Abstract
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, ...effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. To uncover cell type-specific dependencies to LDH, we screened a diverse panel of 94 cancer cell lines for responsiveness to two novel LDH A/B inhibitors developed through the NCI Experimental Therapeutics Program (NExT). We found that Ewing sarcoma (EWS) cell lines were exquisitely sensitive, with IC50 values approximately ten-fold below the median IC50 of the panel. To understand the mechanism behind this sensitivity, we genetically knocked down LDHA and LDHB using siRNA, and discovered that EWS cell lines were sensitive to loss of LDHA only, which inhibited proliferation and induced apoptosis. Notably, treatment of EWS cells with the LDH inhibitors phenocopied these effects. Additionally, genetic knockdown of EWS-FLI1, the oncogenic driver of EWS, resulted in loss of LDHA, but not LDHB. Analysis of publicly available ChIP-seq data generated using shFLI1-transfected EWS cells revealed that LDHA, but not LDHB, is directly regulated by EWS-FLI1. Functional mechanistic studies of glycolytic intermediates and cellular bioenergetics in EWS cells treated with the LDH inhibitors demonstrated that loss of viability was due to impairment of glycolysis, which occurred both in vitro and in vivo, and perturbation of the NAD+/NADH ratio. The translational potential of these compounds was next evaluated using in vivo analyses of pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Intravenous administration of the LDH inhibitors resulted in diminished LDH activity, reduction of the lactate-to-pyruvate ratio, tumor cell necrosis, and a decrease in tumor growth rate in aggressive xenograft models of EWS. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, our data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as EWS that exhibit oncogene-dependent expression of LDH and increased glycolytic activity.
This abstract is also being presented as Poster B33.
Citation Format: Choh Yeung, Anna E. Gibson, Sameer H. Issaq, Nobu Oshima, Marielle E. Yohe, Haiyan Lei, Ganesha Rai, Daniel J. Urban, Michelle S. Johnson, Gloria A. Benevides, Giuseppe L. Squadrito, Sandy Eldridge, John Hamre III, Arnulfo Mendoza, Jack F. Shern, Lee J. Helman, Murali C. Krishna, Matthew D. Hall, Victor M. Darley-Usmar, Leonard M. Neckers, Christine M. Heske. Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma abstract. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR08.
Abstract Polysaccharides extracted from brown marine algae represent a source of marine compounds with potential applications in medicine. Heparin-like compounds, fucoidans, have been proposed as ...alternatives to the anticoagulant heparin, which is prepared from mucous membrane of mammals. In this study, the activity of anticoagulant in activated partial thromboplastin time (APTT) and prothrombin time (PT) tests was assessed in the fucoidan (TF), from seaweed Fucus vesiculosus , partially desulfated fucoidans (PDF), desulfated fucoidans (DF) and purified fractions F1, F2 and F3 in acetone. Studies were also conducted to assess these polysaccharides for platelet aggregation and hemorrhagic activity. The APTT test showed high activity at 5 μg (≥240 s) for TF, F1 and F2 ( P < 0.001). PT test showed high anticoagulant activity at 50 μg (≥120 s) for F1 ( P < 0.001). Fraction F3, with low MW (15.2 kDa) and sulfate content (26.1%), had little effect in these two in vitro tests ( P < 0.001). These compounds demonstrated a two-phase response to platelet aggregation at 50 μg/mL. However, at a concentration of 0.1 mg/mL, a hypoaggregate profile was observed for all fractions tested ( P < 0.001). The analysis showed that fucoidans irreversibly induced platelet aggregation in high concentration. These polymers have low hemorrhagic effect when compared to heparin.
Glyoxyl agarose is constituted by quite thick agarose fibres containing a large number of very stable aldehyde groups attached to the support by very short spacer arms. Under alkaline conditions, ...these activated supports immobilize proteins, via, at least, a two-point reaction involving the region/s of the protein surface with the higher densities of amino groups. These bonds are weak Schiff's bases and the reversibility of the bonds has been used to convert this matrix into a chromatographic one. A more intense multipoint attachment between the immobilized protein and the activated support can be further promoted, with minimal loss of catalytic activity, by a long-term incubation of the protein–support conjugate under suitable conditions. The end-point of the preparation of agarose–protein conjugates is a very mild borohydride reduction. After that reduction, the enzyme remains attached to the support by means of very stable secondary amino bonds (with very similar physical properties to those of the former primary amino ones) and the remaining aldehyde groups on the support are converted into fully inert hydroxyl groups. Very active and highly stabilized derivatives of many enzymes and proteins have been prepared using these supports. The main features of these protein immobilization protocols are discussed here.
The oleaginous yeast
R25L270 was the first yeast able to grow and produce extracellular lipase using Macaúba (
) cake as substrate. The novel lipase was recently identified, and presented promising ...features for biotechnological applications. The
R25L270 lipase efficiently hydrolyzed vegetable and animal oils, and showed selectivity for generating
-5,8,11,15,17-eicosapentaenoic acid from sardine oil. The enzyme can act in a wide range of temperatures (25-48 °C) and pH (6.5-8.4). The present study deals with the immobilization of
R25L270 lipase on hydrophobic, covalent and ionic supports to select the most active biocatalyst capable to obtain omega-3 fatty acids (PUFA) from sardine oil. Nine immobilized agarose derivatives were prepared and biochemically characterized for thermostability, pH stability and catalytic properties (K
and V
). Ionic supports improved the enzyme-substrate affinity; however, it was not an effective strategy to increase the
R25L270 lipase stability against pH and temperature. Covalent support resulted in a biocatalyst with decreased activity, but high thermostability. The enzyme was most stabilized when immobilized on hydrophobic supports, especially Octyl-Sepharose. Compared with the free enzyme, the half-life of the Octyl-Sepharose derivative at 60 °C increased 10-fold, and lipase stability under acidic conditions was achieved. The Octyl-Sepharose derivative was selected to obtain omega-3 fatty acids from sardine oil, and the maximal enzyme selectivity was achieved at pH 5.0.
Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. ...A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.
The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a ...layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). Stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase).
Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme ...structure consequently influencing its activity, selectivity, and stability. The lipase from
sp. section
(CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of
-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties.
Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in ...the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from
Aspergillus niger
and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.
Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are ...capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative.
Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from
was efficiently linked to four chemical supports: agarose activated with cyanogen ...bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol,
-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (
), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the
-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the
-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the
-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the
-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate
-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.
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