Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Differential Impact of the rpoB Mutant on Rifampin and Rifabutin Resistance Signatures of Mycobacterium tuberculosis Is Revealed Using a Whole-Genome Sequencing Assay.

Drug resistance in Mycobacterium tuberculosis (MTB) has long been a serious health issue worldwide. Most drug-resistant MTB isolates were identified due to treatment failure or in clinical examinations 3~6 months postinfection. In this study, we propose a whole-genome sequencing (WGS) pipeline via the Nanopore MinION platform to facilitate the efficacy of phenotypic identification of clinical isolates. We used the Nanopore MinION platform to perform WGS of clinical MTB isolates, including susceptible (n = 30) and rifampin- (RIF) or rifabutin (RFB)-resistant isolates (n = 20) according to results of a susceptibility test. Nonsynonymous variants within the rpoB gene associated with RIF resistance were identified using the WGS analytical pipeline. In total, 131 variants within the rpoB gene in RIF-resistant isolates were identified. The presence of the emergent Asp531Gly or His445Gln was first identified to be associated with the rifampin and rifabutin resistance signatures of clinical isolates. The results of the minimum inhibitory concentration (MIC) test further indicated that the Ser450Leu or the mutant within the rifampin resistance-determining region (RRDR)-associated rifabutin-resistant signature was diminished in the presence of novel mutants, including Phe669Val, Leu206Ile, or Met148Leu, identified in this study. IMPORTANCE Current approaches to diagnose drug-resistant MTB are time-consuming, consequently leading to inefficient intervention or further disease transmission. In this study, we curated lists of coding variants associated with differential rifampin and rifabutin resistant signatures using a single molecule real-time (SMRT) sequencing platform with a shorter hands-on time. Accordingly, the emerging WGS pipeline constitutes a potential platform for efficacious and accurate diagnosis of drug-resistant MTB isolates.

Histone Deacetylase Inhibitors Downregulate Calcium Pyrophosphate Crystal Formation in Human Articular Chondrocytes.

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-ß1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5'-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-ß1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.

H1-Antihistamines Reduce the Risk of Hepatocellular Carcinoma in Patients With Hepatitis B Virus, Hepatitis C Virus, or Dual Hepatitis B Virus-Hepatitis C Virus Infection.

PURPOSE: H1-antihistamines (AHs) may exert protective effects against cancer. This study investigated the association of AH use with the risk of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV), hepatitis C virus (HCV), or dual HBV-HCV virus infection. MATERIALS AND METHODS: Patients with HBV, HCV, or dual HBV-HCV infection were enrolled from Taiwan's National Health Insurance Research Database and examined for the period from January 1, 2006, to December 31, 2015. We used the Kaplan-Meier method and Cox proportional hazards regression to evaluate the association between AH use and HCC risk. RESULTS: We included patients with HBV infection (n = 521,071), HCV (n = 169,159), and dual HBV-HCV (n = 39,016). Patients with HBV, HCV, or dual virus infection who used AHs exhibited significantly lower risk of HCC relative to patients who did not use AH, with their adjusted hazard ratio being 0.489 (95% CI, 0.455 to 0.524), 0.484 (95% CI, 0.450 to 0.522), and 0.469 (95% CI, 0.416 to 0.529), respectively. Furthermore, there was a dose-response relationship between AH use and the risk of HCC in the HBV cohort. The adjusted hazard ratios were 0.597 (95% CI, 0.530 to 0.674), 0.528 (0.465 to 0.600), 0.470 (0.416 to 0.531), and 0.407 (0.362 to 0.457) for AH use of 28-42, 43-63, 64-119, and ≥ 120 cumulative defined daily doses, respectively, relative to no AH use. Additionally, there was also a dose-response relationship between AH use and the risk of HCC in the HCV and dual HBV-HCV cohorts. CONCLUSION: AH use may reduce the risk for HCC among patients with HBV, HCV, or dual infection in a dose-dependent manner. Further mechanistic research is needed.

A machine learning-driven approach for prioritizing food contact chemicals of carcinogenic concern based on complementary in silico methods.

Carcinogenicity is one of the most critical endpoints for the risk assessment of food contact chemicals (FCCs). However, the carcinogenicity of FCCs remains insufficiently investigated. To fill the data gap, the application of standard experimental methods for identifying chemicals of carcinogenic concerns from a large set of FCCs is impractical due to their resource-intensive nature. In contrast, computational methods provide an efficient way to quickly screen chemicals with carcinogenic potential for subsequent experimental validation. Since every model was developed based on a limited number of training samples, the use of single models for carcinogenicity assessment may not cover the complex mechanisms of carcinogenesis. This study proposed a novel machine learning-based weight-of-evidence (WoE) model for prioritizing chemical carcinogenesis. The WoE model can nonlinearly integrate complementary computational methods of structural alerts, quantitative structure-activity relationship models and in silico toxicogenomics models into a WoE-score. Compared to the best single method, the WoE model gained 8% and 19.7% improvement in the area under the receiver operating characteristic curve (AUC) value and chemical coverage, respectively. The prioritization of 1623 FCCs concludes 44 chemicals of high carcinogenic concern. The machine learning-based WoE approach provides a fast and comprehensive way for prioritizing chemicals of carcinogenic concern.

Integrative utility of long read sequencing-based whole genome analysis and phenotypic assay on differentiating isoniazid-resistant signature of Mycobacterium tuberculosis.

BACKGROUND: With the advancement of next generation sequencing technologies (NGS), whole-genome sequencing (WGS) has been deployed to a wide range of clinical scenarios. Rapid and accurate classification of drug-resistant Mycobacterium tuberculosis (MTB) would be advantageous in reducing the amplification of additional drug resistance and disease transmission. METHODS: In this study, a long-read sequencing approach was subjected to the whole-genome sequencing of clinical MTB clones with susceptibility test profiles, including isoniazid (INH) susceptible clones (n = 10) and INH resistant clones (n = 42) isolated from clinical specimens. Non-synonymous variants within the katG or inhA gene associated with INH resistance was identified using Nanopore sequencing coupled with a corresponding analytical workflow. RESULTS: In total, 54 nucleotide variants within the katG gene and 39 variants within the inhA gene associated with INH resistance were identified. Consistency among the results of genotypic profiles, susceptibility test, and minimal inhibitory concentration, the high-INH resistance signature was estimated using the area under the receiver operating characteristic curve with the existence of Ser315Thr (AUC = 0.822) or Thr579Asn (AUC = 0.875). CONCLUSIONS: Taken together, we curated lists of coding variants associated with differential INH resistance using Nanopore sequencing, which may constitute an emerging platform for rapid and accurate identification of drug-resistant MTB clones.

MCPIP1 Enhances TNF-α-Mediated Apoptosis through Downregulation of the NF-κB/cFLIP Axis.

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.

Ugonin J improves metabolic disorder and ameliorates nonalcoholic fatty liver disease by regulating the AMPK/AKT signaling pathway.

Closely associated with visceral obesity, hepatic steatosis resulting from non-alcoholic fatty liver disease (NAFLD) exacerbates insulin resistance. Developing effective drugs to treat NAFLD is imperative. Here, we investigated the pharmacological mechanism of ugonin J (UJ) in controlling metabolic disorder and ameliorating NAFLD pathophysiology in diet-induced obese mice. The effects of UJ were assessed in 5-week-old C57BL/6 J mice fed a high-fat diet (HFD) for 12 weeks. UJ treatment averted HFD-induced body weight gain by reducing fat deposition in adipose tissues and reduced HFD-induced hyperlipidemia and hepatic inflammation. UJ also improved HFD-induced glucose tolerance and insulin resistance. Moreover, the mode of action of UJ was analyzed in palmitate (PA)-induced steatotic human HuS-E/2 hepatocytes and in hyperglycemia-simulating rat BRIN-BD11 pancreatic ß cells. In PA-induced steatotic human hepatocytes, UJ treatment promoted lipid clearance via pAMPK, pACC and CPT-1 upregulation and SREBP-1c downregulation. Interestingly, UJ upregulated Akt activity in hepatocytes and increased insulin secretion from ß cells in acute insulin secretion tests. Taken together, UJ improved adipocyte hypertrophy, hyperinsulinemia, hyperglycemia, hyperlipidemia and fat deposition in livers. UJ also reduced fatty acid accumulation by modulating key metabolic regulators. Our findings demonstrated the therapeutic potential of UJ for the treatment of NAFLD and diet-induced metabolic disorders.

The Antihistamine Deptropine Induces Hepatoma Cell Death through Blocking Autophagosome-Lysosome Fusion.

Some antihistamines have exhibited significant antitumor activity alone or in combination with other therapies in in vitro and clinical studies. However, the underlying mechanisms of how antihistamines inhibit hepatocellular carcinoma proliferation are still unknown. We first screened the antiproliferation activity of 12 benzocycloheptene structural-analogue drugs, and results showed that deptropine was the most potent inhibitor of both Hep3B and HepG2 human hepatoma cells. Deptropine significantly increased light chain 3B-II (LC3B-II) expression but did not induce sequestosome 1 (SQSTM1/p62) degradation in either cell line. Interestingly, other autophagy-related proteins, such as autophagy-related 7 (ATG7), vacuolar protein sorting 34 (VPS34), phosphorylated adenosine 5'-monophosphate-activated protein kinase (AMPK), and phosphorylated protein kinase B (PKB, also known as Akt), exhibited no significant change in either deptropine-treated cell line. Deptropine also inhibited the processing of cathepsin L from its precursor form to its mature form. Immunofluorescence microscopy showed an increase of autophagosomes in deptropine-treated cells, but deptropine blocked the fusion between autophagosomes and lysosomes. In a xenograft nude mice model, 2.5 mg/kg deptropine showed a great inhibitory effect on Hep3B tumor growth. These results suggest that deptropine can induce in vitro and in vivo hepatoma cell death, and the underlying mechanisms might be mediated through inhibiting autophagy by blocking autophagosome-lysosome fusion.

Development of Nanosome-Encapsulated Honokiol for Intravenous Therapy Against Experimental Autoimmune Encephalomyelitis.

BACKGROUND: Honokiol has been reported to possess anti-inflammatory and neuroprotective activities. However, the poor aqueous solubility of honokiol limits its clinical application for systemic administration. PURPOSE: This study aims to develop a novel formulation of nanosome-encapsulated honokiol (NHNK) for intravenous therapy against mouse experimental autoimmune encephalomyelitis (EAE) that mimics human multiple sclerosis. METHODS: Nanosomes and NHNK were prepared by using an ultra-high pressure homogenization (UHPH) method. Mice were treated with NHNK or empty nanosomes during the peak phase of EAE symptoms. Symptoms of EAE were monitored and samples of the spinal cord were obtained for histopathological examinations. RESULTS: The stock of NHNK containing honokiol in the nanosome formulation, which showed the structure of single phospholipid bilayer membranes, was well formulated with the particle size of 48.0 ± 0.1 nm and the encapsulation efficiency 58.1 ± 4.2%. Intravenous administration of NHNK ameliorated the severity of EAE accompanied by a significant reduction of demyelination and inflammation in the spinal cord. Furthermore, NHNK decreased the number of IL-6+, Iba-1+TNF +, Iba-1+IL-12 p40+, and CD3+IFN-γ+ cells infiltrating the spinal cord. CONCLUSION: The UHPH method simplified the preparation of NHNK with uniformly distributed nanosize and high encapsulation efficiency. Intravenous administration of NHNK ameliorated the severity of EAE by suppressing the infiltration of activated microglia and Th1 cells into the spinal cord. Collectively, these results suggest that the formulation of NHNK is a prospective therapeutic approach for inflammatory CNS diseases, such as multiple sclerosis.

Helminthostachys zeylanica alleviates hepatic steatosis and insulin resistance in diet-induced obese mice.

BACKGROUND: Obesity and its associated health conditions, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are worldwide health problems. It has been shown that insulin resistance is associated with increased hepatic lipid and causes hepatic steatosis through a myriad of mechanisms, including inflammatory signaling. METHODS: Helminthostachys zeylanica (HZ) is used widely as a common herbal medicine to relieve fever symptoms and inflammatory diseases in Asia. In the present study, we evaluated whether HZ has therapeutic effects on obesity, NAFLD and insulin resistance. The protective effects of HZ extract were examined using free fatty acid-induced steatosis in human HuS-E/2 cells and a high-fat diet-induced NAFLD in mice. RESULTS: The major components of the HZ extract are ugonins J and K, confirmed by HPLC. Incubation of human hepatocytes, HuS-E/2 cells, with palmitate markedly increased lipid accumulation and treatment with the HZ extract significantly decreased lipid deposition and facilitated AMPK and ACC activation. After 12 weeks of a high-fat diet with HZ extract treatment, the HFD mice were protected from hyperlipidemia and hyperglycemia. HZ extract prevented body weight gain, adipose tissue expansion and adipocyte hypertrophy in the HFD mice. In addition, fat accumulation was reduced in mice livers. Moreover, the insulin sensitivity-associated index, which evaluates insulin function, was also significantly restored. CONCLUSIONS: These results suggest that HZ has a promising pharmacological effect on high-fat diet-induced obesity, hepatic steatosis and insulin resistance, which may have the potential for clinical application.

Author Correction: ZFP36L1 and ZFP36L2 inhibit cell proliferation in a cyclin D-dependent and p53-independent manner.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis.

An increase in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) reportedly attenuates insulin-mediated signaling which participates in the development of brown adipose tissues (BATs). Nevertheless, the effect of MAP4K4 on brown adipogenesis remains largely uncharacterized. In this study, results of a transcriptome analysis (also referred as RNA-sequencing) showed differential expressions of MAP4K4 or SRSF3 transcripts isolated from distinct stages of embryonic BATs. The discriminative splicing profiles of MAP4K4 or SRSF3 were noted as well in brown adipocytes (BAs) with RNA-binding motif protein 4-knockout (RBM4-/-) compared to the wild-type counterparts. Moreover, the relatively high expressions of authentic SRSF3 transcripts encoding the splicing factor functioned as a novel regulator toward MAP4K4 splicing during brown adipogenesis. The presence of alternatively spliced MAP4K4 variants exerted differential effects on the phosphorylation of c-Jun N-terminal protein kinase (JNK) which was correlated with the differentiation or metabolic signature of BAs. Collectively, the RBM4-SRSF3-MAP4K4 splicing cascade constitutes a novel molecular mechanism in manipulating the development of BAs through related signaling pathways.

MCPIP3 as a Potential Metastasis Suppressor Gene in Human Colorectal Cancer.

Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cys⁻Cys⁻Cys⁻His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (VCAM)-1, in human endothelial cells, but the roles and functions of MCPIP3 in cancer cells are still unknown. In human colorectal cancer specimens, we found that the messenger RNA expression of MCPIP3 was significantly downregulated in cancer tissues compared to adjacent normal tissues (18/25; average fold change of 8.18). Two cell models were used to demonstrate the anti-migration activity of MCPIP3. First, Tet-on T-REx-293/HA-MCPIP3 cells were used to examine whether MCPIP3 can change epithelial⁻mesenchymal transition (EMT)-related gene expressions. Second, we used two human colorectal cancer cell lines, SW620 and HCT116, to prove the role of MCPIP3 in regulating EMT-related gene expressions. We found that overexpression of MCPIP3 inhibited cell migration according to a wound-healing assay and Transwell invasion assay and vimentin expression, and increased E-cadherin expression in these two cell lines. These results suggest that MCPIP3 might play a negative role in cell migration of human colorectal cancer cells.

ZFP36L1 and ZFP36L2 inhibit cell proliferation in a cyclin D-dependent and p53-independent manner.

ZFP36 family members include ZFP36, ZFP36L1, and ZFP36L2, which belong to CCCH-type zinc finger proteins with two tandem zinc finger (TZF) regions. Whether ZFP36L1 and ZFP36L2 have antiproliferative activities similar to that of ZFP36 is unclear. In this study, when ZFP36L1 or ZFP36L2 was overexpressed in T-REx-293 cells, cell proliferation was dramatically inhibited and the cell cycle was arrested at the G1 phase. The levels of cell-cycle-related proteins, including cyclin B, cyclin D, cyclin A, and p21, decreased; however, p53 increased in ZFP36L1-or ZFP36L2-overexpressing T-REx-293 cells. Forced expression of ZFP36L1 or ZFP36L2 also inhibited cell proliferation and cyclin D gene expression in three human colorectal cancer cell lines: HCT116 p53+/+, HCT116 p53-/-, and SW620 (mutated p53) cells. However, it increased p53 and p21 expression only in HCT116 p53+/+ cells. Knockdown of ZFP36L1 or ZFP36L2 increased cell proliferation and cyclin D expression; furthermore, the mutation of the TZF of ZFP36L1 or ZFP36L2 caused them to lose their antiproliferative ability, to the extent that they could not inhibit cyclin D expression in these three cell lines. The results indicated that ZFP36L1 and ZFP36L2 play a negative role in cell proliferation; the underlying mechanisms might be mediated through a cyclin D-dependent and p53-independent pathway.

RBM4-SRSF3-MAP4K4 splicing cascade modulates the metastatic signature of colorectal cancer cell.

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.

Correction: Combined Treatment with Troglitazone and Lovastatin Inhibited Epidermal Growth Factor-Induced Migration through the Downregulation of Cysteine-Rich Protein 61 in Human Anaplastic Thyroid Cancer Cells.

[This corrects the article DOI: 10.1371/journal.pone.0118674.].

The impact of the RBM4-initiated splicing cascade on modulating the carcinogenic signature of colorectal cancer cells.

A growing body of studies has demonstrated that dysregulated splicing profiles constitute pivotal mechanisms for carcinogenesis. In this study, we identified discriminative splicing profiles of colorectal cancer (CRC) cells compared to adjacent normal tissues using deep RNA-sequencing (RNA-seq). The RNA-seq results and cohort studies indicated a relatively high ratio of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1-4) and intron 2-retained SRSF6 (SRSF6+intron 2) transcripts in CRC tissues and cell lines. Nova1 variants exhibited differential effects on eliminating SRSF6 expression in CRC cells by inducing SRSF6+intron 2 transcripts which were considered to be the putative target of alternative splicing-coupled nonsense-mediated decay mechanism. Moreover, the splicing profile of vascular endothelial growth factor (VEGF)165/VEGF165b transcripts was relevant to SRSF6 expression, which manipulates the progression of CRC calls. These results highlight the novel and hierarchical role of an alternative splicing cascade that is involved in the development of CRC.

Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression.

We studied the potential mechanisms of valproic acid (VPA) in the treatment of glioblastoma multiforme (GBM). Using the human U87, GBM8401, and DBTRG-05MG GBM-derived cell lines, VPA at concentrations of 5 to 20 mM induced G2/M cell cycle arrest and increased the production of reactive oxygen species (ROS). Stress-related molecules such as paraoxonase 2 (PON2), cyclin B1, cdc2, and Bcl-xL were downregulated, but p27, p21 and Bim were upregulated by VPA treatment. VPA response element on the PON2 promoter was localized at position -400/-1. PON2 protein expression was increased in GBM cells compared with normal brain tissue and there was a negative correlation between the expression of PON2 and Bim. These findings were confirmed by the public Bredel GBM microarray (Gene Expression Omnibus accession: GSE2223) and the Cancer Genome Atlas GBM microarray datasets. Overexpression of PON2 in GBM cells significantly decreased intracellular ROS levels, and PON2 expression was decreased after VPA stimulation compared with controls. Bim expression was significantly induced by VPA in GBM cells with PON2 silencing. These observations were further shown in the subcutaneous GBM8401 cell xenograft of BALB/c nude mice. Our results suggest that VPA reduces PON2 expression in GBM cells, which in turn increases ROS production and induces Bim production that inhibits cancer progression via the PON2-Bim cascade.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver.

BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.