Activation of the MEK/ERK Pathway Mediates the Inhibitory Effects of Silvestrol on Nasopharyngeal Carcinoma Cells via RAP1A, HK2, and GADD45A.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.

Regulation of c-Jun N-Terminal Protein Kinase (JNK) Pathway in Apoptosis of Endothelial Outgrowth Cells Induced by Asymmetric Dimethylarginine.

BACKGROUND Endothelial outgrowth cells (EOCs) are terminal endothelial progenitor cells (EPCs). Asymmetric dimethylarginine (ADMA) has been identified as a novel risk factor for cardiovascular diseases. Our aim in the present study was to investigate the effect of regulation of asymmetric dimethylarginine (ADMA) on EOCs apoptosis and to explore the underlining mechanisms of c-Jun N-terminal protein kinase (JNK) pathway in the process. MATERIAL AND METHODS EOCs were harvested from umbilical cord blood and obtained by using density gradient centrifugation and adhesive culture methods. Endothelial characteristics were identified by immunohistochemistry and fluorescence staining. EOCs were treated with different concentrations of ADMA and detected by flow cytometry. After JNK specific inhibitor (SP600125) was added, EOCs apoptosis protein expressions were measured by Western blot analysis. Proliferation, migration, and vascularization were detected by CCK-8 assay, wound healing assay, and tube-like formation assay, respectively. RESULTS EOCs were successfully extracted from umbilical cord blood and different concentrations of ADMA aggravated EOCs apoptosis. ADMA distinctly activates the phosphorylation activity of JNK. Supplementation of JNK-specific inhibitor (SP600125) decreased expression of Bax and cleaved caspase 3/9, and alleviated ADMA-induced apoptosis. SP600125 also promoted angiogenesis viability. CONCLUSIONS The JNK pathway participates in the apoptosis-promoting process of EOCs, and targeted inhibition of the JNK pathway can alleviate ADMA-induced injury, which I s the potential underlying mechanism of vascular endothelium injury in ischemic stroke.

Mechanisms elevating ORMDL3 expression in recurrent wheeze patients: role of Ets-1, p300 and CREB.

The first genetic factor identified for childhood asthma by genome-wide association study (GWAS) is the locus on chromosome 17q21, harboring the Orosomucoid 1-like 3 (ORMDL3) gene. ORMDL3 is implicated in facilitation of endoplasmic reticulum-mediated inflammatory responses, believed to underlie its association with asthma. In the present study, we demonstrated that mRNA expression of ORMDL3 is significantly increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects by real-time RT-PCR. To elucidate the molecular mechanisms involved in human ORMDL3 regulation, we cloned and characterized the promoter region of ORMDL3. Applying 5'-rapid amplification of cDNA end analysis (RACE), we revealed that ORMDL3 gene used multiple transcriptional start sites (TSSs). Using a series of 5' deletion promoter plasmids in luciferase reporter assays, we identified that the proximal minimal promoter of ORMDL3 was located within the region -84/+58 relative to the TSS. Mutational analysis, RNA interference experiments and sequential chromatin immunoprecipitation (ChIP) assay demonstrated that transcriptional activity of the ORMDL3 gene was cooperatively regulated by multiple transcription factors, including Ets-1, p300 and CREB. The expression levels of Ets-1, p300 and CREB were increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects and showed a strong linear correlation with the expression of ORMDL3. Our findings indicate that Ets-1, p300 and CREB binding to the promoter region drive the ORMDL3 transcription.

Characterization of a novel isoform of the human ORMDL3 gene.

The locus on chromosome 17q21, that encompasses the orosomucoid 1-like protein 3 (ORMDL3) gene, was considered a susceptibility locus associated to asthma, ulcerative colitis and ankylosing spondylitis, and polymorphisms within this locus were thought to be associated to an increased expression of the ORMDL3 gene. Several phosphorylation sites in the N-terminal regions of Orm proteins played crucial roles in the course of sphingolipid synthesis. To provide insight into our understanding of the expression of the ORMDL3 gene, we isolated and characterized a splicing isoform of ORMDL3, ORMDL3 V1, from Hela cells by 5'and 3'-rapid amplification of cDNA end analysis (RACE) and RT-PCR. ORMDL3 V1 skipped the second exon of the wild-type ORMDL3 gene. The predicted protein sequences of this isoform lacked 59 amino acids in the N-terminus of the wild-type ORMDL3 protein. RT-PCR assay showed that the mRNA levels of ORMDL3 V1 were higher in leukocytes, spleen, thymus, and Hela cells, lower in liver, brain, colon, lung, kidney, ovary, and testis. No mRNA expression was found in pancreas, heart, placenta, skeletal muscle, prostate, and small intestine. ORMDL3 V1 open reading frame was subcloned into pEGFP-C1 vector and it was found that the protein synthesis had been followed in transfected Hela cells. Western blot analysis detected a ∼38 kDa EGFP-ORMDL3 V1 fusion protein. Fluorescence microscopy demonstrated that both ORMDL3 V1 and ORMDL3 were almost exclusively expressed and localized in the cytoplasm of HEK293 cells. This study reveals the presence of a novel ORMDL3 splicing isoform, ORMDL3 V1 in human.