Pharmacological Inhibition of Spermine Oxidase Reduces Neurodegeneration and Improves Retinal Function in Diabetic Mice

Diabetic retinopathy (DR) is a significant cause of blindness in working-age adults worldwide. Lack of effective strategies to prevent or reduce vision loss is a major problem. Since the degeneration of retinal neurons is an early event in the diabetic retina, studies to characterize the molecular mechanisms of diabetes-induced retinal neuronal damage and dysfunction are of high significance. We have demonstrated that spermine oxidase (SMOX), a mediator of polyamine oxidation is critically involved in causing neurovascular damage in the retina. The involvement of SMOX in diabetes-induced retinal neuronal damage is completely unknown. Utilizing the streptozotocin-induced mouse model of diabetes, the impact of the SMOX inhibitor, MDL 72527, on neuronal damage and dysfunction in the diabetic retina was investigated. Retinal function was assessed by electroretinography (ERG) and retinal architecture was evaluated using spectral domain-optical coherence tomography. Retinal cryosections were prepared for immunolabeling of inner retinal neurons and retinal lysates were used for Western blotting. We observed a marked decrease in retinal function in diabetic mice compared to the non-diabetic controls. Treatment with MDL 72527 significantly improved the ERG responses in diabetic retinas. Diabetes-induced retinal thinning was also inhibited by the MDL 72527 treatment. Our analysis further showed that diabetes-induced retinal ganglion cell damage and neurodegeneration were markedly attenuated by MDL 72527 treatment. These results strongly implicate SMOX in diabetes-induced retinal neurodegeneration and visual dysfunction.

In vivo inhibition of polyamine oxidase by a spermine analogue, MDL-72527, in tomato exposed to sublethal and lethal salt stress

The spermine analogue N1,N4-bis-(2,3-butadienyl)-1,4-butanediamine (MDL-72527), an effective inhibitor of polyamine oxidases (PAOs), triggers a systemic response in tomato (Solanum lycopersicum L.) exposed to sublethal (100 mM) and lethal (250 mM) NaCl concentrations. The accumulation of free polyamines (PAs), the terminal oxidation of PAs by diamine oxidases (DAOs) and PAOs, and the production of H2O2 by PA oxidases depends on the intensity of salt stress. Spermidine and spermine content increased significantly under sublethal salt concentrations, but remained low under lethal salt stress. Along with increased expression of the selected SlDAO1 and SlPAO1 genes in the leaves and roots, respectively, DAO and PAO activities and their product, H2O2, increased and initiated cell death by irreversible loss of electrolytes at 250 mM NaCl. MDL-72527 significantly increased spermine, spermidine and/or putrescine contents as a result of reduced activity of PA oxidases; furthermore, it inhibited H2O2 and NO production during salt treatment. These results indicate that PAO contributed to H2O2 and NO production under salt stress, and the terminal activities of DAO and PAO play a role in cell death induction at 250 mM NaCl. However, the inhibition of PAO by MDL-72527 does not increase the salt tolerance of plants, since electrolyte leakage increased significantly in the presence of the inhibitor.

Effect of Lysosomotropic Polyamineoxidase Inhibitor MDL-72527 on Platelet Activation

Background/Aims: The polyamine oxidase inhibitor MDL-72527 (N1,N4-bis(2,3-butadienyl)-1,4-butanediamine) were expected to increase the abundance of spermine, a powerful inhibitor of platelet activation. Nothing is known, however, on the sensitivity of platelet function and survival to MDL-72527 exposure. The present study thus explored whether MDL-72527 modifies function and survival of platelets without and with platelet activation by collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to MDL-72527 (100 µM) with or without subsequent activation with CRP (2-5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, exposure of platelets to MDL-72527 did not significantly modify [Ca2+]i, P-selectin abundance, αIIbβ3 integrin abundance, ROS, annexin-V-binding, and forward scatter. The addition of 2-5 µg/ml CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activation, ROS abundance, annexin-V-binding, and aggregation as well as a significant decrease of forward scatter, all effects significantly blunted or virtually abolished in the presence of MDL-72527. Conclusions: MDL-72527 is a powerful inhibitor of platelet activation, apoptosis and aggregation.

MDL 72527 and spermine oxidation products induce a lysosomotropic effect and mitochondrial alterations in tumour cells

Cytotoxic products of polyamines generated in situ by an enzyme-catalysed reaction may be useful as a new avenue in combating cancer. This study demonstrated that MDR (multidrug-resistant) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding WT (wild-type) ones to H2O2 and aldehydes, the products of BSAO (bovine serum amine oxidase)-catalysed oxidation of spermine. Moreover, cytotoxicity was considerably greater when the treatment was carried out at 42°C than at 37°C. TEM (transmission electron microscopy) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. After treatment with BSAO/spermine, a higher mitochondrial membrane depolarization and an increased mitochondrial activity in drug-resistant cells were observed.