TY - JOUR

T1 - The potent and novel thiosemicarbazone chelators di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone and 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone affect crucial thiol systems required for ribonucleotide reductase activity

AU - Yu, Yu

AU - Suryo Rahmanto, Yohan

AU - Hawkins, Clare L

AU - Richardson, Des R

PY - 2011/6

Y1 - 2011/6

N2 - Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP(total) ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems.

AB - Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP(total) ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems.

KW - Blotting, Western

KW - Cell Line, Tumor

KW - Electron Spin Resonance Spectroscopy

KW - Glutathione

KW - Glutathione Reductase

KW - Humans

KW - Iron Chelating Agents

KW - Pyridines

KW - Ribonucleotide Reductases

KW - Sulfhydryl Compounds

KW - Thiosemicarbazones

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1124/mol.111.071324

DO - 10.1124/mol.111.071324

M3 - Journal article

C2 - 21389104

VL - 79

SP - 921

EP - 931

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 6

ER -