Role of 3-Mercaptopyruvate Sulfurtransferase (3-MST) in Physiology and Disease.

3-mercaptopyruvate sulfurtransferase (3-MST) plays the important role of producing hydrogen sulfide. Conserved from bacteria to Mammalia, this enzyme is localized in mitochondria as well as the cytoplasm. 3-MST mediates the reaction of 3-mercaptopyruvate with dihydrolipoic acid and thioredoxin to produce hydrogen sulfide. Hydrogen sulfide is also produced through cystathionine beta-synthase and cystathionine gamma-lyase, along with 3-MST, and is known to alleviate a variety of illnesses such as cancer, heart disease, and neurological conditions. The importance of cystathionine beta-synthase and cystathionine gamma-lyase in hydrogen sulfide biogenesis is well-described, but documentation of the 3-MST pathway is limited. This account compiles the current state of knowledge about the role of 3-MST in physiology and pathology. Attempts at targeting the 3-MST pathway for therapeutic benefit are discussed, highlighting the potential of 3-MST as a therapeutic target.

Deletion of Glyoxalase 1 exacerbates acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC) has a narrow therapeutic window and early treatment is essential for satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late-presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end-products (AGEs) and consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase-1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in APAP mediated activation of RAGE and downstream cell-death cascades. Constitutive Glo-1 knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were employed as tools. Our findings show elevated oxidative stress, activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild type controls. A unique feature of the hepatic necrosis in GKO mice is the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than inflammation seen in wild type. The GSH surrogate and general antioxidant, ψ-GSH alleviated APAP toxicity irrespective of Glo-1 status, suggesting that oxidative stress being the primary driver of APAP toxicity. Overall, exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against initial stages of APAP overdose.

Sulfanegen stimulates 3-mercaptopyruvate sulfurtransferase activity and ameliorates Alzheimer's disease pathology and oxidative stress in vivo.

Increased oxidative stress and inflammation are implicated in the pathogenesis of Alzheimer's disease. Treatment with hydrogen sulfide (H2S) and H2S donors such as sodium hydrosulfide (NaSH) can reduce oxidative stress in preclinical studies, however clinical benefits of such treatments are rather ambiguous. This is partly due to poor stability and bioavailability of the H2S donors, requiring impractically large doses that are associated with dose-limiting toxicity. Herein, we identified a bioavailable 3-mercaptopyruvate prodrug, sulfanegen, which is able to pose as a sacrificial redox substrate for 3-mercaptopyruvate sulfurtransferase (3MST), one of the H2S biosynthetic enzymes in the brain. Sulfanegen is able to mitigate toxicity emanating from oxidative insults and the Aß1-42 peptide by releasing H2S through the 3MST pathway. When administered to symptomatic transgenic mouse model of AD (APP/PS1; 7 and 12 months) and mice that were intracerebroventricularly administered with the Aß1-42 peptide, sulfanegen was able to reverse oxidative and neuroinflammatory consequences of AD pathology by restoring 3MST function. Quantitative neuropathological analyses confirmed significant disease modifying effect of the compound on amyloid plaque burden and brain inflammatory markers. More importantly, sulfanegen treatment attenuated progressive neurodegeneration in these mice, as evident from the restoration of TH+ neurons in the locus coeruleus. This study demonstrates a previously unknown concept that supplementation of 3MST function in the brain may be a viable approach for the management of AD. Finally, brought into the spotlight is the potential of sulfanegen as a promising AD therapeutic for future drug development efforts.

Embelin averts MPTP-induced dysfunction in mitochondrial bioenergetics and biogenesis via activation of SIRT1.

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by the death of dopamine neurons of Substantia nigra pars compacta (SNpc) leading to motor deficits. Amongst the mechanisms proposed, mitochondrial dysfunction, reduced complex-I and PGC1α levels were found to correlate with the pathology of PD. As embelin is a natural product with structural resemblance to ubiquinone, exhibits mitochondrial uncoupling and antioxidant effects, in the present study, we sought to examine its role in the mechanisms mediating PD. Results indicate that embelin protects from MPP+-induced oxidative stress and apoptosis in a time and dose-dependent manner in N27 dopaminergic cells. Cells treated with embelin exhibited increased levels of pAMPK, SIRT1 and PGC1α leading to enhanced mitochondrial biogenesis. Though treatment of cells with MPP+ also increased pAMPK levels, but, SIRT1 and PGC1α levels decreased substantially, possibly due to the block in the mitochondrial electron transport chain and reduced NAD/NADH levels. The mitochondrial uncoupling effects of embelin leading to increased NAD/NADH levels followed by enhanced SIRT1, PGC1α and mitochondrial biogenesis were found to confer embelin mediated protection as treatment of cells with SIRT1 inhibitor or siRNA nullified this effect. Embelin (10 mg/kg) also conferred protection in vivo in MPTP mouse model of PD, wherein, MPTP-induced loss of TH staining, reduced striatal dopamine and markers of mitochondrial biogenesis pathway were averted by embelin.

The deglycase activity of DJ-1 mitigates α-synuclein glycation and aggregation in dopaminergic cells: Role of oxidative stress mediated downregulation of DJ-1 in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the degeneration of dopamine neurons of the substantia nigra pars compacta (SNpc) and the presence of intra-neuronal aggregates of α-synuclein and its post-translational products. Based on emerging reports on the association between glycated α-synuclein and PD; and the newly identified deglycase activity of DJ-1, we sought to find the relevance of deglycase activity of DJ-1 on glycation of α-synuclein and its plausible role in PD. Our results demonstrate that DJ-1 has a higher affinity towards the substrate methylglyoxal (MGO) (Km = 900 mM) as compared to its familial mutant, L166P (Km = 1900 mM). Also, CML α-synuclein (CML-syn) served as a substrate for the deglycase activity of DJ-1. Treatment of cells with Parkinsonian mimetic, 1-methyl-4-phenylpyridinium ion (MPP+); oxidants, such as H2O2 and methylglyoxal (MGO) lead to a dose-dependent decrease in the levels of DJ-1 with a concomitant increase in CML-syn. Also, MGO induced cytosolic α-synuclein aggregates in cells which stained positive with the anti-CML antibody. Further, unilateral stereotaxic administration of MGO into the SNpc of mice induced α-synuclein aggregates and CML-syn with a concomitant reduction in the number of TH positive neurons, protein levels of TH and DJ-1 at the site of injection. Interestingly, overexpression of DJ-1 enhanced the clearance of preformed CML-syn in cells, mitigated MGO induced CML-syn and intracellular α-synuclein aggregates. Overall, the findings of our present study demonstrate that DJ-1 plays a pivotal role in the glycation and aggregation of α-synuclein. Reduced DJ-1 activity due to mutations or oxidative stress may lead to the accumulation of glycated α-synuclein and its aggregates.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells.

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvß3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

Vascular Targeting of a Gold Nanoparticle to Breast Cancer Metastasis.

The vast majority of breast cancer deaths are due to metastatic disease. Although deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle (AuNP) to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the AuNPs, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Because of the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles.

Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells.

On-command drug release from nanochains inhibits growth of breast tumors.

PURPOSE: To evaluate the ability of radiofrequency (RF)-triggered drug release from a multicomponent chain-shaped nanoparticle to inhibit the growth of an aggressive breast tumor. METHODS: A two-step solid phase chemistry was employed to synthesize doxorubicin-loaded nanochains, which were composed of three iron oxide nanospheres and one doxorubicin-loaded liposome assembled in a 100-nm-long linear nanochain. The nanochains were tested in the 4T1-LUC-GFP orthotopic mouse model, which is a highly aggressive breast cancer model. The 4T1-LUC-GFP cell line stably expresses firefly luciferase, which allowed the non-invasive in vivo imaging of tumor response to the treatment using bioluminescence imaging (BLI). RESULTS: Longitudinal BLI imaging showed that a single nanochain treatment followed by application of RF resulted in an at least 100-fold lower BLI signal compared to the groups treated with nanochains (without RF) or free doxorubicin followed by RF. A statistically significant increase in survival time of the nanochain-treated animals followed by RF (64.3 days) was observed when compared to the nanochain-treated group without RF (35.7 days), free doxorubicin-treated group followed by RF (38.5 days), and the untreated group (30.5 days; n=5 animals per group). CONCLUSIONS: These studies showed that the combination of RF and nanochains has the potential to effectively treat highly aggressive cancers and prolong survival.