Patries M. Herst

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Metabolic flexibility and cell hierarchy in metastatic cancer.

Cancer is characterized by disturbed homeostasis of self-renewing cell populations, and their ability to seed and grow in multiple microenvironments. This overarching cellular property of metastatic cancer emerges from the contentious cancer stem cell hypothesis that underpins the more generic hallmarks of cancer (Hanahan and Weinberg, 2000) and its subsequent add-ons. An additional characteristic, metabolic flexibility, is related to concepts developed by Warburg and to subsequent work by mid 20th century biochemists who elucidated the bioenergetic workings of mitochondria. Metabolic flexibility may circumvent limitations inherent in the increasingly popular but erroneous view that aerobic glycolysis is a universal property of cancer cells. Cancer research in the second half of the 20th century was largely the domain of geneticists and molecular biologists using reductionist approaches. Integrated approaches that address cancer cell hierarchy and complexity, and how cancer cells adapt their metabolism according to their changing environment are now beginning to emerge, and these approaches promise to address the poor mortality statistics of metastatic cancer.

The level of glycolytic metabolism in acute myeloid leukemia blasts at diagnosis is prognostic for clinical outcome

This research investigated the level of glycolytic metabolism in leukemic blasts as a prognostic marker in AML. Using an in vitro dye‐reduction assay, we determined the level of glycolytic metabolism in 26 BM samples taken from 23 adult patients with newly diagnosed (n=19) or relapsed (n=4) AML, and AML blasts stratified into two distinct cohorts of moderate (<70%) or high (>80%) levels of glycolytic metabolism. All samples taken at relapse were moderately glycolytic. However, nine of the 19 samples taken at diagnosis were highly glycolytic, and 10 were moderately glycolytic. Three patients had paired samples taken at diagnosis and relapse, and the glycolytic metabolism of these samples did not alter between the two time‐points. The level of glycolytic metabolism did not correlate with the percentage of marrow blasts, patient age, or CG/molecular risk group. Highly glycolytic AML blasts were more resistant to apoptosis induced by ATRA and/or ATO in vitro, suggesting potential resistance to induction chemotherapy, as has been observed in solid tumors. Despite this, high levels of glycolytic metabolism at diagnosis were predictive of a significantly improved duration of CR1 and OS following AML remission induction chemotherapy. In conclusion, we found that the extent of myeloblast glycolysis may be an effective and easily applied method to determine the pretreatment prognosis of AML.

Targeting mitochondrial permeability in cancer drug development

The past decade has seen the emergence of a new mechanistic paradigm of cancer therapeutics. Not only have mitochondria taken centre stage as key cellular organelles mediating intrinsic pathways of cell death by apoptosis, but nonapoptotic pathways have also been shown to involve mitochondrial mechanisms. Both pathways of cell death involve permeabilization of mitochondrial membranes, but the exact nature of the molecular complexes involved at the inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) remains uncertain in the light of recent gene knockout studies. Consequently, the boundary between mitochondrially-mediated apoptotic and nonapoptotic cell death is controversial. Here, we discuss the nature of the pore complexes involved in permeabilization of the IMM and OMM. Several compounds that interact directly with components of these pore complexes and have been shown to exhibit anticancer activity are discussed while other compounds appear to act indirectly through stress-related pathways.

The anti-cancer drug, phenoxodiol, kills primary myeloid and lymphoid leukemic blasts and rapidly proliferating T cells

The plasma electron transport system is a relatively newly-discovered potential target for anti-leukemia drugs. In this paper Herst and coworkers describe the effects of phenoxodiol, an inhibitor of this pathway on leukemia cell lines and primary as well as on resting and activated T cells. The ability of phenoxodiol to kill rapidly proliferating lymphocytes might make this drug a promising candidate for the treatment of pathologically-activated lymphocytes. Background The redox-active isoflavene anti-cancer drug, phenoxodiol, has previously been shown to inhibit plasma membrane electron transport and cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes but not in non-transformed WI-38 cells and human umbilical vein endothelial cells. Design and Methods We determined the effects of phenoxodiol on plasma membrane electron transport, MTT responses and viability of activated and resting human T cells. In addition, we evaluated the effect of phenoxodiol on the viability of leukemic cell lines and primary myeloid and lymphoid leukemic blasts. Results We demonstrated that phenoxodiol inhibited plasma membrane electron transport and cell proliferation (IC50 46 μM and 5.4 μM, respectively) and promoted apoptosis of rapidly proliferating human T cells but did not affect resting T cells. Phenoxodiol also induced apoptosis in T cells stimulated in HLA-mismatched allogeneic mixed lymphocyte reactions. Conversely, non-proliferating T cells in the mixed lymphocyte reaction remained viable and could be restimulated in a third party mixed lymphocyte reaction, in the absence of phenoxodiol. In addition, we demonstrated that leukemic blasts from patients with primary acute myeloid leukemia (n=22) and acute lymphocytic leukemia (n=8) were sensitive to phenoxodiol. The lymphocytic leukemic blasts were more sensitive than the myeloid leukemic blasts to 10 μM phenoxodiol exposure for 24h (viability of 23±4% and 64±5%, respectively, p=0.0002). Conclusions The ability of phenoxodiol to kill rapidly proliferating lymphocytes makes this drug a promising candidate for the treatment of pathologically-activated lymphocytes such as those in acute lymphoid leukemia, or diseases driven by T-cell proliferation such as auto-immune diseases and graft-versus-host disease.

The novel phloroglucinol PMT7 kills glycolytic cancer cells by blocking autophagy and sensitizing to nutrient stress

The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox‐active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild‐type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild‐type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1‐mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy. J. Cell. Biochem. 112: 1869–1879, 2011.

Cell hierarchy, metabolic flexibility and systems approaches to cancer treatment.

The proliferative cancer cell paradigm that has driven cancer drug development for the past 50 years has failed to generate treatments that cure most metastatic adult cancers. This view is supported not only by cumulative experience with conventional cytotoxic anticancer drugs, but also by the application of highly-targeted anticancer compounds against, for example, BCR-ABL in CML and mutant BRAF in metastatic melanoma. Such drugs often send their respective cancers into complete molecular remission but fail to effect cures because a small population of quiescent or slowly selfrenewing cancer cells that are drug and radiation resistant survive treatment indefinitely. This review explores the grounds for an emerging cancer paradigm that views cancer as a disorganized tissue with hierarchical cellular compartments in which the boudaries are less well-defined than in normal tissues with plasticity controlled by epigenetic changes mediated by the local microenvironment. Increased metabolic flexibility and adaptability give cancer cells an additional survival advantage that may be able to be targeted with drugs like metformin. Combining approaches that target the increased metabolic flexibility of cancer cells as well as ablating rapidly-proliferating cells and self-renewing cancer stem cells in individual cancers are needed to address the holy grail of cancer cure.

Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits

Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae, required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.

Perfluorocarbon emulsions radiosensitise brain tumors in carbogen breathing mice with orthotopic GL261 gliomas

Background Tumour hypoxia limits the effectiveness of radiation therapy. Delivering normobaric or hyperbaric oxygen therapy elevates pO2 in both tumour and normal brain tissue. However, pO2 levels return to baseline within 15 minutes of stopping therapy. Aim To investigate the effect of perfluorocarbon (PFC) emulsions on hypoxia in subcutaneous and intracranial mouse gliomas and their radiosensitising effect in orthotopic gliomas in mice breathing carbogen (95%O2 and 5%CO2). Results PFC emulsions completely abrogated hypoxia in both subcutaneous and intracranial GL261 models and conferred a significant survival advantage orthotopically (Mantel Cox: p = 0.048) in carbogen breathing mice injected intravenously (IV) with PFC emulsions before radiation versus mice receiving radiation alone. Carbogen alone decreased hypoxia levels substantially and conferred a smaller but not statistically significant survival advantage over and above radiation alone. Conclusion IV injections of PFC emulsions followed by 1h carbogen breathing, radiosensitises GL261 intracranial tumors.

High Dose Ascorbate Causes Both Genotoxic and Metabolic Stress in Glioma Cells

We have previously shown that exposure to high dose ascorbate causes double stranded breaks (DSBs) and a build-up in S-phase in glioblastoma (GBM) cell lines. Here we investigated whether or not this was due to genotoxic stress as well as metabolic stress generated by exposure to high dose ascorbate, radiation, ascorbate plus radiation and H2O2 in established and primary GBM cell lines. Genotoxic stress was measured as phosphorylation of the variant histone protein, H2AX, 8-oxo-7,8-dihydroguanine (8OH-dG) positive cells and cells with comet tails. Metabolic stress was measured as a decrease in NADH flux, mitochondrial membrane potential (by CMXRos), ATP levels (by ATP luminescence) and mitochondrial superoxide production (by mitoSOX). High dose ascorbate, ascorbate plus radiation, and H2O2 treatments induced both genotoxic and metabolic stress. Exposure to high dose ascorbate blocked DNA synthesis in both DNA damaged and undamaged cell of ascorbate sensitive GBM cell lines. H2O2 treatment blocked DNA synthesis in all cell lines with and without DNA damage. DNA synthesis arrest in cells with damaged DNA is likely due to both genotoxic and metabolic stress. However, arrest in DNA synthesis in cells with undamaged DNA is likely due to oxidative damage to components of the mitochondrial energy metabolism pathway.