RESUMO
The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.
Assuntos
Proteína BRCA2 , Rad51 Recombinase , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Reparo do DNA , Recombinação Homóloga , Peptídeos/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismoRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isocumarinas/química , Isocumarinas/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Inibidores Enzimáticos/síntese química , Humanos , Isocumarinas/síntese química , Isoenzimas/antagonistas & inibidores , Lactato Desidrogenase 5 , Ácido Láctico/biossínteseRESUMO
Activation of the myc oncogene in cancer cells upregulates lactate dehydrogenase A (LDH-A) expression, leading to a sustained glycolytic flux that is needed to produce ATP under hypoxic conditions. We studied the effects of galloflavin (GF), a recently identified LDH inhibitor, on myc overexpressing Burkitt lymphoma (BL) cells. Epstein-Barr virus-infected lymphoblasts were used as a non-neoplastic control. Our results showed that myc overactivation induced a two- to seven-fold increase in LDH-A expression in BL cells compared with non-neoplastic lymphoblasts; this result is consistent with previously reported data. Moreover, GF treatment suppressed LDH activity and inhibited BL cell replication but did not affect lymphoblast viability. Surprisingly, we found that increased levels of the MYC and LDH-A proteins did not lead to a metabolic shift in BL cells toward glycolytic ATP generation. BL cells were treated with GF at doses that achieved 50% inhibition of cell growth and lactate production, and ATP levels were scarcely affected after GF treatment. The same results were also obtained by suppressing LDH activity with oxamate, an LDH specific inhibitor. Our data suggest that LDH activity is important for maintaining a correct NAD/NADH balance in BL cells. LDH inhibition led to decreased NAD cellular levels, which resulted in sirtuin-1 inhibition. Confirming previous studies, sirtuin-1 inhibition caused a reduction in MYC protein levels, depriving BL cells of their most important survival signal. This study further describes the biological functions of the LDH enzyme and suggests that LDH inhibition could be useful for the treatment of cancer.
Assuntos
Linfoma de Burkitt/enzimologia , Inibidores Enzimáticos/farmacologia , Isocumarinas/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , NAD/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuína 1/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Ácido Láctico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Fatores de TempoRESUMO
Protein kinase inhibitors are a relatively new class of promising anticancer drugs, most of which exert their action by binding to the ATP site on the targeted kinases. We hypothesized that a decrease in ATP levels in neoplastic cells could reduce the competition for the same enzymatic site, thus increasing the efficacy of kinase inhibitors. Using oxamic acid, an inhibitor of lactic dehydrogenase (LDH) which hinders aerobic glycolysis, we decreased ATP levels in PLC/PRF/5 cells (a line from a hepatocellular carcinoma). We found that in these cells oxamic acid potentiated the antiproliferative activity of sorafenib, imatinib and sunitinib, three kinase inhibitors. When aerobic glycolysis was shut down by culturing the cells in the absence of glucose, oxamic acid did not reduce the ATP levels, suggesting that in normal tissues, which do not rely on aerobic glycolysis for their ATP synthesis, the block of LDH should not impair cellular metabolism. In conclusion, the inhibition of LDH could enhance anticancer activity of sorafenib, imatinib and sunitinib without increasing their side effects on normal cells, which in conditions of normal functional activity and sufficient oxygen supply do not need the activity of this enzyme.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Ácido Oxâmico/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glicólise/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia , Sorafenibe , Timidina/metabolismoRESUMO
Intrinsic or acquired drug resistance is one of the major problems compromising the success of antineoplastic treatments. Several evidences correlated some therapeutic failures with changes in cell metabolic asset and in line with these findings, hindering the glycolytic metabolism of cancer cells via lactate dehydrogenase (LDH) inhibition was found to overcome the resistance to chemotherapeutic agents. Lactate, the product of LDH reaction, was shown to be involved in epigenetic regulation of gene expression. The experiments described in this paper were aimed at highlighting a possible direct effect of lactate in modifying the response of cancer cells to a chemotherapeutic treatment. To discriminate between the effects potentially caused by glycolytic metabolism from those directly referable to lactate, we selected cancer cell lines able to grow in glucose deprived conditions and evaluated the impact of lactate on the cellular response to cisplatin-induced DNA damage. In lactate-exposed cells we observed a reduced efficacy of cisplatin, which was associated with reduced signatures of DNA damage, enhanced DNA recombination competence and increased expression of a panel of genes involved in DNA repair. The identified genes take part in mismatch and nucleotide excision repair pathways, which were found to contribute in restoring the cisplatin-induced DNA damage. The obtained results suggest that this metabolite could play a role in reducing the efficacy of antineoplastic treatments.
Assuntos
Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Ácido Láctico/metabolismo , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Humanos , Regulação para CimaRESUMO
BACKGROUND: Cancer cells show highly increased glucose utilization which, among other cancer-essential functions, was found to facilitate DNA repair. Lactate dehydrogenase (LDH) activity is pivotal for supporting the high glycolytic flux of cancer cells; to our knowledge, a direct contribution of this enzyme in the control of DNA integrity was never investigated. In this paper, we looked into a possible LDH-mediated regulation of homologous recombination (HR) repair. METHODS: We identified two cancer cell lines with different assets in energy metabolism: either based on glycolytic ATP or on oxidative reactions. In cells with inhibited LDH, we assessed HR function by applying four different procedures. RESULTS: Our findings revealed an LDH-mediated control of HR, which was observed independently of cell metabolic asset. Since HR inhibition is known to make cancer cells responsive to PARP inhibitors, in both the cellular models we finally explored the effects of a combined inhibition of LDH and PARP. CONCLUSIONS: The obtained results suggest for LDH a central role in cancer cell biology, not merely linked to the control of energy metabolism. The involvement of LDH in the DNA damage response could suggest new drug combinations to obtain improved antineoplastic effects. GENERAL SIGNIFICANCE: Several evidences have correlated the metabolic features of cancer cells with drug resistance and LDH inhibition has been repeatedly shown to increase the antineoplastic power of chemotherapeutics. By shedding light on the processes linking cell metabolism to the control of DNA integrity, our findings also give a mechanistic explanation to these data.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
BACKGROUND: Alzheimer's disease (AD) is one of the most frequent cause of neurodegenerative disorder in the elderly. Inflammation has been implicated in brain degenerative processes and peripheral markers of brain AD related impairment would be useful. Plasma levels of alpha-1-antichymotrypsin (ACT), an acute phase protein and a secondary component of amyloid plaques, are often increased in AD patients and high blood ACT levels correlate with progressive cognitive deterioration. During inflammatory responses changes in the micro-heterogeneity of ACT sugar chains have been described. METHODS: N-Glycanase digestion from Flavobacterium meningosepticum (PNGase F) was performed on both native and denatured purified ACT condition and resolved to Western blot with the purpose to revealed the ACT de-glycosylation pattern.Further characterization of the ACT glycan profile was obtained by a glycoarray; each lectin group in the assay specifically recognizes one or two glycans/epitopes. Lectin-bound ACT produced a glyco-fingerprint and mayor differences between AD and controls samples were assessed by a specific algorithms. RESULTS: Western blot analysis of purified ACT after PNGase F treatment and analysis of sugar composition of ACT showed significantly difference in "glyco-fingerprints" patterns from controls (CTR) and AD; ACT from AD showing significantly reduced levels of sialic acid. A difference in terminal GlcNac residues appeared to be related with progressive cognitive deterioration. CONCLUSIONS: Low content of terminal GlcNac and sialic acid in peripheral ACT in AD patients suggests that a different pattern of glycosylation might be a marker of brain inflammation. Moreover ACT glycosylation analysis could be used to predict AD clinical progression and used in clinical trials as surrogate marker of clinical efficacy.
RESUMO
BACKGROUND/AIMS: by reducing the number of ATP molecules produced via aerobic glycolysis, the inhibition of lactic dehydrogenase (LDH) should hinder the growth of neoplastic cells without damaging the normal cells which do not rely on this metabolic pathway for their energetic needs. Here, we studied the effect of oxamic and tartronic acids, 2 inhibitors of LDH, on aerobic glycolysis and cell replication of HepG2 and PLC/PRF/5 cells, 2 lines from human hepatocellular carcinomas. METHODS: aerobic glycolysis was measured by calculating the amounts of lactic acid formed. The effect on replication was assessed by culturing the cells in both standard conditions and glucose-deprived medium, which was used to shut down aerobic glycolysis. RESULTS: the oxamic and tartronic acids inhibited aerobic glycolysis, impaired the growth of both cell lines and also induced an increased expression of p53-upregulated modulator of apoptosis, a signal of cell death. A strong impairment of cell replication by oxamic acid was only found when the cells were cultured in the presence of glucose, indicating that it was for the most part owing to inhibition of aerobic glycolysis. CONCLUSIONS: inhibition of aerobic glycolysis achieved by blocking LDH could be useful in the treatment of human hepatocellular carcinomas. Without interfering with glucose metabolism in normal cells, it could hinder cell growth by itself and could also enhance the chemotherapeutic index of associated anticancer agents by decreasing the levels of ATP selectively in neoplastic cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glicólise/efeitos dos fármacos , L-Lactato Desidrogenase/antagonistas & inibidores , Ácido Láctico/metabolismo , Ácido Oxâmico/farmacologia , Tartronatos/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo do Ácido Cítrico , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Células Hep G2 , Humanos , Consumo de Oxigênio/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proteína BRCA2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/química , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Descoberta de Drogas , Sinergismo Farmacológico , Recombinação Homóloga/efeitos dos fármacos , Humanos , Modelos Moleculares , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ftalazinas/química , Piperazinas/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacosRESUMO
BACKGROUND: Altered energy metabolism is a biochemical fingerprint of cancer cells, widely recognized as one of the "hallmarks of cancer". Cancer cells show highly increased rates of glucose uptake and glycolysis, after which the resulting pyruvate is converted to lactate. The maintenance of this metabolic asset is warranted by lactate dehydrogenase A (LDH-A) and for this reason the development of novel LDH-targeted anticancer therapeutics is underway. However, possible interference in cancer cell metabolism could also arise from cAMP signaling pathway, which could be activated by either oncogenic induction or exogenously, as a result of microenvironment-derived stimuli, increasing cellular cAMP levels. This study aimed at evaluating the impact of activated cAMP signaling pathway on the efficacy of an LDH-targeted anticancer approach. METHODS: We exogenously activated cAMP signaling in MCF-7 human breast cancer cells and explored the metabolic interplay between LDH-A and cAMP pathway. RESULTS: In cAMP-activated cells, we evidenced changes in energy metabolism which reduced their response to LDH inhibition. Interestingly, these experiments also highlighted a potential vulnerability state of treated cells. CONCLUSIONS: cAMP-induced metabolic changes made MCF-7 cells a preferential target of a drug combination treatment which should not affect normal cell viability. GENERAL SIGNIFICANCE: cAMP is a well-recognized second messenger of the pro-inflammatory cascade. The obtained results are relevant in consideration of the crucial role played by inflammation in normal breast cell transformation and in cancer progression. Furthermore, they corroborate the idea of exploiting the metabolic changes observed in cancer cells to obtain a therapeutic advantage.
Assuntos
Ativação Metabólica , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Microambiente TumoralRESUMO
Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.
Assuntos
Antineoplásicos/química , Proteína BRCA2/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Rad51 Recombinase/metabolismo , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteína BRCA2/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Mimetismo Molecular , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Triazóis/síntese químicaRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , L-Lactato Desidrogenase/genética , Serina-Treonina Quinases TOR/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Proliferação de Células/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Isoenzimas/genética , Lactato Desidrogenase 5 , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Neoplasias do Colo/metabolismo , Inflamação/metabolismo , L-Lactato Desidrogenase/metabolismo , Células CACO-2 , Transição Epitelial-Mesenquimal , Humanos , Interleucina-17/farmacologia , Isocumarinas/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Ácido Oxâmico/farmacologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.
Assuntos
Resposta ao Choque Térmico , L-Lactato Desidrogenase/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Isocumarinas/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Neoplasias Hepáticas/metabolismo , Compostos Orgânicos/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , alfa-Fetoproteínas/genéticaRESUMO
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.
Assuntos
Proteína BRCA2/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Modelos Moleculares , Mutação , Ftalazinas/química , Piperazinas/química , Conformação Proteica , Rad51 Recombinase/metabolismoRESUMO
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.
Assuntos
Antineoplásicos , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Descoberta de Drogas , Metabolismo Energético , Epilepsia/tratamento farmacológico , Epilepsia/enzimologia , Glicólise , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/imunologia , Metástase Neoplásica , Neoplasias/enzimologia , Oxirredução , Fenômenos Fisiológicos ViraisRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Linfócitos/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Linfoma de Burkitt/enzimologia , Linfoma de Burkitt/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Glicólise/efeitos dos fármacos , Humanos , Isocumarinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Linfócitos/enzimologia , Linfócitos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Compostos Orgânicos/farmacologia , Concentração Osmolar , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Hidrazonas/síntese química , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: This review deals with the use of serum albumin (SA) as a carrier for the selective delivery of drugs to liver cells. AREAS COVERED: The synthesis and properties of the SA conjugates prepared to enhance the performance of the drugs used in the treatment of viral hepatitis, hepatocellular carcinoma (HCC), liver micrometastases and hepatic fibrosis are reported. EXPERT OPINION: Studies in humans and laboratory animals demonstrated the capacity of SA conjugates to accomplish a liver targeting of the drugs, but at the same time underscored their limits and drawbacks, which can explain why to date these complexes did not reach a practical application. The major drawback is the need of administration by intravenous route, which prevents long-term daily treatments as required by some liver pathologies, such as chronic virus hepatitis and fibrosis. At present, only a conjugate carrying doxorubicin and addressed to the treatment of HCC showed in laboratory animals a solid potentiality to improve the value of the coupled drug. In the future, conjugation to SA could remain a successful strategy to permit the administration of drugs with rapid resolutive effects inside liver cells without causing severe extrahepatic adverse reactions.
Assuntos
Antineoplásicos/química , Doxorrubicina/química , Sistemas de Liberação de Medicamentos , Hepatopatias/tratamento farmacológico , Albumina Sérica/química , Animais , Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Química Farmacêutica , Doxorrubicina/administração & dosagem , Composição de Medicamentos , Hepatite Viral Humana/tratamento farmacológico , Humanos , Cirrose Hepática/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Albumina Sérica/administração & dosagemRESUMO
In the attempt of developing innovative anticancer treatments, growing interest has recently focused on the peculiar metabolic properties of cancer cells. In this context, LDH, which converts pyruvate to lactate at the end of glycolysis, is emerging as one of the most interesting molecular targets for the development of new inhibitors. In fact, because LDH activity is not needed for pyruvate metabolism through the TCA cycle, inhibitors of this enzyme should spare glucose metabolism of normal non-proliferating cells, which usually completely degrade the glucose molecule to CO2. This review is aimed at summarizing the available data on LDH biology in normal and neoplastic cells, which support the anticancer therapeutic approach based on LDH inhibition. These data encouraged pharmaceutical industries and academic institutions in the search of small-molecule inhibitors and promising candidates have recently been identified. The availability of inhibitors with drug-like properties will allow the evaluation in the near future of the real potential of LDH inhibition in anticancer treatment, also making the identification of the most responsive neoplastic conditions possible.