Takafumi Ochi

C-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism

Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.

Glutathione and metallothioneins as cellular defense against cadmium toxicity in cultured Chinese hamster cells

To evaluate the protective role of cell glutathione (GSH) against the toxicity of cadmium, the effect of GSH depletion on the metal toxicity was investigated and the role of glutathione was compared with that of zinc-induced metallothioneins (MTs). A 6-h incubation of cultured Chinese hamster V79 cells with 0.2 mM L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, resulted in approx. 95% depletion of GSH in the cells. The depletion of GSH did not influence the rate of cell growth, the amount of cell protein or the chromosome structure during culture for at least 24 h. Cells depleted or not depleted of GSH were challenged with (1-5).10(-5) M CdCl2 for 2 h and subsequent cell growth was evaluated. The cytotoxicity of cadmium was enhanced with increasing duration of BSO pretreatment and was correlated with the decrease of cell GSH, indicating that GSH constitutes a cellular defense against toxicity by cadmium. Inducibility of MTs by zinc was investigated in cultured V79 cells. Incubation of the cells with 1.10(-4) M zinc acetate did not result in accumulation of MTs over the control values for up to 2 h. Thereafter, however, the synthesis of MTs increased with increasing duration of zinc treatment and an approx. 9-fold increase in the amount of MTs was observed 10 h after addition of zinc. Depletion of cell GSH by BSO did not much influence the increased accumulation of MTs by zinc. In contrast, zinc at the same concentration did not influence the level of cell glutathione up to 12 h. The cytotoxicity of cadmium was markedly reduced in the cells pretreated with zinc and the protective effect of zinc was dependent upon duration of pretreatment, being parallel with the increased accumulation of MTs. Protection of cells from cadmium toxicity by zinc pretreatment was as or a little more effective in the cells depleted of GSH as in those not depleted. Thus, glutathione appears to be an intrinsic protector against cadmium toxicity, while MTs serve as an induced cellular defense that is mobilized against heavy metal stress, but takes more than 2 h to accumulate in significant amounts. Accordingly, it is suggested that GSH and MTs have cooperative protective roles against cadmium toxicity, as an initial defense for the former and a second-stage defense for the latter.

Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction

The effects of scavengers of active oxygen species on cadmium chloride (CdCl2)-induced inhibition of cell growth and DNA synthesis and on the metal-induced clastogenesis were investigated to evaluate whether cadmium could induce a prooxidant state in cultured Chinese hamster V79 cells. Inhibition by CdCl2 of cell growth and [3H]thymidine incorporation into the acid-insoluble fraction of cells and the metal-induced clastogenesis were suppressed in part by the presence of the diffusible radical scavenger, butylated hydroxytoluene (BHT). The action of BHT was concentration-dependent and did not affect the intracellular level of cadmium. D-Mannitol, a hydroxyl radical scavenger, also significantly suppressed Cd-induced inhibition of cell growth and [3H]thymidine incorporation. Catalase was marginally suppressive on Cd-induced inhibition of cell growth. These results suggest that cadmium can induce a prooxidant state in cultured mammalian cells. The mechanism by which cadmium induces a prooxidant state was investigated by measuring the effect of cadmium on those enzymes which constitute a cellular defense against active oxygen and on the level of the intracellular antioxidant, glutathione (GSH). 2-h treatments with CdCl2 over a concentration range of 2-10 X 10(-5) M did not influence superoxide dismutase, catalase, GSH peroxidase or GSSG reductase. In contrast, the level of glutathione was decreased to approximately 40% by treatment with 2 X 10(-5) M cadmium. The decrease in glutathione level may be responsible for a role by active oxygen in Cd-induced inhibition of cell growth and DNA synthesis and the metal-induced clastogenesis.

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As3+)>arsenate (As5+)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As3+, As5+, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As3+, As5+, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.

Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Incidences of chromosomal aberrations by CdCl2 were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and butylated hydroxytoluene (BHT, an antioxidant). These findings may indicate participation of the active oxygen species such as hydrogen peroxide (H2O2) or hydroxyl radicals in the clastogenicity of cadmium. In contrast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCl2. These results suggest that superoxide anion and singlet oxygen are not directly involved in the clastogenicity of the metal. The presence of aminotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCl2. This emphasizes participation of H2O2 in the clastogenicity of cadmium.

Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells

A mechanism for the induction of DNA single-strand scissions in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated by use of the technique of alkaline elution. Inducibility of DNA single-strand scissions by cadmium was examined under an aerobic or anaerobic culture condition. About 62% of the total cellular DNA was eluted throughout the filter within 10 h of elution time by treatment with 4 X 10(-5) M CdCl2 for 2 h in our usual aerobic medium. In contrast, no difference in elution profiles of DNA was observed between untreated control cells and the cells treated with CdCl2 in the anaerobic medium which was prepared by N2 gas bubbling of aerobic medium for 60 min. Furthermore, elution of DNA from cells treated with cadmium decreased markedly in the presence of superoxide dismutase (SOD) when compared with that in the absence of SOD. Inhibition of the cell growth by cadmium was significantly protected by the presence of SOD in the medium although the cell growth was not restored to the control level. These results indicate that active oxygen species participate in Cd-induced DNA single-strand scissions and also in the growth inhibition of the cells by the metal.

Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione.

Abstract Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarisinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of γ-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound.

Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinate in cultured human cells and the role of glutathione

Thio-dimethylarsinate (thio-DMA), a recently discovered urine metabolite in humans, was investigated for its cytotoxic, genotoxic and cell-cycle disruptive effects in the cultured human hepatocarcinoma cell line, HepG2, and Syrian hamster embryo cells. In addition, the role of glutathione (GSH) on the cytotoxic effects of thio-DMA was investigated in terms of the effects of GSH depletion and the effects of exogenously added GSH. LC50 values of arsenicals for cells incubated for 48 h were 0.026 mM for thio-DMA, 0.343 mM for DMA and 3.66 mM for dithio-DMA. Depletion of cell GSH reduced the cytotoxic effects of thio-DMA. The cytotoxic effects of 0.02 mM and 0.05 mM thio-DMA were enhanced markedly when used in combination with 1 to 3 mM GSH, but decreased again when combined with 5 mM GSH. These results suggested that cytotoxic intermediates were generated by the interaction of thio-DMA with GSH, while an excessive amount of GSH suppressed the generation of these intermediates. Flow-cytometry showed that thio-DMA was an inducer of cells with 4N DNA and hypo 2N DNA. The results also demonstrated that cells arrested in the mitotic phase had abnormalities in their spindle organization and centrosome integrity. In addition, cells arrested in mitosis by thio-DMA had chromosome structural aberrations, such as chromatid gaps, chromatid breaks and chromatid exchanges. Moreover, the cytotoxic effects of thio-DMA may in part be associated with an apoptotic mode of cell death that was evaluated by the appearance of nucleosome level DNA fragmentations and an 85-kDa cleavage fragment of poly (ADP-ribose) polymerase. These findings suggest that the presence of thio-DMA in human urine has implications for human health in terms of arsenic metabolism and toxicity.

Induction of 6-thioguanine-resistant mutants and single-strand scission of DNA by cadmium chloride in cultured Chinese hamster cells

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.

Hydrogen peroxide increases the activity of γ-glutamylcysteine synthetase in cultured Chinese hamster V79 cells

Hydrogen peroxide (H2O2) caused a rapid and a concentration-dependent increase in the activity of γ-glutamylcysteine synthetase (γ-GCS) in cultured Chinese hamster V79 cells. The increase in the activity was transient and declined rapidly during post-treatment incubation. Inhibition of protein synthesis by cycloheximide, chelation of divalent iron byo-phenanthroline, and scavenging of free radicals by butyl-4-hydroxyanisole failed to suppress the increase in activity of γ-GCS caused by H2O2. However, catalase completely inhibited the increase in the activity of the enzyme. H2O2 did not change the level of total glutathione (GSH+GSSG) but is oxidized GSH. The increased in levels of GSSG caused by H2O2 was enhanced byo-phenanthroline.These results suggest that the increase in activity of γ-GCS caused by H2O2 is not an inducible phenomenon, nor it is attributable to the action of free radicals generated by an iron-catalyzed Fenton reaction. Furthermore, the changes in levels of GSH and GSSG caused by H2O2 appear not to be responsible for the increase in activity of γ-GCS caused by the hydroperoxide. However, chemical reduction of the enzyme, the activity of which had been increased by H2O2, resulted in a decrease, in the activity, suggesting the involvement of oxidation of the enzyme in the increased activity of γ-GCS caused by H2O2. The results also suggest that the activity of γ-GCS in cultured V79 cells can be regulated by the cellular oxidation-reduction state.