Quantitative Proteomics Reveal the Mechanism of MiR-138-5p Suppressing Cervical Cancer via Targeting ZNF385A.

MicroRNAs are short, noncoding RNA molecules that exert pivotal roles in cancer development and progression by modulating various target genes. There is growing evidence that miR-138-5p is significantly involved in cervical cancer (CC). However, its precise molecular mechanism has yet to be fully understood. In the current investigation, a quantitative proteomics approach was utilized to detect possible miR-138-5p targets in HeLa cells systematically. In total, 364 proteins were downregulated, and 150 were upregulated after miR-138-5p overexpression. Bioinformatic analysis of these differentially expressed proteins (DEPs) revealed significant enrichment in several cancer-related pathways. Zinc finger protein 385A (ZNF385A) was determined as a novel direct target of miR-138-5p and discovered to facilitate the proliferation, migration, and cell cycle progression of HeLa cells. SFN and Fas cell surface death receptor(FAS) were then identified as functional downstream effectors of ZNF385A and miR-138-5p. Moreover, a tumor xenograft experiment was conducted to validate the association of miR-138-5p-ZNF385A-SFN/FAS axis with the development of CC in vivo. Our findings have collectively established a catalog of proteins mediated by miR-138-5p and have provided an in-depth comprehension of the molecular mechanisms responsible for the inhibitory effect of miR-138-5p on CC. The miR-138-5p-ZNF385A-SFN/FAS axis could also be beneficial to the identification of new therapeutic targets.

Gut microbiota-mediated ursodeoxycholic acids regulate the inflammation of microglia through TGR5 signaling after MCAO.

Ischemic stroke has been demonstrated to cause an imbalance of gut microbiota. However, the change in gut microbiota-mediated bile acids (BAs) metabolites remains unclear. Here, we observed a decrease in gut microbiota-mediated BAs, especially ursodeoxycholic acid (UDCA), in the serum of stroke patients as well as in the intestine, serum and brain of stroke mice. Restoration of UDCA could decrease the area of infarction and improve the neurological function and cognitive function in mice in association with inhibition of NLRP3-related pro-inflammatory cytokines through TGR5/PKA pathway. Furthermore, knocking out TGR5 and inhibiting PKA activity reduce the protective effect of UDCA. Taken together, our results suggest that microbiota-mediated UDCA plays an important role in alleviating inflammatory responses and might be a promising therapeutic target in ischemic stroke.

Lysyl oxidase-like 2 promotes stemness and enhances antitumor effects of gefitinib in head and neck cancer via IFIT1 and IFIT3.

Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has recently been identified as an important regulator of tumor progression and metastasis. This study discovered that LOXL2 expression in oral squamous cell carcinoma (OSCC) tissues was significantly associated with tumor clinical stage, lymph node metastasis and patients' overall survival time. LOXL2-overexpressing human buccal SCC TW2.6 (TW2.6/LOXL2) and hypopharyngeal SCC FaDu (FaDu/LOXL2) cells exhibited enhanced migration, invasion, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) phenotypes, independently of its enzymatic activity. Moreover, TW2.6/LOXL2 significantly increased tumor-initiating frequency in SCID mice. We further demonstrated that LOXL2 increased the levels of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and IFIT3 in TW2.6/LOXL2 and FaDu/LOXL2 cells. We also identified IFIT1 and IFIT3 as key downstream components of LOXL2 action in migration, invasion, EMT, and CSC phenotypes in TW2.6 and FaDu cells. Furthermore, a significant positive correlation between LOXL2 expression and IFIT1 and IFIT3 overexpression in human OSCC tissues was observed. In addition, TW2.6/LOXL2 and FaDu/LOXL2 cells were 3.3- to 3.6-fold more susceptible to the epidermal growth factor receptor (EGFR) inhibitor gefitinib than were their respective control cells. The antitumor effect of gefitinib on orthotopic TW2.6/LOXL2 xenograft tumor was fourfold higher than that on controls. Our results indicate that LOXL2 expression is a strong prognostic factor for OSCC and may be used as a marker to identify patients most likely to respond to EGFR-targeted therapy.

Association between oral microbial dysbiosis and poor functional outcomes in stroke-associated pneumonia patients.

BACKGROUND: Despite advances in our understanding of the critical role of the microbiota in stroke patients, the oral microbiome has rarely been reported to be associated with stroke-associated pneumonia (SAP). We sought to profile the oral microbial composition of SAP patients and to determine whether microbiome temporal instability and special taxa are associated with pneumonia progression and functional outcomes. METHODS: This is a prospective, observational, single-center cohort study that examined patients with acute ischemic stroke (AIS) who were admitted within 24 h of experiencing a stroke event. The patients were divided into three groups based on the occurrence of pneumonia and the use of mechanical ventilation: nonpneumonia group, SAP group, and ventilator-associated pneumonia (VAP) group. We collected oral swabs at different time points post-admission and analyzed the microbiota using 16 S rRNA high-throughput sequencing. The microbiota was then compared among the three groups. RESULTS: In total, 104 nonpneumonia, 50 SAP and 10 VAP patients were included in the analysis. We found that SAP and VAP patients exhibited significant dynamic differences in the diversity and composition of the oral microbiota and that the magnitude of this dysbiosis and instability increased during hospitalization. Then, by controlling the potential effect of all latent confounding variables, we assessed the changes associated with pneumonia after stroke and explored patients with a lower abundance of Streptococcus were more likely to suffer from SAP. The logistic regression analysis revealed that an increase in specific taxa in the phylum Actinobacteriota was linked to a higher risk of poor outcomes. A model for SAP patients based on oral microbiota could accurately predict 30-day clinical outcomes after stroke onset. CONCLUSIONS: We concluded that specific oral microbiota signatures could be used to predict illness development and clinical outcomes in SAP patients. We proposed the potential of the oral microbiota as a non-invasive diagnostic biomarker in the clinical management of SAP patients. CLINICAL TRIAL REGISTRATION: NCT04688138. Registered 29/12/2020, https://clinicaltrials.gov/ct2/show/NCT04688138 .

Up-regulating microRNA-214-3p relieves hypoxic-ischemic brain damage through inhibiting TXNIP expression.

A list of microRNAs (miRs) has been referred to involve in the development of hypoxic-ischemic brain damage (HIBD). Based on that, we probed the concrete role of miR-214-3p regulating thioredoxin-interacting protein (TXNIP) in the illness. A neonatal HIBD mouse model was established using the Rice-Vannucci method, followed by measurements of miR-214-3p and TXNIP levels in brain tissues. After modeling, mice were given brain injection of the compounds that could alter miR-214-3p and TXNIP expression. Afterward, neurological function, neuronal inflammation, neuronal apoptosis, neuron morphology, and the number of Nissl body were assessed in HIBD mice. The binding of miR-214-3p to TXNIP was analyzed. Lower miR-214-3p and higher TXNIP were analyzed in brain tissues of mice with HIBD. Up-regulating miR-214-3p or depleting TXNIP improved neurological function, reduced neuronal inflammation and neuronal apoptosis, attenuated morphological damage of neurons, and increased the number of Nissl bodies in mice with HIBD. TXNIP was targeted by miR-214-3p and overexpressing TXNIP reversed the therapeutic effect of miR-214-3p on HIBD mice. It is noted that promotion of miR-214-3p relieves HIBD in mice through inhibiting TXNIP expression.

Using the Random Forest for Identifying Key Physicochemical Properties of Amino Acids to Discriminate Anticancer and Non-Anticancer Peptides.

Anticancer peptides (ACPs) represent a promising new therapeutic approach in cancer treatment. They can target cancer cells without affecting healthy tissues or altering normal physiological functions. Machine learning algorithms have increasingly been utilized for predicting peptide sequences with potential ACP effects. This study analyzed four benchmark datasets based on a well-established random forest (RF) algorithm. The peptide sequences were converted into 566 physicochemical features extracted from the amino acid index (AAindex) library, which were then subjected to feature selection using four methods: light gradient-boosting machine (LGBM), analysis of variance (ANOVA), chi-squared test (Chi2), and mutual information (MI). Presenting and merging the identified features using Venn diagrams, 19 key amino acid physicochemical properties were identified that can be used to predict the likelihood of a peptide sequence functioning as an ACP. The results were quantified by performance evaluation metrics to determine the accuracy of predictions. This study aims to enhance the efficiency of designing peptide sequences for cancer treatment.

ZNF385A and ZNF346 Serve as Prognostic Biomarkers Associated with an Inflamed Immunosuppressive Tumor Microenvironment in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and there are still many problems in the early diagnosis, molecular targeted therapy, and immunotherapy. It is necessary to explore valuable diagnostic markers and new therapeutic targets in HCC. Zinc finger protein 385A (ZNF385A) and zinc finger protein 346 (ZNF346) represent a unique class of RNA-binding Cys2 His2 (C2H2) zinc finger proteins that are involved in the regulation of cell cycle and apoptosis, but little is known of their roles in HCC. Based on multiple databases and analysis tools, we explored the expression, clinical relation, prognostic value, possible biological function, and pathways of ZNF385A and ZNF346, and their relationship with immune infiltration. Our results revealed that ZNF385A and ZNF346 were highly expressed and were associated with poor prognosis in HCC. Hepatitis B virus (HBV) infection may lead to the overexpression of ZNF385A and ZNF346, which was accompanied by elevated apoptosis and chronic inflammation. Moreover, ZNF385A and ZNF346 were positively correlated with immune-suppressive cells, inflammatory cytokines, immune checkpoint genes, and poor immunotherapy efficacy. Finally, the knockdown of ZNF385A and ZNF346 was observed to negatively affect the proliferation and migration of HepG2 cells in vitro. In conclusion, ZNF385A and ZNF346 are promising candidate biomarkers for the diagnosis, prognosis, and response to immunotherapy in HCC, and this study may help to understand the tumor microenvironment (TME) of liver cancer, and to develop new therapeutic targets.

Adhesion G Protein-Coupled Receptor G2 Promotes Hepatocellular Carcinoma Progression and Serves as a Neutrophil-Related Prognostic Biomarker.

Adhesion G protein-coupled receptor G2 (ADGRG2) is an orphan adhesion G protein-coupled receptor (GPCR), which performs a tumor-promoting role in certain cancers; however, it has not been systematically investigated in hepatocellular carcinoma (HCC). In the current study, we utilized multiple databases to analyze the expression and diagnostic and prognostic value of ADGRG2 in HCC and its correlation with immune infiltration and inflammatory factors. The function and upstream regulatory miRNA of ADGRG2 were validated through qPCR, Western blot, CCK8, wound healing, and dual luciferase assays. It turned out that ADGRG2 was significantly higher in HCC and had a poor survival rate, especially in AFP ≤ 400 ng/mL subgroups. Functional enrichment analysis suggested that ADGRG2 may be involved in cancer pathways and immune-related pathways. In vitro, siRNA-mediated ADGRG2 silencing could inhibit the proliferation and migration of Huh7 and HepG2 cells. There was a highly significant positive correlation between ADGRG2 and neutrophils. Moreover, NET-related genes were filtered and confirmed, such as ENO1 and S100A9. Meanwhile, the high expression of ADGRG2 was also accompanied by the highest number of inflammatory cytokines, chemokines, and chemokine receptors and good immunotherapy efficacy. Finally, AGDGR2 may be sensitive to two drugs (PIK-93 and NPK76-II-72-1) and can be targeted by miR-326. In conclusion, ADGRG2 may serve as a novel biomarker and drug target for HCC diagnosis, immunotherapy, and prognosis and was related to neutrophils and the inflammatory process of liver cancer development.

Phenytoin induces connective tissue growth factor (CTGF/CCN2) production through NADPH oxidase 4-mediated latent TGFß1 activation in human gingiva fibroblasts: Suppression by curcumin.

OBJECTIVE AND BACKGROUND: Gingival overgrowth (GO) is a common side effect of some drugs such as anticonvulsants, immunosuppressant, and calcium channel blockers. Among them, the antiepileptic agent phenytoin is the most common agent related to this condition due to its high incidence. Transforming growth factor ß (TGFß) importantly contributes to the pathogenesis of GO. Connective tissue growth factor (CTGF or CCN2) is a key mediator of tissue fibrosis and is positively associated with the degree of fibrosis in GO. We previously showed that Src, c-jun N-terminal kinase, and Smad3 mediate TGFß1-induced CCN2 protein expression in human gingival fibroblasts (HGFs). This study investigates whether phenytoin can induce CCN2 synthesis through activated latent TGFß in HGFs and its mechanisms. METHODS: CCN2 synthesis, latent TGFß1 activation, and cellular reactive oxygen species (ROS) generation in HGFs were studied using western blot analysis, a TGFß1 Emax® ImmunoAssay System, and 2',7'-dichlorodihydrofluorescein diacetate (an oxidation-sensitive fluorescent probe), respectively. RESULTS: Phenytoin significantly stimulated CCN2 synthesis, latent TGFß1 activation, and ROS generation in HGFs. Addition of an TGFß-neutralizing antibody, TGFß receptor kinase inhibitor SB431542, and Smad3 inhibitor SIS3 completely inhibited phenytoin-induced CCN2 synthesis. General antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor diphenylene iodonium, and specific NOX4 inhibitor plumbagin almost completely suppressed phenytoin-induced total cellular ROS and latent TGFß1 activation. Curcumin dose-dependently decreased phenytoin-induced TGFß1 activation and CCN2 synthesis in HGFs. CONCLUSIONS: Our findings indicated that NOX4-derived ROS play pivotal roles in phenytoin-induced latent TGFß1 activation. Molecular targeting the phenytoin/NOX4/ROS/TGFß1 pathway may provide promising strategies for the prevention and treatment of GO. Curcumin-inhibited phenytoin-induced CCN2 synthesis is caused by the suppression of latent TGFß1 activation.

The impact of COVID-19 on the ride-sharing industry and its recovery: Causal evidence from China.

The COVID-19 pandemic has brought unprecedented disruptions to many industries, and the transportation industry is among the most disrupted ones. We seek to address, in the context of a ride-sharing platform, the response of drivers to the pandemic and the post-pandemic recovery. We collected comprehensive trip data from one of the leading ride-sharing companies in China from September 2019 to August 2020, which cover pre-, during-, and post-pandemic phases in three major Chinese cities, and investigate the causal effect of the COVID-19 pandemic on driver behavior. We find that drivers only slightly reduce their number of shifts in response to increased COVID-19 cases, likely because they have to make a living from providing ride-sharing services. Nevertheless, conditional on working, drivers exhibit strong risk aversion: As the number of new cases increases, drivers strategically adjust the scope of their search for passengers, complete fewer trips, and as a result, make lower daily earnings. Finally, our heterogeneity analyses indicate that the effects appear to vary both across drivers and over time, with generally stronger effects on drivers who are older, more experienced, more active before the pandemic, and higher-status within the firm. Our findings have strong policy implications: These drivers tend to contribute more to the focal company, and also rely more on providing ride-sharing services to make a living. Therefore, they should be prioritized in stimulus plans offered by the government or the ride-sharing company.

Transactivation of Sus1 and Sus2 by Opaque2 is an essential supplement to sucrose synthase-mediated endosperm filling in maize.

The endosperm-specific transcription factor Opaque2 (O2) acts as a central regulator for endosperm filling, but its functions have not been fully defined. Regular o2 mutants exhibit a non-vitreous phenotype, so we used its vitreous variety Quality Protein Maize to create EMS-mutagenesis mutants for screening o2 enhancers (oen). A mutant (oen1) restored non-vitreousness and produced a large cavity in the seed due to severely depleted endosperm filling. When oen1 was introgressed into inbred W64A with a normal O2 gene, the seeds appeared vitreous but had a shrunken crown. oen1 was determined to encode Shrunken1 (Sh1), a sucrose synthase (SUS, EC 2.4.1.13). Maize contains three SUS-encoding genes (Sh1, Sus1, and Sus2) with Sh1 contributing predominantly to the endosperm. We determined SUS activity and found a major and minor reduction in oen1 and o2, respectively. In o2;oen1-1, SUS activity was further decreased. We found all Sus gene promoters contain at least one O2 binding element that can be specifically recognized and be transactivated by O2. Sus1 and Sus2 promoters had a much stronger O2 transactivation than Sh1, consistent with their transcript reduction in o2 endosperm. Although sus1 and sus2 alone or in combination had no perceptible phenotype, either of them could dramatically enhance seed opacity and cavity in sh1, indicating that transactivation of Sus1 and Sus2 by O2 supplements SUS-mediated endosperm filling in maize. Our findings demonstrate that O2 transcriptionally regulates the metabolic source entry for protein and starch synthesis during endosperm filling.

Towards coeliac-safe bread.

Gluten-free foods cannot substitute for products made from wheat flour. When wheat products are digested, the remaining peptides can trigger an autoimmune disease in 1% of the North American and European population, called coeliac disease. Because wheat proteins are encoded by a large gene family, it has been impossible to use conventional breeding to select wheat varieties that are coeliac-safe. However, one can test the properties of protein variants by expressing single genes in coeliac-safe cereals like maize. One source of protein that can be considered as coeliac-safe and has bread-making properties is teff (Eragrostis tef), a grain consumed in Ethiopia. Here, we show that teff α-globulin3 (Etglo3) forms storage vacuoles in maize that are morphologically similar to those of wheat. Using transmission electron microscopy, immunogold labelling shows that Etglo3 is almost exclusively deposited in the storage vacuole as electron-dense aggregates. Of maize seed storage proteins, 27-kDa γ-zein is co-deposited with Etglo3. Etglo3 polymerizes via intermolecular disulphide bonds in maize, similar to wheat HMW glutenins under non-reducing conditions. Crossing maize Etglo3 transgenic lines with α-, ß- and γ-zein RNA interference (RNAi) lines reveals that Etglo3 accumulation is only dramatically reduced in γ-zein RNAi background. This suggests that Etglo3 and 27-kDa γ-zein together cause storage vacuole formation and behave similar to the interactions of glutenins and gliadins in wheat. Therefore, expression of teff α-globulins in maize presents a major step in the development of a coeliac-safe grain with bread-making properties.

Regulation of immune-driven pathogenesis in Parkinson's disease by gut microbiota.

Parkinson's disease (PD) is one of the most significant medical and social burdens of our time. The prevalence of PD increases with age and the number of individuals diagnosed with PD is expected to double from 6.9 million in 2015 to 14.2 million in 2040. To date, no drugs can stop the ongoing neurodegeneration caused by PD due to its unclear and complex pathogenic mechanisms. It has been wildly recognized that both gut microbiota and neuro-immunity are involved in the pathology of PD. In this review, we intend to provide a comprehensive overview of current knowledge on how gut microbiota involved in immune-driven pathogenesis of PD, and its potential as a new target of dietary and/or therapeutic interventions for PD.

Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize.

The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

Arecoline activates latent transforming growth factor ß1 via mitochondrial reactive oxygen species in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFß in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFß1 signaling and to inspect the detailed mechanisms involved. METHODS: Western blot and TGFß1 Emax® ImmunoAssay were used to measure the effect of arecoline on the TGFß signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS). RESULTS: Arecoline induced latent TGFß1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFß-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFß1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFß1 activation and mitochondrial ROS in BMFs. CONCLUSION: Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFß1 leading to the initiation of TGFß1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF.

Connective tissue growth factor decreases mitochondrial metabolism through ubiquitin-mediated degradation of mitochondrial transcription factor A in oral squamous cell carcinoma.

BACKGROUND/PURPOSE: Deregulation of metabolic pathways is one of the hallmarks of cancer progression. Connective tissue growth factor (CTGF/CCN2) acts as a tumor suppressor in oral squamous cell carcinoma (OSCC). However, the role of CTGF in modulating cancer metabolism is still unclear. METHODS: OSCC cells stably overexpressing CTGF (SAS/CTGF) and shRNA against CTGF (TW2.6/shCTGF) were established. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were examined by the Seahorse XF24 analyzer. The expression of CTGF and mitochondrial biogenesis related genes was measured by real-time polymerase chain reaction or Western blot analysis. RESULTS: CTGF decreased OCR, ECAR, adenosine triphosphate (ATP) generation, mitochondrial DNA (mtDNA), and mitochondrial transcription factor A (mtTFA) protein expression in OSCC cells. Overexpression of mtTFA restored CTGF-decreased OCR, ECAR, mtDNA copy number, migration and invasion of SAS/CTGF cells. Immunoprecipitation assay showed a higher level of ubiquitinated mtTFA protein after CTGF treatment. MG132, an inhibitor of proteasomal degradation, reversed the effect of CTGF on mtTFA protein expression in SAS cells. CONCLUSION: CTGF can decrease glycolysis, mitochondrial oxidative phosphorylation, ATP generation, and mtDNA copy number by increasing mtTFA protein degradation through ubiquitin proteasome pathway and in turn reduces migration and invasion of OSCC cells. Therefore, CTGF may be developed as a potential additive therapeutic drug for oral cancer in the near future.

Phenethyl isothiocyanate enhances TRAIL-induced apoptosis in oral cancer cells and xenografts.

OBJECTIVES: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been regarded as a promising candidate for cancer therapy. However, most of oral cancer cell lines are resistant to the TRAIL-induced cytotoxicity. The aim of this study was to investigate the ability of phenethyl isothiocyanate (PEITC) to sensitize TRAIL-induced apoptosis in TRAIL-resistant oral cancer cells and xenografts. MATERIALS AND METHODS: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, Western blotting, and a mouse xenograft model were used to study the effects of PEITC and TRAIL on two TRAIL-resistant human oral cancer cells, SAS and Ca9-22. RESULTS: PEITC upregulated death receptor 4 (DR4) and DR5 protein expression and increased reactive oxygen species (ROS) production in both SAS and Ca9-22 cells. Antioxidant N-acetyl-L-cysteine (NAC) and c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 inhibited PEITC-induced DR4 and DR5 expression. Inhibitor experiments showed that PEITC induced apoptosis through ROS-mediated JNK activation and upregulation of DR4 and DR5. Furthermore, treatment with PEITC significantly increased TRAIL-induced apoptosis in both cells. Combined treatment with PEITC and TRAIL had greater effect on the inhibition of tumor growth than either agent alone. CONCLUSIONS: We showed for the first time that PEITC overcomes TRAIL resistance in oral cancer cells and enhance the therapeutic potential of TRAIL in vivo. CLINICAL RELEVANCE: PEITC, either alone or in combination with TRAIL, can be used as a new therapeutic approach for the treatment of oral cancers.

Sphingosine-1-phosphate stimulated connective tissue growth factor expression in human buccal fibroblasts: Inhibition by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Connective tissue growth factor (CCN2) has been associated with the pathogenesis of various fibrotic diseases, including oral submucous fibrosis (OSF). The chemical constituents of areca nut along with the mechanical trauma cause OSF. The coarse fibers of areca nut injure the mucosa and hence sphingosine-1-phosphate (S1P) is released at the wounded sites. Recent studies have shown that S1P is involved in wound healing and the development of fibrosis. The aims of this study were to investigate the effects of S1P on CCN2 expression in human buccal fibroblasts (HBFs) and identify the potential targets for drug intervention or chemoprevention of OSF. METHODS: Western blot analyses were used to study the effects of S1P on CCN2 expression and its signaling pathways in HBFs and whether epigallocatechin-3-gallate (EGCG), the main and most significant polyphenol in green tea, could inhibit this pathway. RESULTS: S1P significantly enhanced CCN2 synthesis in HBFs. This effect can be inhibited by c-Jun NH2-terminal kinase (JNK) inhibitor and extracellular signal-regulated kinase inhibitor but not by P38 mitogen-activated protein kinase inhibitor. Interestingly, EGCG completely blocked S1P-induced CCN2 expression via suppressing S1P-induced JNK phosphorylation. CONCLUSION: S1P released by repetitive mechanical trauma during AN chewing may contribute to the pathogenesis of OSF through upregulating CCN2 expression in HBFs. EGCG could be an adjuvant to the current offered therapy options or the prevention of OSF through suppression of JNK activation.

Cyclosporine A induces connective tissue growth factor expression in human gingival fibroblasts: suppression by epigallocatechin-3-gallate.

BACKGROUND/PURPOSE: Transforming growth factor-ß (TGF-ß) plays an important role in the pathogenesis of cyclosporine A (CsA)-induced gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) acts as a cofactor with TGF-ß to induce the maximal profibrotic effects of TGF-ß. We investigated the effects of CsA on CCN2 expression in human gingival fibroblasts (HGFs) and the potential chemopreventive agent for CsA-induced GO. METHODS: Western blot analyses were used to examine the signaling pathways of CsA-induced CCN2 expression in HGFs and whether epigallocatechin-3-gallate (EGCG), curcumin, or lovastatin can inhibit CsA-induced CCN2 expression. RESULTS: CsA significantly stimulated CCN2 synthesis in HGFs. This effect can be inhibited by c-Jun NH(2)-terminal kinase (JNK) and Smad3 inhibitors but not by TGF-ß neutralizing antibody and TGF-ß type I receptor inhibitor. Furthermore, EGCG completely blocked CsA-induced CCN2 expression. CONCLUSION: CsA-induced CCN2 protein expression is mediated through JNK and Smad signaling. CsA may contribute to the pathogenesis of GO through upregulation of CCN2 expression in HGFs. EGCG could be an adjuvant for the prevention of CsA-induced GO.

Epigallocatechin-3-gallate inhibits lysophosphatidic acid-stimulated connective tissue growth factor via JNK and Smad3 suppression in human gingival fibroblasts.

BACKGROUND/PURPOSE: Connective tissue growth factor (CTGF/CCN2) is involved in the development and progression of fibrotic diseases, including gingival overgrowth (GO). Recent studies indicate that lysophosphatidic acid (LPA) is also significantly involved in wound healing and the development of fibrosis. This study investigated whether epigallocatechin-3-gallate (EGCG) can inhibit LPA-induced CCN2 expression in human gingival fibroblast (GF) and its mechanism. METHODS: Western blot analyses were used to study the signaling pathways of LPA-induced CCN2 expression in human GFs and the effects of EGCG on this pathway. RESULTS: LPA stimulated CCN2 synthesis in human GFs. This effect can be significantly inhibited bytransforming growth factor-ß type I receptor/ALK5, Smad3, and JNK inhibitors but not ERK, P38, and MAPK inhibitors. EGCG completely inhibited LPA-induced CCN2 expression through attenuating the LPA-induced JNK and Smad3 phosphorylation in human GFs. CONCLUSION: LPA produced at the surgical wound may contribute to the recurrence of GO by upregulating CCN2 expression in human GFs. This effect was mediated by Smad3 and JNK activation and ALK5 transactivation. EGCG could be a useful agent for reducing the recurrence of GO after surgery through suppression of JNK and Smad3 activations.