Stanisław Ułaszewski

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.

Butyltin(IV) 2-sulfobenzoates: Synthesis, structural characterization and their cytostatic and antibacterial activities

Three butyltin complexes with 2-sulfobenzoic acid [Sn(C(4)H(9))(2){O(3)SC(6)H(4)COO-2}(H(2)O)]·(C(2)H(5)OH) (1), [Sn(C(4)H(9))(3){O(3)SC(6)H(4)COOH-2}] (2) and [Sn(2)(C(4)H(9))(6){μ-O(3)SC(6)H(4)COO-2}] (3) have been synthesized and characterized by IR and (1)H, (13)C and (119)Sn NMR spectra. They show interesting properties in solid state and solutions because there are many modes of coordination of the Sbz ligand. The structure of complex 1 has been determined by X-ray crystallography. It is a chain compound with 2-sulfonatobenzoate coordinated to Sn atoms as a bridging and chelate ligand via O atoms of COO and SO(3) groups. In solutions the chains dissociate giving mainly mononuclear complexes. The NMR spectra and calculation at the DFT B3LYP/3-21G** level indicate that in solutions of compounds 1, 2 and 3 in polar solvents, many complexes showing dynamic properties are formed. Density functional theory (DFT) calculations showed that many five- and six-coordinate isomers and conformers can exist in equilibrium. All compounds effectively interact with AMP and ATP. The NMR spectra showed that nucleotides are coordinated to Sn atoms via PO(4) groups. The complexes are very active cytostatic agents against tumor strains. They are more effective than cisplatin. It is interesting that activity of 3 against non-tumor cell NHDF is lower than against tumor cells. Antibacterial activity of 1 and 2 has been investigated. Compound 2 is a very effective agent against Gram-positive bacteria. Antibacterial activity of 1 is lower than that of 2. Activity of 1 both against Gram-positive and Gram-negative bacteria is similar.

The anticancer agent 3-bromopyruvate: a simple but powerful molecule taken from the lab to the bedside

At the beginning of the twenty-first century, 3-bromopyruvate (3BP), a simple alkylating chemical compound was presented to the scientific community as a potent anticancer agent, able to cause rapid toxicity to cancer cells without bystander effects on normal tissues. The altered metabolism of cancers, an essential hallmark for their progression, also became their Achilles heel by facilitating 3BP’s selective entry and specific targeting. Treatment with 3BP has been administered in several cancer type models both in vitro and in vivo, either alone or in combination with other anticancer therapeutic approaches. These studies clearly demonstrate 3BP’s broad action against multiple cancer types. Clinical trials using 3BP are needed to further support its anticancer efficacy against multiple cancer types thus making it available to more than 30 million patients living with cancer worldwide. This review discusses current knowledge about 3BP related to cancer and discusses also the possibility of its use in future clinical applications as it relates to safety and treatment issues.

Antifungal activity of organotin compounds with functionalized carboxylates evaluated by the microdilution bioassay in vitro

We investigated the susceptibility of 96 well-characterized strains of yeast-like and filamentous fungi towards new organotin compounds: (1) [Sn(C4H9)3(OOCC6H4SO3H-2)], (2) Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br, and (3) [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl. In the case of yeast-like fungi, the in vitro susceptibility tests were carried out according to the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference method M27-A2, while for filamentous fungi the investigations were conducted according to the M38-A and M38-P methods. The organotin complexes 1, 2 and 3 are active antifungal agents. Minimal inhibitory concentrations (MIC) were in the range of 0.25-4.68 microg/ml for all tested fungal strains. Considerably larger differences were found for minimal fungicidal concentrations (MFC). In the case of yeast-like fungi, the fungicidal effect was generally observed at organotin compounds concentrations of 2.34-9.37 microg/ml. The MFC values for filamentous fungi were considerably higher and were in the range of 18.74-50 microg/ml. In conclusion, organotin compounds 1, 2 and 3 showed high fungistatic and fungicidal activities against different species of pathogenic and nonpathogenic fungi. However, they were also highly cytotoxic towards two mammalian cell lines.

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.

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IMR) mutant in the presence of the QAS. The IMR mutant showed increased sensitivity to CaCl2 and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IMR mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.

Transport of 3-bromopyruvate across the human erythrocyte membrane

Abstract3-Bromopyruvic acid (3-BP) is a promising anticancer compound because it is a strong inhibitor of glycolytic enzymes, especially glyceraldehyde 3-phosphate dehydrogenase. The Warburg effect means that malignant cells are much more dependent on glycolysis than normal cells. Potential complications of anticancer therapy with 3-BP are side effects due to its interaction with normal cells, especially erythrocytes. Transport into cells is critical for 3-BP to have intracellular effects. The aim of our study was the kinetic characterization of 3-BP transport into human erythrocytes. 3-BP uptake by erythrocytes was linear within the first 3 min and pH-dependent. The transport rate decreased with increasing pH in the range of 6.0–8.0. The Km and Vm values for 3-BP transport were 0.89 mM and 0.94 mmol/(l cells x min), respectively. The transport was inhibited competitively by pyruvate and significantly inhibited by DIDS, SITS, and 1-cyano-4-hydroxycinnamic acid. Flavonoids also inhibited 3-BP transport: the most potent inhibition was found for luteolin and quercetin.

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.

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).