Modulation of two-dimensional palladium nanozyme activity to enhance chemodynamic/photothermal combined therapy for melanoma.

Nanozymes are artificial enzymes that mimic natural enzyme-like activities and exhibit tremendous potential for tumor chemodynamic therapy. However, the development of novel nanozymes with superior catalytic activities for nanotheranostics remains a formidable challenge. Herein, we report a facile synthesis of monodisperse palladium nanosheets (Pd nanosheets) and their assembly on graphene oxide (GO) that enhances the catalytic activities of Pd nanoparticles. Simultaneously, the obtained nanocomposites (rGO-Pd) could be applied as a smart near-infrared (NIR) light-responsive nanotheranostic for near infrared imaging-guided chemodynamic/photothermal combined therapy. Notably, rGO-Pd exhibited high peroxidase mimicking activities, which could catalyze the conversion of intratumoral H2O2 to ˙OH. Impressively, the reactive oxygen species (ROS) generation of rGO-Pd was further remarkably enhanced by the endogenous acidity of the tumor microenvironment and the exogenous NIR light-responsive photothermal effect. These collective properties of the rGO-Pd nanozyme enabled it to be a ROS generation accelerator for photothermally enhanced tumor chemodynamic therapy. Thus, the as-developed rGO-Pd may represent a promising new type of high-performance nanozyme for multifunctional nanotheranostics toward cancer.

Displays of 18 F-FAPI PET/CT in Myocarditis With Variable Symptom Durations.

ABSTRACT: We report cardiac 18 F-FAPI PET/CT findings in 3 cases with myocarditis of varying duration (7 hours, 1 week, and 1 month). Myocarditis with varying symptom durations showed different 18 F-FAPI uptake, suggesting that the 18 F-FAPI PET/CT may be helpful in evaluating the extent of fibrosis caused by myocarditis. This information may assist in treatment decision-making for patients with myocarditis.

Mutations in V-ATPase in follicular lymphoma activate autophagic flux creating a targetable dependency.

The recent discovery of recurrent gene mutations in chaperones or components of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) was an unexpected finding. The application of whole exome sequencing and targeted gene re-sequencing has resulted in the identification of mutations in ATP6AP1, ATP6V1B2 and VMA21 in a combined 30% of FL, together constituting a major novel mutated pathway in this disease. Interestingly, no other human hematological malignancy carries these mutations at more than sporadic occurrences, implicating unique aspects of FL biology requiring these mutations. The mutations in ATP6V1B2 and VMA21 through separate mechanisms impair lysosomal V-ATPase activity resulting in an elevated lysosomal pH. The elevated lysosomal pH impairs protein and peptide hydrolysis and associates with reduced cytoplasmic amino acid concentrations resulting in compensatory activation of autophagic flux. The elevated autophagic flux constitutes a survival dependency for FL cells and can be targeted with inhibitors to ULK1 and multiple recently identified cyclin-dependent kinase inhibitors. Targeting autophagy alone or in combination with other targeted therapies constitutes a novel therapeutic opportunity for FL patients.

Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase activity prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.Abbreviations: ALs: autolysosomes; APs: autophagosomes; ER: endoplasmic reticulum; FL: follicular lymphoma; GFP: green fluorescent protein; IP: immunoprecipitation; LE/LY: late endosomes/lysosomes; Lyso-IP: lysosomal immunoprecipitation; OST: oligosaccharide transferase; prApe1: precursor aminopeptidase I; SEP: super ecliptic pHluorin; V-ATPase: vacuolar-type H+-translocating ATPase.

Chronic hypoperfusion due to intracranial large artery stenosis is not associated with cerebral ß-amyloid deposition and brain atrophy.

BACKGROUND: Insufficient cerebral perfusion is suggested to play a role in the development of Alzheimer disease (AD). However, there is a lack of direct evidence indicating whether hypoperfusion causes or aggravates AD pathology. We investigated the effect of chronic cerebral hypoperfusion on AD-related pathology in humans. METHODS: We enrolled a group of cognitively normal patients (median age: 64 years) with unilateral chronic cerebral hypoperfusion. Regions of interest with the most pronounced hypoperfusion changes were chosen in the hypoperfused region and were then mirrored in the contralateral hemisphere to create a control region with normal perfusion. 11C-Pittsburgh compound-positron emission tomography standard uptake ratios and brain atrophy indices were calculated from the computed tomography images of each patient. RESULTS: The median age of the 10 participants, consisting of 4 males and 6 females, was 64 years (47-76 years). We found that there were no differences in standard uptake ratios of the cortex (volume of interest [VOI]: P = 0.721, region of interest [ROI]: P = 0.241) and grey/white ratio (VOI: P = 0.333, ROI: P = 0.445) and brain atrophy indices (Bicaudate, Bifrontal, Evans, Cella, Cella media, and Ventricular index, P > 0.05) between the hypoperfused regions and contralateral normally perfused regions in patients with unilateral chronic cerebral hypoperfusion. CONCLUSION: Our findings suggest that chronic hypoperfusion due to large vessel stenosis may not directly induce cerebral ß-amyloid deposition and neurodegeneration in humans.

Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation.

PURPOSE: On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. EXPERIMENTAL DESIGN: We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. RESULTS: We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. CONCLUSIONS: Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

Follicular lymphoma-associated mutations in vacuolar ATPase ATP6V1B2 activate autophagic flux and mTOR.

The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to mTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the human v-ATPase subunit ATP6V1B2 (also known as Vma2 in yeast) activate autophagic flux and maintain mTOR/TOR in an active state. Engineered lymphoma cell lines and primary FL B cells carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutations. These data support the idea of mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a possible new therapeutically targetable pathway.

PAC-1 and its derivative WF-210 Inhibit Angiogenesis by inhibiting VEGF/VEGFR pathway.

Procaspase Activating Compound-1 (PAC-1) and its derivative WF-210 induce apoptosis in cancer cells by activating procaspase-3 to caspase-3. The aim of this study was to extend current knowledge about the mechanisms of PAC-1 and WF-210, particularly about their effects on tumor angiogenesis. PAC-1 and WF-210 restrained VEGF-induced human umbilical vascular endothelial cells (HUVECs) proliferation, invasion, and tube formation. PAC-1 and WF-210 abrogated VEGF-induced vessel sprouting from rat aortic rings and inhibited vascular formation in the Matrigel plug assay. PAC-1 and WF-210 suppressed phosphorylation of VEGFR2 and its downstream protein kinases c-Src, FAK, and AKT in both HUVECs and U-87 cells. When given to mice bearing subcutaneous or orthotopic xenograft, PAC-1 and WF-210 inhibited the tumor growth and tumor angiogenesis. Further tests showed that PAC-1 and WF-210 inhibited stemness and induced autophagy flux of U-87 cells. This study revealed mechanisms of PAC-1 and WF-210 other than inducing apoptosis, which provides additional support for their using in the clinic.

The roles of conserved aromatic residues (Tyr5 and Tyr42) in interaction of scorpion toxin BmK AGP-SYPU1 with human Nav1.7.

Scorpion toxins are invaluable source of therapeutic leads and pharmacological tools which produce influence on the voltage gated sodium channels. In the previous study, our group has reported BmK AGP-SYPU1 (64 amino acids), one scorpion toxin with both potential α-type and ß-type scorpion characteristics and analgesic activity in vivo, act as an activator to hNav1.4 and hNav1.5. Additionally, conserved aromatic amino acids Tyr5 and Tyr42 played important roles in bioactivity of BmK AGP-SYPU1 on hNav1.4 and hNav1.5. However, the anti-nonceptitor mechanisms of BmK AGP-SYPU1 referred in vivo have not been clarified yet. The roles of Tyr5 and Tyr42 have still kept unclear in the interaction of BmK AGP-SYPU1 with other VGSCs. In this study, in order to give a deep insight into the relationship of structure and function, the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNav1.7, which has emerged as a key molecule involved in pain processing, were examined by using Na+-specialized fluorescent dye and the whole-cell patch clamp. The data showed that BmK AGP-SYPU1 also displayed as an activator to hNav1.7 with the potential characteristic of α-type and ß-type scorpion toxin. Both Tyr5 and Tyr42 were the key amino acids to the functional properties of BmK AGP-SYPU1 when interacting with hNav1.7, and the Tyr42 was involved in the preferences of the toxin to distinct action sites of hNav1.7. On the whole, these data provided more information about the structure of BmK AGP-SYPU1 related to its function upon hNav1.7, and may bring some clues for engineering scorpion toxins to achieve high bioactivity with lower side effects.

Synthesis and evaluation of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives as non-purine xanthine oxidase inhibitors.

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid, whose overproduction leads to the gout-causing hyperuricemia. In this study, a series of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives (4a-4k and 6a-6k) was synthesized and evaluated for their inhibitory potency against xanthine oxidase. The 1-hydroxyl substituted derivatives 4a-4k showed excellent inhibitory potency with IC50 values ranging from 0.003 µM to 1.2 µM, with compounds 4d (IC50 = 0.003 µM), 4e (IC50 = 0.003 µM), and 4f (IC50 = 0.006 µM) manifesting the most potent xanthine oxidase inhibitory potency that were comparable with that of Febuxostat (IC50 = 0.01 µM). Lineweaver-Burk plot analysis revealed that representative compound 4f acted as a mixed-type inhibitor for xanthine oxidase. The basis of significant inhibition of xanthine oxidase by 4f was rationalized by its molecular docking into the active site of xanthine dehydrogenase.

Copper improves the anti-angiogenic activity of disulfiram through the EGFR/Src/VEGF pathway in gliomas.

Disulfiram (DSF) possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. DSF also potently inhibits angiogenesis, but the effect of Cu on this anti-angiogenic activity is unknown. Here we show that DSF inhibits the proliferation, migration, invasion, adhesion and complex tube formation of human umbilical vascular endothelial cells (HUVECs). Aortic ring assays and Matrigel plug assays revealed that DSF significantly inhibited the formation of microvessels. Importantly, Cu improved the anti-angiogenic activity of DSF in all these assays, while copper alone had no effect. DSF/Cu treatment of U87 human glioblastoma cells resulted in suppression of VEGF secretion through the EGFR/c-Src/VEGF pathway. Reduction of EGFR phosphorylation disables recruitment of multiple Src homology 2 (SH2) domains, resulting in transcriptional down-regulation of VEGF. The role of EGFR/c-Src/VEGF pathway was further confirmed by using specific inhibitor, which significantly improved the anti-angiogenic activity of DSF/Cu. DSF/Cu also exerted increased anti-tumor effects on subcutaneous and intracerebral U87 xenograft models by reducing microvessel density (MVD) and VEGF expression. These results indicate that Cu improves the anti-angiogenic activity of DSF by targeting the EGFR/Src/VEGF signaling pathway, thus providing a rationale for the use of DSF/Cu rather than DSF alone as an angiogenesis inhibitor in clinical applications.

The Functional Property Changes of Muscular Na(v)1.4 and Cardiac Na(v)1.5 Induced by Scorpion Toxin BmK AGP-SYPU1 Mutants Y42F and Y5F.

Scorpion toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure-function relationship of scorpion toxins on VGSC subtypes. In the previous study, we reported one α-type scorpion toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNa(v)1.4 and hNa(v)1.5 heterologously expressed CHO cell lines by using Na⁺-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNa(v)1.4 and hNa(v)1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and ß-type scorpion toxin. Notably, Y5F mutant exhibited lower activatory effects on hNa(v)1.4 and hNa(v)1.5 compared with BmK AGP-SYPU1. Y42F was an enhanced activator and confirmed that the conserved Tyr42 was the key amino acid involved in bioactivity or biotoxicity. These data provided a deep insight into the structure-function relationship of BmK AGP-SYPU1, which may be the guidance for engineering α-toxin with high selectivity on VGSC subtypes.

Targeting procaspase-3 with WF-208, a novel PAC-1 derivative, causes selective cancer cell apoptosis.

Caspase-3 is a critical effector caspase in apoptosis cascade, and is often over-expressed in many cancer tissues. The first synthesized procaspase-3 activator, PAC-1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase-3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF-208 stood out by its high cytotoxicity against procaspase-3 overexpressed HL-60 cells. Compared with PAC-1, WF-208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF-208 activated procaspase-3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase-3-dependent cell death in tumour cells, which possibly because of the zinc-chelating properties. WF-208 also showed greater antitumour activity than PAC-1 in murine xenograft model. In conclusion, we have discovered WF-208 as a promising procaspase-3 activating compound, with higher activity and higher cell selectivity than PAC-1.

A novel small-molecule activator of procaspase-3 induces apoptosis in cancer cells and reduces tumor growth in human breast, liver and gallbladder cancer xenografts.

PURPOSE: Procaspase-3, a proenzyme of apoptotic executioner caspase-3, is overexpressed in numerous tumors. We aimed to characterize a novel procaspase-3 activator, WF-210, which may have potential as an anticancer drug. EXPERIMENTAL DESIGN: The procaspase-3 activating ability, antitumor efficacy, mechanisms of action, and toxicity profiles of WF-210 were investigated in vitro and in vivo, using normal cells, cancer cells, and mouse xenograft models. The role of procaspase-3 in WF-210-induced apoptosis was explored by manipulating procaspase-3 expression in cultured cells. RESULTS: WF-210 activated procaspase-3 with an EC50 of 0.95 µM, less than half that of its mother compound PAC-1 (2.08 µM). The mechanism involved the chelation of inhibitory zinc ions, subsequently resulting in an auto-activation of procaspase-3. WF-210 was more cytotoxic than PAC-1 to human cancer cells, but less cytotoxic to normal cells. Cancer cells with high procaspase-3 expression, like HL-60 and U-937, were particularly sensitive. WF-210-induced the apoptosis of HL-60 and U-937 cells by activating procaspases and promoting proteasome-dependent degradation of XIAP and Survivin. The level of WF-210-induced apoptosis in cultured cells was related to the level of procaspase-3 expression. Finally, WF-210 was superior to PAC-1 in retarding the in vivo growth of breast, liver and gallbladder xenograft tumors which overexpress procaspase-3, and induced no substantial weight loss or neurotoxicity. WF-210 and PAC-1 had no effect on the growth of MCF-7 xenograft tumors, which do not express procaspase-3. CONCLUSION: We identified WF-210 as a potent small-molecule activator of procaspase-3. The favorable antitumor activity and acceptable toxicity profile of WF-210 provide a strong rationale for its clinical evaluation in the treatment of tumors with high procaspase-3 expression.

NMDA receptors in the medial prefrontal cortex and the dorsal hippocampus regulate methamphetamine-induced hyperactivity and extracellular amino acid release in mice.

The medial prefrontal cortex (mPFC) and the dorsal hippocampus (DHC) play significant roles in stimulant-induced neurobehavioral effects. Methamphetamine (MAP)-induced hyperactivity has been reported to be involved in the regulation of the glutamatergic system. The present study examined whether the glutamatergic and GABAergic systems in the mPFC and DHC were involved in MAP-induced hyperactivity in mice. A combined kainic acid (KA) or N-methyl-d-aspartate (NMDA) lesion and microdialysis technique targeting both the mPFC and DHC were used. The results showed that both KA- and NMDA-induced lesions of the mPFC facilitated MAP-induced hyperactivity, while neither KA- nor NMDA-induced lesions of the DHC had a similar effect. MAP increased the extracellular glutamate (Glu) levels in the mPFC and reduced Glu levels in the DHC. GABA levels in both of these regions were reduced. A KA or NMDA lesion of the mPFC inhibited the Glu reduction in the DHC, and the same lesion of the DHC inhibited the Glu increase in the mPFC induced by MAP. A NMDA lesion of the mPFC blocked GABA reduction in the DHC, but a lesion of DHC enhanced the GABA decrease in the mPFC induced by MAP. Furthermore, a NMDA lesion of DHC increased the vesicular glutamate transporter-2 (VGLUT2) expression in the mPFC following MAP-administration. These findings indicate that glutamatergic as well as GABAergic systems in these two regions are involved in MAP-induced hyperactivity. Moreover, there may be an inhibitory role in these two regions, especially mediated by NMDA receptors, in MAP-induced abnormal behavior and neurotransmission responses.