The HK2 Dependent "Warburg Effect" and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate.

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

Glutathione may have implications in the design of 3-bromopyruvate treatment protocols for both fungal and algal infections as well as multiple myeloma.

In different fungal and algal species, the intracellular concentration of reduced glutathione (GSH) correlates closely with their susceptibility to killing by the small molecule alkylating agent 3-bromopyruvate (3BP). Additionally, in the case of Cryptococcus neoformans cells 3BP exhibits a synergistic effect with buthionine sulfoximine (BSO), a known GSH depletion agent. This effect was observed when 3BP and BSO were used together at concentrations respectively of 4-5 and almost 8 times lower than their Minimal Inhibitory Concentration (MIC). Finally, at different concentrations of 3BP (equal to the half-MIC, MIC and double-MIC in a case of fungi, 1 mM and 2.5 mM for microalgae and 25, 50, 100 µM for human multiple myeloma (MM) cells), a significant decrease in GSH concentration is observed inside microorganisms as well as tumor cells. In contrast to the GSH concentration decrease, the presence of 3BP at concentrations corresponding to sub-MIC values or half maximal inhibitory concentration (IC50) clearly results in increasing the expression of genes encoding enzymes involved in the synthesis of GSH in Cryptococcus neoformans and MM cells. Moreover, as shown for the first time in the MM cell model, the drastic decrease in the ATP level and GSH concentration and the increase in the amount of ROS caused by 3BP ultimately results in cell death.

Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate.

In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.

The complete genome of Blastobotrys (Arxula) adeninivorans LS3 - a yeast of biotechnological interest.

BACKGROUND: The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant. RESULTS: The sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid. CONCLUSIONS: The high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications.

Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

3-Bromopyruvate: a novel antifungal agent against the human pathogen Cryptococcus neoformans.

We have investigated the antifungal activity of the pyruvic acid analogue: 3-bromopyruvate (3-BP). Growth inhibition by 3-BP of 110 strains of yeast-like and filamentous fungi was tested by standard spot tests or microdilution method. The human pathogen Cryptococcus neoformans exhibited a low Minimal Inhibitory Concentration (MIC) of 0.12-0.15 mM 3-BP. The high toxicity of 3-BP toward C. neoformans correlated with high intracellular accumulation of 3-BP and also with low levels of intracellular ATP and glutathione. Weak cytotoxicity towards mammalian cells and lack of resistance conferred by the PDR (Pleiotropic Drug Resistance) network in the yeast Saccharomyces cerevisiae, are other properties of 3-BP that makes it a novel promising anticryptococcal drug.

Yeast ATP-binding cassette transporters conferring multidrug resistance.

Overexpression of the ATP-binding cassette (ABC) drug transporter P-glycoprotein (P-gp) is often responsible for the failure of chemotherapy as a treatment for human tumors. The presence of proteins homologous to P-gp in organisms ranging from prokaryotes to eukaryotes indicates that drug export is a general mechanism of multidrug resistance. Yeasts are no exception. They have developed a large subfamily of ABC exporters involved in pleiotropic drug resistance (PDR) and in the cellular efflux of a wide variety of drugs. The PDR transporters Pdr5p of Saccharomyces cerevisiae and Cdr1p of Candida albicans are important members of this PDR subfamily, which comprises up to 10 phylogenetic clusters in fungi. Here, we review current achievements concerning the structure, molecular mechanism, and physiological functions of yeast Pdr transporters.

Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae.

We have investigated the cytotoxicity in Saccharomyces cerevisiae of the novel antitumor agent 3-bromopyruvate (3-BP). 3-BP enters the yeast cells through the lactate/pyruvate H(+) symporter Jen1p and inhibits cell growth at minimal inhibitory concentration of 1.8 mM when grown on non-glucose conditions. It is not submitted to the efflux pumps conferring Pleiotropic Drug Resistance in yeast. Yeast growth is more sensitive to 3-BP than Gleevec (Imatinib methanesulfonate) which in contrast to 3-BP is submitted to the PDR network of efflux pumps. The sensitivity of yeast to 3-BP is increased considerably by mutations or chemical treatment by buthionine sulfoximine that decrease the intracellular concentration of glutathione.

The mammalian ABC transporter ABCA1 induces lipid-dependent drug sensitivity in yeast.

ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Evolution of the 12-spanner drug:H+ antiporter DHA1 family in hemiascomycetous yeasts.

Frequently, although not exclusively, multidrug resistance (MDR) results from the action of drug-efflux pumps, which are thought to be able to catalyze the active expulsion of several unrelated cytotoxic compounds out of the cell or their intracellular partitioning. The transporters of the major facilitator superfamily (MFS) presumably involved in MDR belong to the 12-spanner drug:H(+) antiporter DHA1 or to the 14- spanner drug:H(+) antiporter DHA2 families. The expression of most Saccharomyces cerevisiae DHA1 family members was found to confer broad chemoprotection. The evolution of the hemiascomycetous DHA1 proteins, belonging to the Génolevures GL3C007 family, was studied using a combined phylogenetic and gene neighborhood approach. The phylogenetic analysis of 189 DHA1 proteins belonging to the genome of 13 hemiascomycetous species identified 20 clusters. Eleven clusters contained no S. cerevisiae members. The phylogenetic clusters were analyzed by the IONS method developed for Identification of Orthologues by Neighborhood and Similarity. This allowed reconstructing the evolutionary history of most DHA1 members within 10 main gene lineages, spanning the whole hemiascomycetes clade, encompassing an evolutionary history of about 350 million years. In addition, five other more species specific lineages, spanning only two hemiascomycetous species, were identified. It is concluded that 57 out of the 143 members of the DHA1 hemiascomycetous members originated from gene duplication events. In half of these duplicates, the two members belong to different phylogenetic clusters, indicating that at least one of them has sufficiently differentiated to provide potential novel functions to this complex family from which most physiological substrates remain unknown.

Vmr 1p is a novel vacuolar multidrug resistance ABC transporter in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Yhl035p/Vmr1p is an ABC transporter of the MRP subfamily that is conserved in all post Whole Genome Duplication species. The deletion of the YHL035 gene caused growth sensitivity to several amphiphilic drugs such as cycloheximide, 2,4-dichlorophenoxyacetic acid, 2,4-dinitrophenol as well as to cadmium and other toxic metals. Vmr1p-GFP was located in the vacuolar membrane. The ATP-dependent transport of a DNP-S-glutathione conjugate was reduced in a vesicular fraction from the VMR1 deletant. The energy-dependent efflux of rhodamine 6G was increased by VMR1 deletion. Growth sensitivity to cadmium of the VMR1-deleted strain was more pronounced in glycerol/ethanol than in glucose-grown cells. The VMR1 promoter had higher activity when grown in glycerol/ethanol compared with glucose. In glucose, the VMR1 promoter was activated by the deletion of the glucose-dependent repressor ADR1.

Fungal PDR transporters: Phylogeny, topology, motifs and function.

The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily frequently correlates with multidrug resistance. Phylogenetic analysis of 349 full-size ( approximately 160kDa) PDR proteins (Pdrps) from 55 fungal species, including major fungal pathogens, identified nine separate protein clusters (A-G, H1a/H1b and H2). Fungal, plant and human ABCG-family Pdrps possess a nucleotide-binding domain [NBD] and a transmembrane domain [TMD] in a family-defining 'reverse' ABC transporter topology [NBD-TMD] that is duplicated [NBD-TMD](2) in full-size fungal and plant Pdrps. Although full-size Pdrps have similar halves indicating early gene duplication/fusion, they show asymmetry of their NBDs and extracellular loops (ELs). Members of cluster F are most symmetric and may be closely related to the evolutionary ancestor of Pdrps. Unique structural elements are predicted, new PDR-specific motifs identified, and the significance of these and other structural features discussed.

Combined phylogeny and neighborhood analysis of the evolution of the ABC transporters conferring multiple drug resistance in hemiascomycete yeasts.

BACKGROUND: Pleiotropic Drug Resistant transporters (PDR) are members of the ATP-Binding Cassette (ABC) subfamily which export antifungals and other xenobiotics in fungi and plants. This subfamily of transmembrane transporters has nine known members in Saccharomyces cerevisiae. We have analyzed the complex evolution of the pleiotropic drug resistance proteins (Pdrp) subfamily where gene duplications and deletions occur independently in individual genomes. This study was carried out on 62 Pdrp from nine hemiascomycetous species, seven of which span 6 of the 14 clades of the Saccharomyces complex while the two others species, Debaryomyces hansenii and Yarrowia lipolytica, are further apart from an evolutive point of view. RESULTS: Combined phylogenetic and neighborhood analyses enabled us to identify five Pdrp clusters in the Saccharomyces complex. Three of them comprise orthologs of the Pdrp sensu stricto, Pdr5p, Pdr10p, Pdr12p, Pdr15p, Snq2p and YNR070wp. The evolutive pathway of the orthologs of Snq2 and YNR070w is particularly complex due to a tandem gene array in Eremothecium gossypii, Kluyveromyces lactis and Saccharomyces (Lachancea) kluyveri. This pathway and different cases of duplications and deletions were clarified by using a neighborhood analysis based on synteny. For the two distant species, Yarrowia lipolytica and Debaryomyces hansenii, no neighborhood evidence is available for these clusters and many homologs of Pdr5 and Pdr15 are phylogenetically assigned to species-based clusters. Two other clusters comprise the orthologs of the sensu lato Pdrp, Aus1p/Pdr11p and YOL075cp respectively. The evolutionary pathway of these clusters is simpler. Nevertheless, orthologs of these genes are missing in some species. CONCLUSION: Numerous duplications were traced among the Hemiascomycetous Pdrp studied. The role of the Whole Genome Duplication (WGD) is sorted out and our analyses confirm the common ancestrality of Pdr5p and Pdr15p. A tandem gene array is observed in Eremothecium gossypii. One of the copies is the ortholog of Snq2 while the other one is lost in the post-WGD species. The neighborhood analysis provides an efficient method to trace the history of genes and disentangle the orthology and paralogy relationships.

Efflux-mediated antifungal drug resistance.

Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

Outwitting multidrug resistance to antifungals.

The economic cost of fungal infection and its mortality associated with multidrug resistance remain unacceptably high. Recent understanding of the transcriptional regulation of plasma membrane efflux pumps of modest specificity provides new avenues for the development of broad-spectrum fungicides. Together with improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux, we envisage multifunctional azole analogs that inhibit not only ergosterol biosynthesis and drug efflux-pump activity but also activation of the transcriptional machinery that induces drug efflux-pump expression.

Compensatory activation of the multidrug transporters Pdr5p, Snq2p, and Yor1p by Pdr1p in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transcription factors Pdr1p and Pdr3p activate the expression of several genes, including PDR5, SNQ2, and YOR1, which encode ATP-binding cassette transporters that extrude dozens of antifungals with overlapping but distinct specificity. In this study, it was observed that growth resistance to specific Pdr5p substrates rose upon disruption of the YOR1 or SNQ2 coding region and was accompanied by increased efflux. Similarly, resistance to Yor1p- and Snq2p-specific substrates increased upon deletion of PDR5. The mRNA and protein levels of the respective transporters increased in parallel to drug resistance. beta-Galactosidase activity fused to the PDR5 or YOR1 promoter required the presence of Pdr1p and its specific binding sites for the compensatory induction, whereas Pdr3p had an inhibitory effect.

A phylogenetic analysis of the sugar porters in hemiascomycetous yeasts.

A total of 214 members of the sugar porter (SP) family (TC 2.A.1.1) from eight hemiascomycetous yeasts: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Ashbya (Eremothecium) gossypii, Debaryomyces hansenii, Yarrowia lipolytica, Candida albicans and Pichia stipitis, were identified. The yeast SPs were classified in 13 different phylogenetic clusters. Specific sugar substrates could be allocated to nine phylogenetic clusters, including two novel TC clusters that are specific to fungi, i.e. the glycerol:H(+) symporter (2.A.1.1.38) and the high-affinity glucose transporter (2.A.1.1.39). Four phylogenetic clusters are identified by the preliminary fifth number Z23, Z24, Z25 and Z26 and the substrates of their members remain undetermined. The amplification of the SP clusters across the Hemiascomycetes reflects adaptation to specific carbon and energy sources available in the habitat of each yeast species.

Evolution of the carboxylate Jen transporters in fungi.

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.

Emergence of species-specific transporters during evolution of the hemiascomycete phylum.

We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named "ubiquitous," "species specific," "phylum gains and losses," or "homoplasic." This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification.