóis/farmacologia , Neoplasias Ovarianas/metabolismo , Neoplasias da Próstata/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Ácidos/metabolismo , Animais , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/patologia , Simportadores/metabolismoRESUMO
Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer related mutations in HCC, integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. messenger RNA, mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry based metabolomics analysis of stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compared with non-tumor liver (p value < 0.0001). In addition, branched-chain amino acid metabolism (p value < 0.0001) and tricarboxylic acid metabolism (p value < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p value = 0.0013), succinate (p value = 0.0001), lactate (p value = 0.0114), glycerophosphoethanolamine (p value = 0.015), and inorganic phosphate (p value = 0.0001). However, (13) C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a (13) C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via (13) C labeling, to investigate targeted therapeutics against these enzymes, and to optimize the in vivo performance of (13) C MRI techniques.
Assuntos
Ácido Aspártico/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Redes e Vias Metabólicas , Metabolômica , Transcriptoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alanina/metabolismo , Isótopos de Carbono , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Ácido Láctico/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Pessoa de Meia-Idade , Fosfatos/metabolismo , Fosfatidiletanolaminas/metabolismo , Análise de Componente Principal , Ácido Succínico/metabolismoRESUMO
Dabrafenib therapy for metastatic melanoma focuses on blocking growth-promoting signals produced by a hyperactive BRAF protein. We report the metabolic differences of four human melanoma cell lines with diverse responses to dabrafenib therapy (30 mg/kg; oral): WM3918 < WM9838BR < WM983B < DB-1. Our goal was to determine if metabolic changes produced by the altered signaling pathway due to BRAF mutations differ in the melanoma models and whether these differences correlate with response to treatment. We assessed metabolic changes in isolated cells using high-resolution proton magnetic resonance spectroscopy (1H MRS) and supplementary biochemical assays. We also noninvasively studied mouse xenografts using proton and phosphorus (1H/31P) MRS. We found consistent changes in lactate and alanine, either in isolated cells or mouse xenografts, correlating with their relative dabrafenib responsiveness. In xenografts, we also observed that a more significant response to dabrafenib correlated with higher bioenergetics (i.e., increased ßNTP/Pi). Notably, our noninvasive assessment of the metabolic status of the human melanoma xenografts by 1H/31P MRS demonstrated early metabolite changes preceding therapy response (i.e., tumor shrinkage). Therefore, this noninvasive methodology could be translated to assess in vivo predictive metabolic biomarkers of response in melanoma patients under dabrafenib and probably other signaling inhibition therapies.
RESUMO
The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady-state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or (18) F-fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin-sensitive mTOR lies downstream of many oncogenic signaling pathways.
Assuntos
Glicólise/efeitos dos fármacos , Ácido Láctico/metabolismo , Linfoma de Células B/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Camundongos , Camundongos SCIDRESUMO
In vivo (31) P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg intraperitoneally) exhibit a decrease in intracellular pH (pH(i) ) from 6.90 ± 0.05 to 6.33 ± 0.10 (p < 0.001), a slight decrease in extracellular pH (pH(e) ) from 7.00 ± 0.04 to 6.80 ± 0.07 (p > 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p < 0.001) relative to the baseline level. Both bioenergetics and pH(i) decreases were sustained for at least 3 h following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p > 0.05) at 20 min post-LND, with no significant change in pH(e) and a small transient decrease in bioenergetics (32.9 ± 10.6%, p > 0.05) at 40 min post-LND. No changes in pH(i) or adenosine triphosphate/inorganic phosphate were detected in the brain (pH(i) , bioenergetics; p > 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p > 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by (1) H MRS with a selective multiquantum pulse sequence with Hadamard localization increased approximately three-fold (p = 0.009). Treatment with LND increased the systemic melanoma response to melphalan (LPAM; 7.5 mg/kg intravenously), producing a growth delay of 19.9 ± 2.0 days (tumor doubling time, 6.15 ± 0.31 days; log(10) cell kill, 0.975 ± 0.110; cell kill, 89.4 ± 2.2%) compared with LND alone of 1.1 ± 0.1 days and LPAM alone of 4.0 ± 0.0 days. The study demonstrates that the effects of LND on tumor pH(i) and bioenergetics may sensitize melanoma to pH-dependent therapeutics, such as chemotherapy with alkylating agents or hyperthermia.
Assuntos
Concentração de Íons de Hidrogênio/efeitos dos fármacos , Indazóis/administração & dosagem , Espectroscopia de Ressonância Magnética/métodos , Melanoma/química , Melanoma/tratamento farmacológico , Melfalan/administração & dosagem , Animais , Antineoplásicos Alquilantes , Linhagem Celular Tumoral , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Melanoma/fisiopatologia , Camundongos , Radioisótopos de Fósforo , Prótons , Resultado do TratamentoRESUMO
PURPOSE: To examine the effect of transvascular (k(be)) and cellular-interstitial water protons exchange (k(ie)) in estimates of the blood-to-tissue contrast agent transfer rate constant (K(trans)), interstitial volume fraction (v(e)), and blood plasma volume fraction (v(p)) using a full three-site two exchange (3S2X) model. MATERIALS AND METHODS: Using the Bloch-McConnell equations, magnetic resonance imaging (MRI) signal arising from a 3S2X system was derived for the T(1)-weighted spoiled gradient-recalled echo (SPGR) pulse sequence. To model the effects of k(be) and k(ie) on estimates of R(1), the MRI-measured arterial input function, the different sets of values of kinetic parameters: K(trans), v(e), and v(p) and water protons exchange rates, k(be) and k(ie), were used. To calculate the tissue water protons R(1), the signal evolving from a 3S2X model was set to a monoexponential function. By comparing the estimated K(trans), v(e), and v(p) with their simulated model truth values, the impact of k(be) and k(ie) on K(trans), v(e), and v(p) was evaluated. RESULTS: v(p) was strongly underestimated and K(trans) and v(e) were much less influenced by k(be), when k(ie) was held constant. When k(be) was held constant, the k(ie) had a significant effect on K(trans) and v(e) and less effect on v(p). CONCLUSION: The full 3S2X model allows accurate estimation of K(trans), v(e), and v(p) and rates of water proton exchange.
Assuntos
Artérias/metabolismo , Determinação do Volume Sanguíneo/métodos , Água Corporal/metabolismo , Gadolínio DTPA/farmacocinética , Imageamento por Ressonância Magnética/métodos , Modelos Cardiovasculares , Simulação por Computador , Meios de Contraste/farmacocinética , Humanos , Volume PlasmáticoRESUMO
Drug treatment may alter the metabolism of cancer cells and may alter the mitochondrial redox state. Using the redox scanner that collects the fluorescence signals from both the oxidized flavoproteins (Fp) and the reduced form of nicotinamide adenine dinucleotide (NADH) in snap-frozen tumor tissues, we investigated the effects of chemotherapy on mouse xenografts of a human diffuse large B-cell lymphoma cell line (DLCL2). The mice in the treatment group were treated with CHOP - cyclophosphamide (C) + hydroxydoxorubicin (H) + Oncovin (O) + prednisone (P) using the following regimen: CHO administration on day 1 followed by prednisone administration on day 1-5. On day 5 the mitochondrial redox state of the treated group was slightly more reduced than that of the control group (p = 0.049), and the Fp content of the treated group was significantly decreased (p = 0.033).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NAD/metabolismo , Oxirredução , Prednisona/administração & dosagem , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lonidamine (LND) is an anti-cancer drug with great potential as a metabolic modulator of chemotherapy, radiotherapy, hyperthermia, and photodynamic therapy in cancer treatment. LND affects several important aspects of cancer cell metabolism: it inhibits Complex I and II of the electron transport chain (ETC) and pyruvate carriers (mitochondrial), and monocarboxylate transporters in the plasma membrane of the cell. Cancer cells are affected by changes in pH on the molecular level, and so are the drugs used to treat cancer, thus it is important to understand how pH affects their structures and LND is no exception. LND dissolves at a pH of 8.3 in tris-glycine buffer but has limited solubility at pH 7. To understand how pH affects the structure of LND, and its effect as a metabolic modulator on cancer therapy, we made up samples of LND at pH 2, pH 7, and pH 13, and analyzed these samples using 1H and 13C NMR. We looked for ionization sites to explain the behavior of LND in solution. Our results showed considerable chemical shifts between the extremes of our experimental pH range. LND was ionized at its indazole α-nitrogen, however, we did not directly observe the protonation of the carboxyl group oxygen that is expected at pH 2, which may be the result of a chemical-exchange phenomenon.
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As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become "addicted" may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically "starve" oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.
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Linfoma Anaplásico de Células Grandes , Receptores Proteína Tirosina Quinases , Humanos , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Transdução de Sinais , Proteínas Nucleares/genética , Linhagem Celular TumoralRESUMO
Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T(1(rho))-MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutter-speed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, K(trans), that measures vascular perfusion/permeability. K(trans) was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T(1(rho)) values that were significantly higher than those of C8161 melanoma. Measurements of T(1) and T(2) relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp + NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential.
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Biomarcadores Tumorais/metabolismo , Diagnóstico por Imagem , Imageamento por Ressonância Magnética , Melanoma/metabolismo , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Meios de Contraste , Humanos , Camundongos , Mitocôndrias/metabolismo , Metástase Neoplásica , Oxirredução , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
1H magnetic resonance spectroscopy (MRS) with the multiple quantum coherence (MQC) technique allows for the detection of lactate, an end product of glycolysis, in the environment of lipids. The method can also be used to detect alanine, a byproduct of glutaminolysis. An issue is that when both lactate and alanine are detected together by the MQC technique, a phase mismatch arises between lactate and alanine signals due to off-resonance rotations and the difference in double quantum coherence frequencies between the two molecules. Such phase mismatch can cause errors in spectral fitting and metabolite quantification. In this study, we designed two pulse sequences that eliminate such phase differences of lactate and alanine while suppressing lipid signals by modifications of the Selective Multiple Quantum Coherence (Sel-MQC) sequence, a well-known MQC technique. Using the product operator formalism and the off-resonance rotation matrices, the phase evolutions of lactate and alanine during the spectrally selective pulses and the free precession times of the sequence at the single quantum, double quantum and zero quantum coherence states of these molecules were calculated. The multiple quantum (MQ) evolution time t1 that can remove the phase difference of lactate and alanine at the echo was calculated and fine-tuned with experiments. The lactate and alanine signal intensities and the editing efficiencies from the two modified Sel-MQC sequences were theoretically predicted by using the product operator evolutions and compared with the experimental data. The J-coupled lipid signals were successfully suppressed by both sequences. One of the two developed sequences was applied to a human body with a phantom of lactate and alanine, which resulted in successful in-phase editing of lactate and alanine and suppression of the lipid signals from the body. The study sets an important foundation for the noninvasive detection of lactate and alanine from tumors of cancer patients.
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Alanina , Ácido Láctico , Humanos , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/métodos , Lipídeos/químicaRESUMO
Objective.The selective multiple quantum coherence (Sel-MQC) sequence is a magnetic resonance spectroscopy (MRS) technique used to detect lactate and suppress co-resonant lipid signalsin vivo. The coherence pathways of J-coupled lipids upon the sequence, however, have not been studied, hindering a logical design of the sequence to fully attenuate lipid signals. The objective of this study is to elucidate the coherence pathways of J-coupled lipids upon the Sel-MQC sequence and find a strategy to effectively suppress lipid signals from these pathways while keeping the lactate signal.Approach.The product operator formalism was used to express the evolutions of the J-coupled spins of lipids and lactate. The transformations of the product operators by the spectrally selective pulses of the sequence were calculated by using the off-resonance rotation matrices. The coherence pathways and the conversion rates of the individual pathways were derived from them. Experiments were performed on phantoms and two human subjects at 3 T.Main results.The coherence pathways contributing to the various lipid resonance signals by the Sel-MQC sequence depending on the gradient ratios and RF pulse lengths were identified. Theoretical calculations of the signals from the determined coherence pathways and signal attenuations by gradients matched the experimental data very well. Lipid signals from fatty tissues of the subjects were successfully suppressed to the noise level by using the gradient ratio -0.8:-1:2 or 1:0.8:2. The new gradient ratios kept the lactate signal the same as with the previously used gradient ratio 0:-1:2.Significance.The study has elucidated the coherence pathways of J-coupled lipids upon the Sel-MQC sequence and demonstrated how lipid signals can be effectively suppressed while keeping lactate signals by using information from the coherence pathway analysis. The findings enable applying the Sel-MQC sequence to lactate detection in an environment of high concentrations of lipids.