Pharmacological Ascorbate Elicits Anti-Cancer Activities against Non-Small Cell Lung Cancer through Hydrogen-Peroxide-Induced-DNA-Damage.

Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH-; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH- on NSCLC and elucidate its underlying mechanisms. P-AscH- treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH- in NSCLC therapy. Treatments with P-AscH- increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH- on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH-. The data from this current research advance our understanding of P-AscH- in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.

mTORC2 interactome and localization determine aggressiveness of high-grade glioma cells through association with gelsolin.

mTOR complex 2 (mTORC2) has been implicated as a key regulator of glioblastoma cell migration. However, the roles of mTORC2 in the migrational control process have not been entirely elucidated. Here, we elaborate that active mTORC2 is crucial for GBM cell motility. Inhibition of mTORC2 impaired cell movement and negatively affected microfilament and microtubule functions. We also aimed to characterize important players involved in the regulation of cell migration and other mTORC2-mediated cellular processes in GBM cells. Therefore, we quantitatively characterized the alteration of the mTORC2 interactome under selective conditions using affinity purification-mass spectrometry in glioblastoma. We demonstrated that changes in cell migration ability specifically altered mTORC2-associated proteins. GSN was identified as one of the most dynamic proteins. The mTORC2-GSN linkage was mostly highlighted in high-grade glioma cells, connecting functional mTORC2 to multiple proteins responsible for directional cell movement in GBM. Loss of GSN disconnected mTORC2 from numerous cytoskeletal proteins and affected the membrane localization of mTORC2. In addition, we reported 86 stable mTORC2-interacting proteins involved in diverse molecular functions, predominantly cytoskeletal remodeling, in GBM. Our findings might help expand future opportunities for predicting the highly migratory phenotype of brain cancers in clinical investigations.

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

Combined proteomic strategies for in-depth venomic analysis of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand.

This study focuses on comprehensive characterization of the venom proteome of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand. H. schistosus can be considered as the deadliest sea snake commonly found in the Pacific and Indian oceans. Their envenomation causes muscular paralysis and rhabdomyolysis. To develop effective treatment for this snakebite, it is necessary to understand the detailed venom composition. In this study, multiple mass spectrometry-based approaches were employed. Bottom-up proteomics revealed that tryptic digestion in-solution provided a higher number of toxin proteins identified and a larger sequence coverage, compared to in-gel digestion. In addition, a venom gland transcriptome-derived database was constructed and used as a reference, which 43 known and novel toxin proteins were identified using this database and the UniProtKB. Three-finger toxin and phospholipase A2 were shown to be top two most abundant protein families. Minor compositions included other toxin families and a number of non-toxin proteins. Moreover, a hybrid de novo sequencing was performed to enhance identification of the small proteins/peptides. Using non-digested samples, there were 46 predicted toxin peptides. The finding from this study could lead to a better understanding in pathological effects of the snakebite and the future development of effective antivenoms. SIGNIFICANCE: This study provides a better understanding of the venom proteome composition of the beaked sea snake (H. schistosus) found in the Gulf of Thailand, using a combination of different sample preparation techniques, Serpentes protein database searching, transcriptome-derived protein database searching, and a hybrid de novo peptide sequencing strategy. It revealed 13 toxin protein families and novel proteins in the beaked sea snake venom including new species of phospholipase A2s (PLA2s) and three-finger toxins (3FTxs). It could serve as a basis for the development of snakebite treatments and for the discovery of novel pharmaceutical drugs from the toxin peptides.

A urinary proteomic study in hypercalciuric dogs with and without calcium oxalate urolithiasis.

Background and Aims: Hypercalciuria is an important predisposing factor commonly found in humans and dogs with calcium oxalate (CaOx) urolithiasis. Calcium oxalate crystals can induce an inflammatory reaction that subsequently produces several proteins that have an inhibitory or stimulatory effect on stone formation. This study aimed to evaluate the differences in urinary proteomic profiles between hypercalciuric CaOx stone dogs and hypercalciuric stone-free dogs (CaOx stone and control groups, respectively). Materials and Methods: Seven dogs with hypercalciuric CaOx urolithiasis and breed-, sex-, and aged-matched controls with hypercalciuria were included in the study. Serum and urine samples were obtained from all dogs to analyze electrolytes. Urinary proteomic profiles were analyzed using liquid chromatography-mass spectrometry. Student's t-test was used to compare the differences between groups. Results: Forty-nine urinary proteins were identified in the stone-free and CaOx stone groups, whereas 19 and 6 proteins were unique in the CaOx stone and stone-free groups, respectively. The urinary thrombomodulin level was significantly higher in the CaOx stone group (relative ratio = 1.8, p < 0.01) than in the stone-free group. Conclusion: This study demonstrated that urinary proteomic profiles may be used as a candidate biomarker for urinary tract injury in CaOx urolithiasis in dogs.

cGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus.

Introduction: Type I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms. Methods: This study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis. Results: The Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages. Conclusion: These findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus.

Vector control, the most efficient tool to reduce mosquito-borne disease transmission, has been compromised by the rise of insecticide resistance. Recent studies suggest the potential of mosquito-associated microbiota as a source for new biocontrol agents or new insecticidal chemotypes. In this study, we identified a strain of Serratia marcescens that has larvicidal activity against Anopheles dirus, an important malaria vector in Southeast Asia. This bacterium secretes heat-labile larvicidal macromolecules when cultured under static condition at 25°C but not 37°C. Two major protein bands of approximately 55 kDa and 110 kDa were present in spent medium cultured at 25°C but not at 37°C. The Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of these two protein bands identified several proteases and chitinases that were previously reported for insecticidal properties against agricultural insect pests. The treatment with protease and chitinase inhibitors led to a reduction in larvicidal activity, confirming that these two groups of enzymes are responsible for the macromolecule's toxicity. Taken together, our results suggest a potential use of these enzymes in the development of larvicidal agents against Anopheles mosquitoes.

Analysis of the Zika and Japanese Encephalitis Virus NS5 Interactomes.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

Urinary sulfated glycosaminoglycan insufficiency and chondroitin sulfate supplement in urolithiasis.

Familial members of urolithiasis have high risk for stone development. We observed the low sulfated glycosaminoglycan (GAG) excretion in urolithiasis patients and their descendants. In this study, we investigated urinary excretion of sulfated GAG, chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) in urolithiasis and their children, and explored the effect of CS and HA supplement in urolithic hyperoxaluric rats. The 24-hour urines were collected from urolithiasis patients (28) and their children (40), as well as healthy controls (45) and their children (33) to measure urinary sulfated GAG, CS, HS and HA excretion rate. Our result showed that urinary sulfated GAG and CS were diminished in both urolithiasis patients and their children, while decreased HS and increased HA were observed only in urolithiasis patients. Percentage of HS per sulfated GAG increased in both urolithiasis patients and their children. In hyperoxaluric rats induced by ethylene glycol and vitamin D, we found that CS supplement could prevent stone formation, while HA supplement had no effect on stone formation. Our study revealed that decreased urinary GAG and CS excretion are common in familial members of urolithiasis patients, and CS supplement might be beneficial in calcium oxalate urolithiasis prophylaxis for hyperoxaluric patients.

Quantitative proteomic analysis of dermal papilla from male androgenetic alopecia comparing before and after treatment with low-level laser therapy.

BACKGROUND: Currently, low-level laser therapy (LLLT) has been approved as a new treatment for androgenetic alopecia (AGA). However, it has not been elucidated how LLLT promotes hair growth in vivo. OBJECTIVES: To investigate the change in protein expression from dermal papilla (DP) tissues in male AGA patients after LLLT treatment using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. METHODS: This is an open-label, prospective, single-arm study obtained punch scalp biopsy specimens from patients with AGA before and after LLLT treatment. Each subject was self-treated with helmet type of LLLT (655 nm, 5 mW) device at home for 25 minutes per treatment every other day for 24 weeks. LC-MS/MS analysis based on the dimethyl labeling strategy for protein quantification was used to identify proteins expressed in DP tissues from AGA patients. RESULTS: Proteomic analysis revealed 11 statistically significant up-regulated and 2 down-regulated proteins in LLLT treated DP compared with baseline (P < 0.05). A bioinformatic analysis signifies that these proteins are involved in several biological processes such as regulation of cellular transcription, protein biosynthesis, cell energy, lipid homeostasis, extracellular matrix (ECM), ECM structural constituent, cell-cell/cell-matrix adhesion as well as angiogenesis. ATP-binding cassette sub-family G member, a transporter involved in cellular lipid homeostasis, was the most up-regulated protein. Additionally, LLLT increased the main ECM proteins in DP which results in a bigger volume of DP and a clinical improvement of hair diameter in AGA patients. CONCLUSION: We identified the proteome set of DP proteins of male patients with AGA treated with LLLT which implicates the role of LLLT in promoting hair growth and reversing of miniaturization process of AGA by enhancing DP cell function. Our results strongly support the benefit of LLLT in the treatment of AGA. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Lime powder treatment reduces urinary excretion of total protein and transferrin but increases uromodulin excretion in patients with urolithiasis.

Our previous study has shown that lime powder (LP) had an inhibitory effect against calcium oxalate stone formation. However, the precise mechanisms underlying such beneficial effect remained unclear. Our present study thus aimed to address the effect of LP on excretory level and compositions of urinary proteins using a proteomics approach. From a total of 80 calcium oxalate stone formers recruited into our 2-year randomized clinical trial of LP effect, 10 patients with comparable age and clinical parameters were selected for this proteomic study. 24-h urine specimens were collected from all subjects, at baseline (before) and after LP treatment for 6 months, and then subjected to quantitative proteomics analysis and subsequent validation by ELISA. Total urinary protein excretion was significantly decreased by LP treatment, but unaffected by placebo. Nanoflow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) followed by quantitative analysis revealed 17 proteins whose levels were significantly altered (16 decreased and 1 increased) exclusively by LP treatment. Among these, the decrease of transferrin and increase of uromodulin were validated by ELISA. Moreover, there was a significant correlation between microalbuminuria and urinary transferrin level by Pearson's correlation test. In summary, LP treatment caused significant reduction in total urinary protein excretion and changes in urinary protein compositions that could be linked to stone inhibitory effects and might be relevant mechanisms responsible for the beneficial effects of LP to prevent kidney stone formation and recurrence.

Plumbagin Enhances Tamoxifen Sensitivity and Inhibits Tumor Invasion in Endocrine Resistant Breast Cancer through EMT Regulation.

Tamoxifen is widely used as the first line drug for estrogen receptor-positive subtype which is expressed in 70% of overall breast cancer patients. However, approximately 50% of these patients develop acquired resistance after 5 years of treatment, which is characterized by tumor recurrence and metastasis. The epithelial mesenchymal transition (EMT) is an important process in breast cancer invasion. Fundamentally, targeting the EMT represents a crucial therapeutic strategy for preventing or treating breast cancer metastasis. Plumbagin (PLB) is a natural naphthoquinone with significant anticancer effects against several types of tumor cells including breast cancer. In this study, we investigated the effect of PLB on human endocrine-resistant breast cancer cell growth, invasion and the possible mechanisms underlying such actions. PLB exhibited potent cytotoxic activity at a micromolar concentration against endocrine-resistant breast cancer cells. Interestingly, a fixed low concentration of PLB and tamoxifen combination resulted in an increase in growth inhibition in endocrine-resistant cells. In addition, PLB also significantly suppressed mesenchymal biomarker expressions that govern the EMT process, resulting in attenuated metastatic capabilities. In conclusion, PLB should be developed as a pharmacological agent for the use as a single treatment or in combination for endocrine-resistant breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.