Structural and Metal Ion Effects on Human Topoisomerase IIα Inhibition by α-(N)-Heterocyclic Thiosemicarbazones.

Our previous research has shown that α-(N)-heterocyclic thiosemicarbazone (TSC) metal complexes inhibit human topoisomerase IIα (TopoIIα), while the ligands without metals do not. To find out the structural elements of TSC that are important for inhibiting TopoIIα, we have synthesized two series of α-(N)-heterocyclic TSCs with various substrate ring segments, side chain substitutions, and metal ions, and we have examined their activities in TopoIIα-mediated plasmid DNA relaxation and cleavage assays. Our goal is to explore the structure-activity relationship of α-(N)-heterocyclic TSCs and their effect on TopoIIα. Our data suggest that, similar to Cu(II)-TSCs, Pd(II)-TSC complexes inhibit plasmid DNA relaxation mediated by TopoIIα. In TopoIIα-mediated plasmid DNA cleavage assays, the Cu(II)-TSC complexes induce higher levels of DNA cleavage than their Pd(II) counterparts. The Cu(II)-TSC complexes with methyl, ethyl, and tert-butyl substitutions are slightly more effective than those with benzyl and phenyl groups. The α-(N)-heterocyclic ring substrates of the TSCs, including benzoylpyridine, acetylpyridine, and acetylthiazole, do not exhibit a significant difference in TopoIIα-mediated DNA cleavage. Our data suggest that the metal ion of TSC complexes plays a predominant role in inhibition of TopoIIα, the side chain substitution of the terminal nitrogen plays a secondary role, while the substrate ring segment has the least effect. Our molecular modeling data support the biochemical data, which together provide a mechanism by which Cu(II)-TSC complexes stabilize TopoIIα-mediated cleavage complexes.

C-peptide and zinc delivery to erythrocytes requires the presence of albumin: implications in diabetes explored with a 3D-printed fluidic device.

People with type 1 diabetes (T1D) must administer insulin exogenously due to the destruction of their pancreatic ß-cells. Endogenous insulin is stored in ß-cell granules along with C-peptide, a 31 amino acid peptide that is secreted from these granules in amounts equal to insulin. Exogenous co-administration of C-peptide with insulin has proven to reduce diabetes-associated complications in animals and humans. The exact mechanism of C-peptide's beneficial effects after secretion from the ß-cell granules is not completely understood, thus hindering its development as an exogenously administered hormone. Monitoring tissue-to-tissue communication using a 3D-printed microfluidic device revealed that zinc and C-peptide are being delivered to erythrocytes by albumin. Upon delivery, erythrocyte-derived ATP increased by >50%, as did endothelium-derived NO, which was measured downstream in the 3D-printed device. Our results suggest that hormone replacement therapy in diabetes may be improved by exogenous administration of a C-peptide ensemble that includes zinc and albumin.

C-peptide-stimulated nitric oxide production in a cultured pulmonary artery endothelium is erythrocyte mediated and requires Zn(2+).

BACKGROUND: C-peptide has been shown to stimulate the production of nitric oxide (NO) in aortic endothelial cells via activation of endothelial nitric oxide synthase (eNOS) through an increased calcium influx. Here, results obtained using cultured bovine pulmonary artery endothelial cells (bPAECs) suggest that C-peptide does not induce eNOS activation directly in cultured pulmonary artery endothelium. However, C-peptide has been shown to stimulate the release of ATP from erythrocytes, a well-documented stimulus of eNOS activity in the pulmonary endothelium. Therefore, studies were performed to examine if C-peptide can indirectly stimulate NO production in a cultured pulmonary endothelium that is erythrocyte mediated. METHODS: NO production and free intracellular calcium changes were monitored in immobilized bPAECs using specific intracellular fluorescent probes after stimulation with adenosine triphosphate (ATP), calcium ionophore A23187, or C-peptide. A microfluidic device enabled immobilized bPAECs to interact with flowing erythrocytes in the presence and absence of C-peptide to determine the role of the erythrocyte in C-peptide-stimulated NO production in cultured bPAECs. RESULTS: ATP and the calcium ionophore stimulate significant increases in both intracellular NO production and influx of free calcium in cultured bPAECs. In contrast, C-peptide, ranging from physiological to above physiological concentrations, was unable to stimulate NO production or calcium influx in the bPAECs. However, when erythrocytes were pre-incubated with a mixture containing physiological concentrations of C-peptide with Zn(2+) and haemodynamically pumped beneath bPAECs cultured on a microfluidic device, an 88.6 ± 7.5% increase in endothelial NO production was observed. CONCLUSIONS: C-peptide does not affect NO production in bPAECs directly but can impact NO production through an erythrocyte-mediated mechanism. Furthermore, in the absence of Zn(2+), C-peptide does not stimulate this NO production directly or indirectly. These results suggest that C-peptide, in the presence of Zn(2+), may be a determinant in purinergic receptor signalling via its ability to stimulate the release of ATP from erythrocytes.

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream.

Inspired by previous reports, our group has recently demonstrated that C-peptide exerts beneficial effects upon interactions with red blood cells (RBCs). These effects can be measured in RBCs obtained from animal models of both type 1 diabetes and type 2 diabetes, though to different extents. To date, the key metrics that have been measured involving C-peptide and RBCs include an increase in glucose uptake by these cells and a subsequent increase in adenosine triphosphate (ATP) release. Importantly, to date, our group has only been able to elicit these beneficial effects when the C-peptide is prepared in the presence of Zn2+. The C-peptide-induced release of ATP is of interest when considering that ATP is a purinergic signaling molecule known to stimulate the production of nitric oxide (NO) in the endothelium and in platelets. This NO production has been shown to participate in smooth muscle relaxation and subsequent vessel dilation. Furthermore, NO is a well-established platelet inhibitor. The objective of this review is to provide information pertaining to C-peptide activity on RBCs. Special attention is paid to the necessity of Zn2+ activation, and the origin of that activation in vivo. Finally, a mechanism is proposed that explains how C-peptide is exerting its effects on other cells in the bloodstream, particularly on endothelial cells and platelets, via its ability to stimulate the release of ATP from RBCs.