Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates.

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.