Jarmila Navrátilová

Acidic pH of Tumor Microenvironment Enhances Cytotoxicity of the Disulfiram/Cu2+ Complex to Breast and Colon Cancer Cells

Background: Resistance of cancer cells to chemotherapeutic agents is a major cause of treatment failure in patients with cancer. The drug resistance of tumor cells can be significantly modified by specific features of tumor microenvironment, such as oxygen depletion (hypoxia), glucose/energy deprivation and acidosis. Methods: The effects of acidic tumor-like microenvironment on cytotoxicity of antabuse (disulfiram, DSF)/Cu2+ complexes to MCF-7 breast carcinoma and HT-29 colon carcinoma cells were studied. Results: We show that acidic pH significantly potentiates toxicity of DSF/Cu2+ complex to breast and colon cancer cells. This phenomenon is associated with changes in cell metabolism, altered Akt kinase and NFκB activity and increased reactive oxygen species production. Conclusion: Specific pH of tumor microenvironment enhances cytotoxicity of DSF/Cu2+ to breast and colon cancer cells.

Low-Glucose Conditions of Tumor Microenvironment Enhance Cytotoxicity of Tetrathiomolybdate to Neuroblastoma Cells

Growth of tumor cells depends on sufficient supply of fermentable substrate, such as glucose. This provokes development of new anticancer therapies based on dietary restrictions. However, some tumor cells can lower their glucose dependency and activate processes of ATP formation/saving to retain viability even in limited glucose supply. In addition, tumor cells often lose sensitivity to many conventional anticancer drugs in the low-glucose conditions. Thus, development of the drugs effectively killing the tumor cells in nutrient-limited conditions is necessary. In this study, we show an enhanced cytotoxicity of tetrathiomolybdate, the drug exhibiting antiangiogenic and tumor-suppressing effects, to neuroblastoma SH-SY5Y and SK-N-BE(2) cells in the low-glucose conditions. This preference results from the tetrathiomolybdate-induced upregulation of cell dependency on glucose. The cells treated with tetrathiomolybdate increase the uptake of glucose, production of lactate, activate the Akt- and AMPK-signaling pathways and downregulate COX IV. In cells growing in the low-glucose conditions, these events result in significant decrease of the intracellular ATP supply and apoptosis. We propose tetrathiomolybdate as suitable agent to be used in combination with dietary restrictions in therapy of neuroblastoma.

Substrate-assisted laser desorption inductively-coupled plasma mass spectrometry for determination of copper in myeloid leukemia cells

This work presents a detailed characterization of substrate-assisted laser desorption inductively coupled plasma mass spectrometry (SALD ICP-MS), a new approach in the determination of trace elements in submicrolitre volumes. SALD ICP-MS allows quantitative analysis of metals in liquid samples with defined volumes deposited onto an absorbing substrate due to complete sample desorption at relatively low laser power density. Analysis of model samples of Cu(II) showed a constant signal over a wide range of laser power density, an independence of signal on desorption modes and a wide linear dynamic range. SALD ICP-MS with its limit of detection for copper below 26 fg in submicrolitre samples was successfully applied to investigate cytotoxic effects of disulfiram in human myeloid leukemia cells U937 depending on intracellular levels of copper.

Transactivation by temperature-dependent p53 mutants in yeast and human cells.

The p53 protein plays an important role in cancer prevention. In response to stress signals, p53 controls essential cell functions by regulating expression of its target genes. Full or partial loss of the p53 function in cancer cells usually results from mutations of the p53 gene. Some of them are temperature-dependent, allowing reactivation of the p53 function in certain temperature. These mutations can alter general transactivation ability of the p53 protein or they modify its transactivation only towards specific genes. We analyzed transactivation of several target genes by 23 temperature-dependent p53 mutants and stratified them into four functional groups. Seventeen p53 mutants exhibited temperature-dependency and discriminative character in human and yeast cells. Despite the differences of yeast and human cells, they allowed similar transactivation rates to the p53 mutants, thus providing evidence that functional analysis of separated alleles in yeast is valuable tool for assessment of the human p53 status.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.