RESUMEN
PURPOSE: The precise determination of energy metabolites is challenged by the heterogeneity of their distribution, their rapid changes after surgical resection and the architectural complexity of malignancies. Induced metabolic bioluminescence imaging (imBI) allows to determine energy metabolites in tissue sections and to co-localize these with histological structures based on consecutive sections stained with HE. In this prospective pilot study patients with suspected advanced ovarian cancer (OC) were enrolled to prove the feasibility of imBI. METHODS: During surgery, suspicious peritoneal metastases were resected and transferred in liquid nitrogen within 30 s. ATP, glucose and lactate concentrations were measured. Furthermore, the expression of monocarboxylate transporters MCT1 and MCT4 was determined by immunofluorescence staining. RESULTS: 16 patients were screened, 12 entered the study. Final histological assessment revealed ten malignant and two benign peritoneal lesions. In all 12 cases high concentrations of ATP suggested that energy metabolism was not altered by the surgical and transport procedures (mean 0.56 µmol/g, range 0.24-1.21 µmol/g). The mean concentration of glucose was 1.95 µmol/g (range 0.58-4.71 µmol/g). The concentration of lactate was drastically higher in the ten OC cases (mean 24.79 µmol/g, range 17.51-37.16 µmol/g) compared to the benign samples (mean 5.98 µmol/g, range 5.43-6.54 µmol/g). Lactate concentrations seem to correlate with MCT1 (spearman rank correlation ρ = 0.624, 0.05 > p > 0.025), but not with MCT4 (spearman rank correlation ρ = 0.018, p > 0.1). CONCLUSIONS: ImBI is feasible in peritoneal metastases of OC and encourages further effort to elucidate the role of glucose, lactate, MCT1 and MCT4 in OC.
Asunto(s)
Mediciones Luminiscentes , Imagen Molecular/métodos , Neoplasias Glandulares y Epiteliales/diagnóstico por imagen , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/patología , Neoplasias Peritoneales/diagnóstico por imagen , Neoplasias Peritoneales/secundario , Anciano , Anciano de 80 o más Años , Carcinoma Epitelial de Ovario , Progresión de la Enfermedad , Metabolismo Energético , Estudios de Factibilidad , Femenino , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Persona de Mediana Edad , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Proyectos Piloto , Estudios Prospectivos , Simportadores/metabolismoRESUMEN
The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with ß1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and ß1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of ß1-integrin and decreased ß1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with ß1-integrin mediating integrin internalization and thus correlates with downstream signaling of ß1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.
Asunto(s)
Movimiento Celular , Endocitosis , Integrina beta1/metabolismo , Receptores de LDL/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Adhesión Celular , Modelos Animales de Enfermedad , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Proteínas Supresoras de Tumor/deficienciaRESUMEN
BACKGROUND: Malignant transformation of cells is often accompanied by up-regulation of glycolysis-related enzymes and transporters, as well as a distortion of mitochondrial respiration. As a consequence, most malignant tumors utilize high amounts of glucose and produce and accumulate high concentrations of lactate, even in the presence of oxygen. This phenomenon has been termed 'Warburg Effect'. Here, we aimed at resolving the interrelation between tumor metabolism, reactive oxygen species, double strand DNA breaks and radio-resistance in ovarian cancer-derived cells. METHODS: As a model system two ovarian cancer-derived cell lines, OC316 and IGROV-1, and its corresponding xenografts were used. First, the metabolic properties of the xenografts were tested to ensure that initial in vitro data might later be transferred to in vivo data. In parallel, three inhibitors of tumor cell metabolism, 2-deoxy-D-glucose, an inhibitor of glycolysis, oxamate, a pyruvate analogue and inhibitor of lactate dehydrogenase, and rotenone, a specific inhibitor of mitochondrial electron complex I, were tested for their effect on the metabolism and radio-sensitivity of the respective ovarian cancer-derived cell lines. RESULTS: We found that all three inhibitors tested led to significant changes in the tumor cell energy metabolism at non-cytotoxic concentrations. Furthermore, we found that inhibition of tumor glycolysis by 2-deoxy-D-glucose in combination with rotenone decreased the radio-resistance at a clinically relevant radiation dose. This apparent radio-sensitizing effect appears to be based on an increased level of double strand DNA breaks 1 h and 24 h after gamma irradiation. Both cancer-derived cell lines maintained their metabolic properties, as well as their protein expression profiles and levels of reactive oxygen species in xenografts, thus providing a suitable model system for further in vivo investigations. CONCLUSION: A combination of metabolic inhibitors and reactive oxygen species-generating therapies, such as irradiation, may effectively enhance the therapeutic response in particularly metabolically highly active (ovarian) tumors.
Asunto(s)
Glucosa/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/terapia , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quimioradioterapia/métodos , Daño del ADN , Desoxiglucosa/farmacología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Femenino , Rayos gamma , Glucólisis/efectos de los fármacos , Glucólisis/efectos de la radiación , Humanos , Ratones SCID , Microscopía Fluorescente , Neoplasias Ováricas/genética , Ácido Oxámico/farmacología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/efectos de la radiación , Rotenona/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown earlier to impair the penetration of dendritic cells in a tumor spheroid model, which is contrary to the response of the malignant cell population in the present study, we included blood monocytes in our assay as a highly motile immune cell type and precursor of tumor-associated macrophages. Interestingly, high levels of L-lactate (20 mM) at a pH of 7.4 inhibited monocyte migration in the Boyden chamber system. In addition, cytokine release of TNF and IL-6 was inhibited. The obtained data suggest that high lactate content promotes tumor progression by contributing to the phenomenon of tumor immune escape and by enhancing the migratory potential of the malignant cell population which may directly be coupled to a higher incidence of metastasis.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Ácido Láctico/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Cadenas beta de Integrinas/metabolismoRESUMEN
Metastasis and recurrences are major problems regarding an effective treatment of solid malignant tumors in clinical oncology. Since the impact of radiation on cell motility is not yet well understood, intrinsic and radiation-induced changes in cell migration have been discussed as possible mechanisms involved in the limitations of radiotherapy. This holds true for conventional radiation treatment and even more for the cellular and molecular effects of therapeutically relevant (12)C heavy ions. The present study is therefore focused on the investigation of tumor cell migration in vitro after irradiation with X-rays and (12)C heavy ions and on radiation-induced changes in the expression of proteins that are potentially relevant for motility. Two colon carcinoma cell lines, HCT116 and HCT116 p21-/-, were chosen for this study, which should be isogenic except for their p21-status. We can show here that cells lacking p21 react almost alike to radiation as wild type cells regarding survival and tumor cell migration 24 h after irradiation. Interestingly, differences in protein expression 24 h after irradiation of beta(1) integrin and protein kinase B isoforms Akt1 and Akt2 seem to exist. We conclude that tumor cell migration is unaffected by the p21-status in colorectal carcinoma cells and that the expression of the aforementioned proteins alone is not accountable for the differences observed.