RESUMEN
Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.
Asunto(s)
Adhesión Bacteriana/fisiología , Colon/metabolismo , Escherichia coli Enterohemorrágica/fisiología , Infecciones por Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Anticuerpos de Dominio Único/farmacología , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana/efectos de los fármacos , Sitios de Unión , Camelus , Colon/microbiología , Colon/patología , Escherichia coli Enterohemorrágica/efectos de los fármacos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Homología de Secuencia , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.
Asunto(s)
L-Lactato Deshidrogenasa/genética , Adenocarcinoma , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Melanoma , Ratones , Fosforilación Oxidativa , Piridonas/farmacología , Tiofenos/farmacologíaRESUMEN
Glycolysis is the main route for energy production in tumors. LDH-A is a key enzyme of this process and its inhibition represents an attractive strategy to hamper cancer cell metabolism. Galloflavin is a reliable LDH-A inhibitor as previously identified by us; however, its poor physicochemical properties and chemical tractability render it unsuitable for further development. Therefore, a rational design was undertaken with the aim to reproduce the pharmacophore of galloflavin on simpler, potentially more soluble and synthetic accessible scaffolds. Following a process of structural simplification, natural urolithin M6 (UM6), which is an ellagitannin metabolite produced by gut microbiota, was identified as a putative galloflavin mimetic. In the present study, the synthesis of UM6 is described for the first time. An efficient synthetic pathway has been developed, which involved five steps from readily accessible starting materials. The key reaction steps, a Suzuki coupling and an intramolecular C-H oxygenation, have been optimized to improve the synthetic feasibility and provide the best conditions in terms of reaction time and yield. Moreover, this route would be suitable to obtain other analogs for SAR studies. Preliminary biological tests revealed that UM6 was able to smoothly reproduce the behavior of galloflavin, confirming that our approach was successful in providing a new and accessible structure in the search for new LDH-A inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Isocumarinas/química , Isocumarinas/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Inhibidores Enzimáticos/síntesis química , Humanos , Isocumarinas/síntesis química , Isoenzimas/antagonistas & inhibidores , Lactato Deshidrogenasa 5 , Ácido Láctico/biosíntesisRESUMEN
mTOR kinase and the A isoform of lactate dehydrogenase (LDH-A) are key players controlling the metabolic characteristics of cancer cells. By using cultured human breast cells as a "metabolic tumor" model, we attempted to explore the correlation between these two factors. "Metabolic tumors" are defined as neoplastic conditions frequently associated with features of the metabolic syndrome, such as hyper-insulinemia and hyper-glycemia. MCF-7 cells (a well differentiated carcinoma) and MCF-10A cells (a widely used model for studying normal breast cell transformation) were used in this study. These cells were exposed to known factors triggering mTOR activation. In both treated cultures, we evaluated the link between mTOR kinase activity and the level of LDH expression / function. Furthermore, we elaborated the metabolic changes produced in cells by the mTOR-directed LDH-A up-regulation. Interestingly, we observed that in the non-neoplastic MCF-10A culture, mTOR-directed up-regulation of LDH-A was followed by a reprogramming of cell metabolism, which showed an increased dependence on glycolysis rather than on oxidative reactions. As a consequence, lactate production appeared to be enhanced and cells began to display increased self-renewal and clonogenic power: signals suggestive of neoplastic change. Enhanced clonogenicity of cells was abolished by rapamycin treatment, and furthermore heavily reduced by LDH enzymatic inhibition. These results highlighted a mechanistic link between metabolic alterations and tumorigenesis, whereby suggesting LDH inhibition as a possible chemo-preventive measure to target the metabolic alterations driving neoplastic change.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , L-Lactato Deshidrogenasa/genética , Serina-Treonina Quinasas TOR/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Proliferación Celular/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glucólisis/genética , Humanos , Isoenzimas/genética , Lactato Deshidrogenasa 5 , Células MCF-7 , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Fosforilación , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The inflammatory microenvironment is an essential component of neoplastic lesions and can significantly impact on tumor progression. Besides facilitating invasive growth, inflammatory cytokines were also found to reprogram cancer cell metabolism and to induce aerobic glycolysis. Previous studies did not consider the possible contribution played in these changes by lactate dehydrogenase (LDH). The A isoform of LDH (LDH-A) is the master regulator of aerobic glycolysis; it actively reduces pyruvate and causes enhanced lactate levels in tumor tissues. In cancer cells, lactate was recently found to directly increase migration ability; moreover, when released in the microenvironment, it can facilitate matrix remodeling. In this paper, we illustrate that treatment of human colon adenocarcinoma cells with TNF-α and IL-17, two pro-inflammatory cytokines, modifies LDH activity, causing a shift toward the A isoform which results in increased lactate production. At the same time, the two cytokines appeared to induce features of epithelial-mesenchymal transition in the treated cells, such as reduction of E-cadherin levels and increased secretion of metalloproteinases. Noteworthy, oxamate and galloflavin, two inhibitors of LDH activity which reduce lactate production in cells, were found to relieve the inflammation-induced effects. These results suggest LDH-A and/or lactate as common elements at the cross-road between cancer cell metabolism, tumor progression and inflammation. At present, LDH inhibitors suitable for clinical use are actively searched as possible anti-proliferative agents; our data lead to hypothesize for these compounds a wider potential in anticancer treatment.
Asunto(s)
Neoplasias del Colon/metabolismo , Inflamación/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Células CACO-2 , Transición Epitelial-Mesenquimal , Humanos , Interleucina-17/farmacología , Isocumarinas/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/genética , Ácido Láctico/metabolismo , Ácido Oxámico/farmacología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In normal cells, heat shock response (HSR) is rapidly induced in response to a variety of harmful conditions and represents one of the most efficient defense mechanism. In cancer tissues, constitutive activation converts HSR into a life-threatening process, which plays a major role in helping cell survival and proliferation. Overexpression of heat shock proteins (HSPs) has been widely reported in human cancers and was found to correlate with tumor progression. Hepatocellular carcinoma is one of the conditions in which HSR activation was shown to have the highest clinical significance. Transcription of HSPs is induced by HSF-1, which also activates glycolytic metabolism and increases the expression of LDH-A, the master regulator of the Warburg effect. In this paper, we tried to explore the relationship between HSR and LDH-A. In cultured hepatocellular carcinoma cells, by using two enzyme inhibitors (oxamate and galloflavin), we found that the reduction of LDH-A activity led to decreased level and function of the major HSPs involved in tumorigenesis. Galloflavin (a polyphenol) also inhibited the ATPase activity of two of the examined HSPs. Finally, hindering HSR markedly lowered the alpha-fetoprotein cellular levels and induced senescence. Specific inhibitors of single HSPs are currently under evaluation in different neoplastic diseases. However, one of the effects usually observed during treatment is a compensatory elevation of other HSPs, which decreases treatment efficacy. Our results highlight a connection between LDH and HSR and suggest LDH inhibition as a way to globally impact on this tumor promoting process.
Asunto(s)
Respuesta al Choque Térmico , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Proteínas de Choque Térmico/genética , Humanos , Isocumarinas/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Neoplasias Hepáticas/metabolismo , Compuestos Orgánicos/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , alfa-Fetoproteínas/genéticaRESUMEN
In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.
Asunto(s)
Proteína BRCA2/antagonistas & inhibidores , Ftalazinas/farmacología , Piperazinas/farmacología , Recombinasa Rad51/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Línea Celular Tumoral , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Modelos Moleculares , Mutación , Ftalazinas/química , Piperazinas/química , Conformación Proteica , Recombinasa Rad51/metabolismoRESUMEN
Lactate dehydrogenase (LDH) inhibition is considered a worthwhile attempt in the development of innovative anticancer strategies. Unfortunately, in spite of the involvement of several research institutions and pharma-companies, the discovery of LDH inhibitors with drug-like properties seems a hardly resolvable challenge. While awaiting new advancements, in the present review we will examine other pathologic conditions characterized by increased glycolysis and LDH activity, which could potentially benefit from LDH inhibition. The rationale for targeting LDH activity in these contexts is the same justifying the LDH-based approach in anticancer therapy: because of the enzyme position at the end of glycolytic pathway, LDH inhibitors are not expected to hinder glucose metabolism of normal cells. Moreover, we will summarize the latest contributions in the discovery of enzyme inhibitors and try to glance over the reasons underlying the complexity of this research.
Asunto(s)
Antineoplásicos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Antiinflamatorios/química , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Descubrimiento de Drogas , Metabolismo Energético , Epilepsia/tratamiento farmacológico , Epilepsia/enzimología , Glucólisis , Humanos , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/inmunología , Metástasis de la Neoplasia , Neoplasias/enzimología , Oxidación-Reducción , Fenómenos Fisiológicos de los VirusRESUMEN
Up-regulation of glycolysis, a well recognized hallmark of cancer cells, was also found to be predictive of poor chemotherapy response. This observation suggested the attempt of sensitizing cancer cells to conventional chemotherapeutic agents by inhibiting glucose metabolism. Lactate dehydrogenase (LDH) inhibition can be a way to hinder glycolysis of cancer cells without affecting the metabolism of normal tissues, which usually does not require this enzymatic activity. In this paper, we showed that two LDH inhibitors (oxamate and galloflavin) can increase the efficacy of cisplatin in cultured Burkitt's lymphoma (BL) cells and that this potentiating effect is not exerted in proliferating normal lymphocytes. This result was explained by the finding that in BL cells LDH inhibition induced reactive oxygen species (ROS) generation, which was not evidenced in proliferating normal lymphocytes. In BL cells treated with the association of cisplatin and LDH inhibitors, these ROS can be a further cause of DNA damage, to be added to that produced by cisplatin, leading to the failure of the response repair. At present LDH inhibitors suitable for clinical use are actively searched; our results can allow a better understanding of the potentiality of LDH as a possible target to develop innovative anticancer treatments.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Burkitt/tratamiento farmacológico , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Linfocitos/efectos de los fármacos , Antineoplásicos/efectos adversos , Linfoma de Burkitt/enzimología , Linfoma de Burkitt/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/efectos adversos , Glucólisis/efectos de los fármacos , Humanos , Isocumarinas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Linfocitos/enzimología , Linfocitos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Compuestos Orgánicos/farmacología , Concentración Osmolar , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glycolysis is drastically increased in tumors and it is the main route to energy production with a minor use of oxidative phosphorylation. Among the key enzymes in the glycolytic process, LDH is emerging as one of the most interesting targets for the development of new inhibitors. In this context, in the present work, we carried out a virtual screening procedure followed by chemical modifications of the identified structures according to a "hit-to-lead" process. The effects of the new molecules were preliminary probed against purified human LDH-A. The compounds active at low micromolar level were additionally characterized for their activity on some cellular metabolic processes by using Raji human cell line. Within the series, 1 was considered the best candidate, and a more detailed characterization of its biological properties was performed. In Raji cells exposed to compound 1 we evidenced the occurrence of effects usually observed in cancer cells after LDH-A inhibition: reduced lactate production and NAD/NADH ratio, apoptosis. The flow cytometry analysis of treated cells also showed cell cycle changes compatible with effects exerted at the glycolytic level. Finally, in agreement with the data obtained with other inhibitors or by silencing LDH-A expression, compound 1 was found to increase Raji cells response to some commonly used chemotherapeutic agents. Taken together, all these finding are in support of the LDH-A inhibiting activity of compound 1.