Marcella Manerba

Galloflavin (CAS 568-80-9): A Novel Inhibitor of Lactate Dehydrogenase

One of the most prominent alterations in cancer cells is their strict dependence on the glycolytic pathway for ATP generation. This observation led to the evaluation of glycolysis inhibitors as potential anticancer agents. The inhibition of lactate dehydrogenase (LDH) is a promising way to inhibit tumor cell glucose metabolism without affecting the energetic balance of normal tissues. However, the success of this approach depends chiefly on the availability of inhibitors that display good selectivity. We identified a compound (galloflavin, CAS 568‐80‐9) which, in contrast to other inhibitors of human LDH, hinders both the A and B isoforms of the enzyme. To determine the mechanism of action, we collected LDH‐A and ‐B inhibition data in competition reactions with pyruvate or NADH and evaluated the results using software for enzyme kinetics analysis. We found that galloflavin inhibits both human LDH isoforms by preferentially binding the free enzyme, without competing with the substrate or cofactor. The calculated Ki values for pyruvate were 5.46 μM (LDH‐A) and 15.06 μM (LDH‐B). In cultured tumor cells, galloflavin blocked aerobic glycolysis at micromolar concentrations, did not interfere with cell respiration, and induced cell death by triggering apoptosis. To our knowledge, the inhibition of LDH is, to date, the only biochemical effect described for galloflavin. Because galloflavin is not commercially available, we also describe herein a procedure for its synthesis and report its first full chemical characterization.

Inhibition of lactate dehydrogenase activity as an approach to cancer therapy

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.

Galloflavin, a new lactate dehydrogenase inhibitor, induces the death of human breast cancer cells with different glycolytic attitude by affecting distinct signaling pathways.

Galloflavin (GF), a recently identified lactate dehydrogenase inhibitor, hinders the proliferation of cancer cells by blocking glycolysis and ATP production. The aim of the present experiments was to study the effect of this compound on breast cancer cell lines reproducing different pathological subtypes of this tumor: MCF-7 (the well differentiated form), MDA-MB-231 (the aggressive triple negative tumor) and MCF-Tam (a sub-line of MCF-7 with acquired tamoxifen resistance). We observed marked differences in the energetic metabolism of these cell lines. Compared to MCF-7 cells, both MDA-MB-231 and MCF-Tam cells exhibited higher LDH levels and glucose uptake and showed lower capacity of oxygen consumption. In spite of these differences, GF exerted similar growth inhibitory effects. This result was explained by the finding of a constitutively activated stress response in MDA-MB-231 and MCF-Tam cells, which reproduce the poor prognosis tumor forms. As a further proof, different signaling pathways were found to be involved in the antiproliferative action of GF. In MCF-7 cells we observed a down regulation of the ERα-mediated signaling needed for cell survival. On the contrary, in MCF-Tam and MDA-MB-231 cells growth inhibition appeared to be contributed by an oxidative stress condition. The prevalent mechanism of cell death was found to be apoptosis induction. Because of the clinical relevance of breast cancer forms having the triple negative and/or chemoresistant phenotype, our results showing comparable effects of GF even on aggressively growing cells encourage further studies to verify the potential of this compound in improving the chemotherapy of breast cancer.

Impairment of Aerobic Glycolysis by Inhibitors of Lactic Dehydrogenase Hinders the Growth of Human Hepatocellular Carcinoma Cell Lines

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.

Inhibition of lactic dehydrogenase as a way to increase the anti-proliferative effect of multi-targeted kinase inhibitors.

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.

Effect of sorafenib on the energy metabolism of hepatocellular carcinoma cells

Recent data demonstrated that sorafenib impaired the oxidative phosphorylation of a rat myogenic cell line and suggested that this biochemical lesion can contribute to the cardiac toxicity caused by the drug. With the experiments reported here, we verified whether sorafenib inhibits oxidative phosphorylation also in cells from human hepatocellular carcinomas (HCCs), which are treated with this drug. By using the HCC cell lines PLC/PRF/5 and SNU-449 we studied the effects of the drug on ATP cellular levels, oxygen consumption and aerobic glycolysis, a metabolic pathway generally used by neoplastic cells to meet their energy demand. The effect of sorafenib on ATP cellular levels was also studied in cells grown in a glucose-free medium, which only derive their energy from oxidative phosphorylation. We found that at clinically relevant concentrations sorafenib hindered oxidative phosphorylation, whereas at the same time stimulated aerobic glycolysis in glucose-grown cells, thus attenuating the cellular ATP depletion. These results support the impairment of oxidative phosphorylation as a mechanism contributing to the antineoplastic activity of sorafenib in the treatment of HCCs.

Altered glycosylation profile of purified plasma ACT from Alzheimer’s disease

BackgroundAlzheimer’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.MethodsN-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.ResultsWestern 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.ConclusionsLow 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.

Lactate dehydrogenase inhibitors sensitize lymphoma cells to cisplatin without enhancing the drug effects on immortalized normal lymphocytes.

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 Burkitts 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.

Lactate dehydrogenase inhibitors can reverse inflammation induced changes in colon cancer cells.

&NA; 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‐&agr; 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. Graphical abstract Figure. No caption available.

Lactate dehydrogenase inhibition: exploring possible applications beyond cancer treatment

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.