Retention mechanism of hypoxia selective nuclear imaging/radiotherapeutic agent Cu-diacetyl-bis(N 4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells

Abstract

The retention mechanism of the novel imaging/radiotherapeutic agent, Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells was clarified in comparison with that in normal tissue in vitro. With Cu-ATSM and reversed phase HPLC analysis, the reductive metabolism of Cu-ATSM in subcellular fractions obtained from Ehrlich ascites tumor cells was examined. As a reference, mouse brain was used. To determine the contribution of enzymes in the retention mechanisms, and specific inhibitor studies were performed. In subcellular fractions of tumor cells, Cu-ATSM was reduced mainly in the microsome/cytosol fraction rather than in the mitochondria. This finding was completely different from that found in normal brain cells. The reduction process in the microsome/cytosol was heat-sensitive and enhanced by adding exogenous NAD(P)H, an indication of enzymatic reduction of Cu-ATSM in tumor cells. Among the known bioreductive enzymes, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase in microsome played a major role in the reductive retention of Cu-ATSM in tumors. This enzymatic reduction was enhanced by the induction of hypoxia. Radiocopper labeled Cu-ATSM provides useful information for the detection of hypoxia as well as the microsomal bioreductive enzyme expression in tumor.

Species dependence of [64Cu]Cu-Bis(thiosemicarbazone) radiopharmaceutical binding to serum albumins

“…However, for Cu-ATSM this redox trapping is sensitive to the level of cell oxygenation, occurring most rapidly in hypoxic cells, leading to investigation of [ 60,64 Cu[Cu-ATSM as a marker of tissue hypoxia [26][27][28][29][30][31].…”

“…1), have shown promise as PET radiopharmaceuticals for imaging tissue perfusion (Cu-PTSM, Cu-ETS) [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and hypoxia (Cu-ATSM) [26][27][28][29][30][31][32]. The clinical utility of Cu-PTSM for quantifying perfusion in high-flow tissues, such as hyperemic myocardium, appears limited by the high affinity of this chelate for HSA, a result not predicted by the promising results from canine models [13].…”

PET imaging of cardiac hypoxia: Opportunities and challenges

“…In brain, 70-80% of Cu-PTSM and Cu-ATSM was reduced by mitochondria, while in a variety of cancer cell types, mitochondrial retention is reported to represent approximately 10% of the total [82,[100][101][102]. Thus far, reduction of these complexes has only been investigated in individual subcellular fractions [100,[103][104][105], rather than intact tissues which have been exposed to copper complexes and then fractionated. Errors in accounting for relative differences in volumes and concentrations of each fraction potentially skew the measured contribution of each fraction to total cellular bioreduction, but some tissue-specific differences have been identified nonetheless.…”

“…They demonstrated that this reduction was inhibited in a dose-dependent manner by the flavin enzyme inhibitor DPIC, and pHMB, a thiolenzyme inhibitor, suggesting that cytosolic electron transport chain enzymes were responsible. Further evidence for this was provided by treatment of microsomal fractions with inhibitors of cytochrome b5 reductase and cytochrome P450 reductase, resulting in inhibition of tracer reduction [101].…”

64Cu-ATSM Reflects pO2 Levels in Human Head and Neck Cancer Xenografts but Not in Colorectal Cancer Xenografts: Comparison with 64CuCl2

“…The mechanism responsible for 64 Cu-ATSM retention is not completely understood, but in vitro studies have indicated that the 64 Cu-ATSM complex undergoes reduction by free diffusion after entering the cells (27)(28)(29). In normoxic cells, 64 Cu-ATSM is rapidly reoxidized and consequently able to leave the cell again by free diffusion.…”