PD-L1 inhibitors combined with whole brain radiotherapy in patients with small cell lung cancer brain metastases: Real-world evidence.

BACKGROUND: Numerous studies have demonstrated that brain metastases patients may benefit from intracranial radiotherapy combined with immune checkpoint inhibitors (ICIs). However, it is unclear whether this treatment is effective for patients with small cell lung cancer brain metastases (SCLC-BMs). METHODS: We conducted a retrospective study by analyzing medical records of patients with SCLC-BMs from January 1, 2017 to June 1, 2022. Data related to median overall survival (mOS), median progression-free survival (mPFS), and intracranial progression-free survival (iPFS) were analyzed. RESULTS: A total of 109 patients were enrolled, of which 60 received WBRT and 49 received WBRT-ICI. Compared to the WBRT alone cohort, the WBRT-ICI cohort showed longer mOS (20.4 months vs. 29.3 months, p = 0.021), mPFS (7.9 months vs. 15.1 months, p < 0.001), and iPFS (8.3 months vs. 16.5 months, p < 0.001). Furthermore, WBRT-ICI cohort had a better response rate for both BMs. (p = 0.035) and extracranial diseases (p < 0.001) compared to those receiving WBRT alone. Notably, the use of WBRT before ICI was associated with longer mOS compared to the use of WBRT after ICI (23.3 months for the ICI-WBRT group vs. 34.8 months for the WBRT-ICI group, p = 0.020). CONCLUSION: Our results indicated that WBRT combined with immunotherapy improved survival in SCLC-BMs patients compared to WBRT monotherapy. Administering WBRT prior to ICI treatment is associated with improved survival outcomes compared to WBRT following ICI treatment, for patients with SCLC-BMs. These findings highlight the significance of conducting further prospective researches on combination strategies of intracranial radiotherapy and ICI in SCLC-BMs patients.

G-CSF improving combined whole brain radiotherapy and immunotherapy prognosis of non-small cell lung cancer brain metastases.

OBJECTIVE: To evaluate the therapeutic advantage of G-CSF to whole brain radiotherapy (WBRT) in combination with immunotherapy as a first-line treatment for non-small cell lung cancer (NSCLC) brain metastases (BMs). METHODS: In this retrospective study, 117 patients (37 in G-CSF group and 80 in no G-CSF group) who underwent first-line WBRT combined with immunotherapy were enrolled. Their survival, intracranial response, BM-related symptoms and toxicity were evaluated. RESULTS: The overall survival (OS) of patients in G-CSF group was significantly improved compared to patients no G-CSF group (median time: 14.8 vs 10.2 months; HR: 0.61, 95 % CI: 0.38-0.97, p = 0.035). However, there were no significant differences in intracranial responses between the two groups (p > 0.05). The G-CSF group exhibited a significantly higher rate of relief from BM-related symptoms compared to the no G-CSF group (91.7 % vs 59.5 %, p = 0.037). Cox proportional hazards regression analyses indicated that after-treatment ALC > 0.9 × 10^9/L (HR 0.57, 95 % CI 0.32-0.99, p = 0.046) and Hb > 110 g/dL (HR 0.41, 95 % CI 0.24-0.71, p = 0.001) were significant potential factors associated with extended OS. The addition of G-CSF was well tolerated and effectively reduced the incidence of neutropenia (0 % vs 5.0 %, p = 0.17). CONCLUSION: Integrating G-CSF with WBRT and immunotherapy as a first-line treatment for NSCLC-BMs has exhibited significant efficacy and favorable tolerability.

(-)-Guaiol inhibit epithelial-mesenchymal transition in lung cancer via suppressing M2 macrophages mediated STAT3 signaling pathway.

In the context of cancer expansion, epithelial-mesenchymal transition (EMT) plays an essential role in driving invasion and metastasis potential of cancer cells. Tumor-associated macrophages (TAMs)-derived factors involved in the initiation and progression of EMT. We assess the role of M2 macrophage in suppressing lung tumors of a natural compound (-)-Guaiol by using macrophage depleted model. Bone marrow-derived monocytes (BMDMs) were extracted and induced to M2-like phenotype in vitro. The co-culture of M2 macrophage and lung cancer cells was established to observe that inhibition of lung tumor growth by (-)-Guaiol requires presence of macrophages. This suppressed effect of (-)-Guaiol was alleviated when mice macrophage was depleted. The expression of M2-like macrophages was strongly reduced by (-)-Guaiol treated mice, but not the changes of M1-like macrophages. In vitro studies, we demonstrated that (-)-Guaiol suppressed M2 polarization of BMDMs, as well as migration, invasion, and EMT of lung cancer cells in co-culture. M2 macrophage-derived interleukin 10 (IL-10) was investigated as a critical signaling molecule between M2 macrophage and lung cancer cells. We have also verified that the mechanism of (-)-Guaiol inhibiting the EMT process of lung cancer is related to the activation of IL-10-mediated signal transducer and activator of transcription 3 (STAT3). These results suggested that the suppressive effect role of (-)-Guaiol in M2 macrophage promoting EMT of lung cancer, which was associated with inhibition of IL-10 mediated STAT3 signaling pathway.

Target and Mechanism of the Xihuang Pill Based on Network Pharmacology for Lung Squamous Cell Carcinoma.

Context: Lung squamous cell carcinoma (LUSC) accounts for 30% of non-small-cell lung cancers (NSCLC), and an effective pharmacological treatment for LUSC isn't yet available. The Xihuang Pill is a potent Chinese medicinal preparation widely prescribed for the management of LUSC. Objective: The study intended to use the network-pharmacology method to ascertain the effective active ingredients, targets of action, and cellular-signal transduction involved in the prevention and treatment of LUSC when using the Xihuang Pill and to identify the mechanism of action of the pills against LUSC, to provide a more adequate scientific basis for subsequent studies. Design: The research team performed a genetic study. Setting: The study took place at Shanghai. Outcome Measures: The research team: (1) created the feature sets, for both the LUSC and normal features, using the Cancer Genome Atlas' (TCGA's) LUSC dataset; (2) performed a weighted correlation network analysis (WGCNA) of the differentially expressed genes (DEGs) using the R package WGCNA; (3) searched for the chemical components of the Xihuang Pill using the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP) and the Herb Group Identification Platform, and (4) selected the novel the Matthews correlation coefficient (MCC) algorithm to screen the hub genes. Results: The study found 8713 DEGs between the LUSC and normal groups. The top ten, important, downregulated genes included: (1) advanced glycosylation end product (AGER), (2) chitinase, acidic pseudogene 2 (CHIAP2), (3) CD300 molecule like family member G (CD300LG), (4) solute carrier family 6 member 4 (SLC6A4), (5) carboxypeptidase B2 (CPB2), (6) claudin 18 (CLDN18), (7) gamma-glutamyltransferase light chain 1 (GGTLC1), (8) gastrokine 2 (GKN2), (9) progastricsin (PGC), and (10) pulmonary surfactant-associated protein C (SFTPC). The top 10 upregulated genes included: (1) cancer susceptibility 9 (CASC9), (2) homeobox C13 (HOXC13), (3) keratin 6a (KRT6A), (4) desmoglein 3 (DSG3), (5) keratin 16 (KRT16), (6) forkhead box E1 (FOXE1), (7) preferentially expressed antigen in melanoma (PRAME), (8) calmodulin-like protein 3 (CALML3), (9) KRT68, and (10) aldo-keto reductase family 1 member B10 (AKR1B10). The study found 41 active ingredients and 843 targets for the Xihuang Pill. The PPI network included 10 hub genes, including cyclin dependent kinase 1 (CDK1), cyclin B1 (CCNB1), cyclin B2 (CCNB2), polo-like kinase 1 (PLK1), aurora kinase B (AURKB), baculoviral IAP repeat containing 5 (BIRC5), cyclin A2 (CCNA2), aurora kinase A (AURKA), centrosome-associated protein E (CENPE), and threonine tyrosine kinase (TTK), which were the principal target genes at the core of the gene-pathway network for the drug compound to central-target relationship. The enrichment analyses used the overlapping genes and the 10 hub genes and found 390 biological processes (BPs), 25 molecular functions (MFs), 43 cellular components (CCs), and 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The main enrichment occurred in the regulation of protein serine-threonine kinase activity, mitotic nuclear division, progesterone-mediated oocyte maturation, and the cell cycle. Conclusions: The study found the targets and relevant pathways of the hub genes of Xihuang Pill using biological analysis and molecular docking and demonstrated the interactions of critical chemical compounds with the hub's targeted genes were. More research is necessary to further determine whether the Xihuang Pill can improve LUSC patients' survival rate by regulation of those genes.

Vitamin C Urinary Loss and Deficiency in Human Immunodeficiency Virus (HIV): Cross-sectional Study of Vitamin C Renal Leak in Women With HIV.

BACKGROUND: Reduced plasma vitamin C (vitC) concentrations in human immunodeficiency virus (HIV) may result from abnormal urinary excretion: a renal leak. VitC renal leak indicates underlying nutritional dysregulation independent of diet. We hypothesized that increased renal leak prevalence in HIV would be associated with deficient vitC concentrations. METHODS: We conducted an outpatient cross-sectional study of 96 women (40 HIV [PWH] and 56 without HIV [PWOH]) at the National Institutes of Health and Georgetown University. Renal leak was defined as abnormal urinary vitC excretion at fasting plasma concentrations <43.2µM, 2 SDs below vitC renal threshold in healthy women. To determine the primary outcome of renal leak prevalence, matched urine and plasma samples were collected the morning after overnight fast. Secondary outcomes assessed group differences in mean plasma vitC concentrations and prevalence of vitC deficiency. Exploratory outcomes assessed clinical parameters associated with renal leak. VitC was measured by high-performance liquid chromatography with coulometric electrochemical detection. RESULTS: PWH had significantly higher renal leak prevalence (73%vs14%; OR (odds ratio):16; P<.001), lower mean plasma vitC concentrations (14µMvs50µM; P<.001), and higher prevalence of vitC deficiency (43%vs7%; OR:10; P<.001) compared with PWOH, unchanged by adjustments for confounding factors. Significant predictors of renal leak included antiretroviral therapy (ART), Black race, older age, and metabolic comorbidities but not viral load or CD4 count. When compared with other chronic disease cohorts, PWH had the highest prevalence of renal leak and vitC deficiency (P<.001). CONCLUSIONS: High prevalence of vitC renal leak in HIV was associated with vitC deficiency, ART use, and race/ethnicity differences.

Vitamin C Urinary Loss in Fabry Disease: Clinical and Genomic Characteristics of Vitamin C Renal Leak.

BACKGROUND: Reduced plasma vitamin C concentrations in chronic diseases may result from abnormal urinary excretion of vitamin C: a renal leak. We hypothesized that vitamin C renal leak may be associated with disease-mediated renal dysregulation, resulting in aberrant vitamin C renal reabsorption and increased urinary loss. OBJECTIVES: We investigated the prevalence, clinical characteristics, and genomic associations of vitamin C renal leak in Fabry disease, an X-linked lysosomal disease associated with renal tubular dysfunction and low plasma vitamin C concentrations. METHODS: We conducted a non-randomized cross-sectional cohort study of men aged 24-42 y, with Fabry disease (n = 34) and controls without acute or chronic disease (n = 33). To match anticipated plasma vitamin C concentrations, controls were placed on a low-vitamin C diet 3 wk before inpatient admission. To determine the primary outcome of vitamin C renal leak prevalence, subjects were fasted overnight, and matched urine and fasting plasma vitamin C measurements were obtained the following morning. Vitamin C renal leak was defined as presence of urinary vitamin C at plasma concentrations below 38 µM. Exploratory outcomes assessed the association between renal leak and clinical parameters, and genomic associations with renal leak using single nucleotide polymorphisms (SNPs) in the vitamin C transporter SLC23A1. RESULTS: Compared with controls, the Fabry cohort had 16-fold higher odds of renal leak (6% vs. 52%; OR: 16; 95% CI: 3.30, 162; P < 0.001). Renal leak was associated with higher protein creatinine ratio (P < 0.01) and lower hemoglobin (P = 0.002), but not estimated glomerular filtration rate (P = 0.54). Renal leak, but not plasma vitamin C, was associated with a nonsynonymous single nucleotide polymorphism in vitamin C transporter SLC23A1 (OR: 15; 95% CI: 1.6, 777; P = 0.01). CONCLUSIONS: Increased prevalence of renal leak in adult men with Fabry disease may result from dysregulated vitamin C renal physiology and is associated with abnormal clinical outcomes and genomic variation.

COL11A1 Was Involved in Cell Proliferation, Apoptosis and Migration in Non-Small Cell Lung Cancer Cells.

BACKGROUND: Type XI collagen (COL11A1) was reported to associate with malignancy in several cancer types, whereas its role in lung cancer is not fully understood. Therefore, the present study aimed to explore the expression level and biological role of COL11A1 in lung cancer cells. METHODS: Gene Expression Omnibus (GEO) database containing 6 lung cancer tissues and normal lung tissues was used to identify potential aberrantly expressed genes. The expression of COL11A1, apoptosis related genes, cell cycle related genes and migration associated genes at the protein level were evaluated by western blot and at the mRNA level was determined by real-time PCR in lung cancer cell lines. Furthermore, the expression of COL11A1 was silenced by siRNA, and cell viability was detected by Cell counting kit-8 (CCK-8) assay. Moreover, cell apoptosis and cell cycle were determined by flow cytometry. In addition, transwell and wound-healing assay were applied to determine cell migration ability. RESULTS: GEO analysis suggests that COL11A1 was highly expressed in patients with lung cancer, which was consistent with the results in lung cancer cell lines. COL11A1 knockdown in lung cancer cells significantly lowered the colony formation ability, augmented cell apoptosis rate and elevated the expression of cleaved caspase-3, cleaved caspase-9, Bax, P21 and the expression of Bcl-2, CyclinD1, CDK2 and CDK-4 was significantly downregulated (all p < 0.05). Moreover, post-COL11A1 knockdown, the cell wound-healing and migration ability was significantly impaired, which also supported by the upregulation of E-Cadherin and downregulation of N-Cadherin, Vimentin and Snail (all p < 0.05). Furthermore, we also found that COL11A1 knockdown decreased the expression of p-AKT, p-PI3K and p-ERK. CONCLUSION: The present study revealed that COL11A1 may contribute to the malignancy and involve in the pathogenesis of lung cancer.

Vitamin E sequestration by liver fat in humans.

BACKGROUNDWe hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls.METHODSCustom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies.RESULTSIn healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3-4 hours; d3- and d6-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d6-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration.CONCLUSIONSThe unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins.TRIAL REGISTRATIONClinicalTrials.gov, NCT00862433.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.

Screening of potential biomarkers and their predictive value in early stage non-small cell lung cancer: a bioinformatics analysis.

BACKGROUND: Non-small cell lung cancer (NSCLC) remains the first leading cause of death in malignancies worldwide. Despite the early screening of NSCLC by low-dose spiral computed tomography (CT) in high-risk individuals caused a 20% reduction in the mortality, there still exists imperative needs for the identification of novel biomarkers for the diagnosis and treatment of lung cancer. METHODS: mRNA microarray datasets GSE19188, GSE33532, and GSE44077 were searched, and the differentially expressed genes (DEGs) were obtained using GEO2R. Functional and pathway enrichment analyses were performed for the DEGs using DAVID database. Protein-protein interaction (PPI) network was plotted with STRING and visualized by Cytoscape. Module analysis of the PPI network was done through MCODE. The overall survival (OS) analysis of genes from MCODE was performed with the Kaplan Meier-plotter. RESULTS: A total of 221 DEGs were obtained, which were mainly enriched in the terms related to cell division, cell proliferation, and signal transduction. A PPI network was constructed, consisting of 221 nodes and 739 edges. A significant module including 27 genes was identified in the PPI network. Elevated expression of these genes was associated with poor OS of NSCLC patients, including UBE2T, UNF2, CDKN3, ANLN, CCNB2, and CKAP2L. The enriched functions and pathways included protein binding, ATP binding, cell cycle, and p53 signaling pathway. CONCLUSIONS: The DEGs in NSCLC have the potential to become useful targets for the diagnosis and treatment of NSCLC.

Vitamin C and Helicobacter pylori Infection: Current Knowledge and Future Prospects.

The gram-negative bacterium, Helicobacter pylori (H. pylori), infection is predominantly known for its strong association with development of gastric diseases, including gastritis, peptic ulcers, and stomach cancer. Numerous clinical reports show that ascorbic acid deficiency has been connect with gastritis. Vitamin C levels both in gastric acid and serum have constantly been affirmed to be low in subjects with H. pylori infected gastritis and peptic ulcers. Ascorbic acid supplementation likely relates to reduced incidences of bleeding from peptic ulcers and gastric cancer. H. pylori eradication is shown to increase vitamin C levels, while the benefits of ascorbic acid oral intake to increase the effectiveness of H. pylori-eradication therapy are controversial. Recent studies suggest that ascorbate intake intravenously, but not orally; pharmacologic ascorbate concentrations up to 30 mmol/L in blood, several millimolar in tissues as well as in interstitial fluid, are easily and safely achieved. Pharmacologic ascorbate can exert pro-oxidant effects locally as a drug by mediating hydrogen peroxide (H2O2) formation, which was applied to animal and clinical trials of cancer, sepsis, and severe burns etc. In this review, we summarize current understanding of the associations of vitamin C and H. pylori infection, and outline some potential strategies for H. pylori intervention from emerging advances on ascorbic acid physiology and pharmacology.

The SLC2A14 gene, encoding the novel glucose/dehydroascorbate transporter GLUT14, is associated with inflammatory bowel disease.

Background: Variations in intestinal antioxidant membrane transporters are implicated in the initiation and progression of inflammatory bowel disease (IBD). Facilitated glucose transporter member 14 (GLUT14), encoded by the solute carrier family 2 member 14 (SLC2A14) gene, is a putative transporter for dehydroascorbic acid and glucose. Although information on the gene is limited, shorter and longer GLUT14 isoforms have been identified. We hypothesized that GLUT14 mediates glucose and dehydroascorbic acid uptake. If this function could be validated, then genetic variations may associate with IBD.Objective: This study aimed to determine the substrate(s) for the GLUT14 protein and interrogated genetic associations of SLC2A14 with IBD.Design: The uptake of radiolabeled substrates into Xenopus laevis oocytes expressing the 2 GLUT14 isoforms was assessed. Examination of gene-targeted genetic association in the Manitoba Inflammatory Bowel Disease Cohort Study was conducted through the genotyping of single nucleotide polymorphisms (SNPs) representing linkage blocks of the SLC2A14 gene.Results: Both GLUT14 isoforms mediated the uptake of dehydroascorbic acid and glucose into X. laevis oocytes. Three alleles in the SLC2A14 gene associated independently with IBD. The odds of having ulcerative colitis (UC) or Crohn disease (CD) were elevated in carriers of the SLC2A14 SNP rs2889504-T allele (OR: 3.60; 95% CI: 1.95, 6.64 and OR: 4.68; 95% CI: 2.78, 8.50, respectively). Similarly, the SNP rs10846086-G allele was associated with an increased risk of both UC and CD (OR: 2.91; 95% CI: 1.49, 5.68 and OR: 3.00; 95% CI: 1.55, 5.78, respectively). Moreover, the SNP rs12815313-T allele associated with increased susceptibility to CD and UC (OR: 2.12; 95% CI: 1.33, 3.36 and OR: 1.61; 95% CI: 1.01, 2.57, respectively).Conclusion: These findings strengthen the hypothesis that genetically determined local dysregulation of dietary vitamin C or antioxidants transport contributes to IBD development. These transporter proteins are targetable by dietary interventions, opening the avenue to a precision intervention for patients of specific genotypes with IBD. This trial was registered at clinicaltrials.gov as NCT03262649.

MicroRNA-21 Regulates Non-Small Cell Lung Cancer Cell Invasion and Chemo-Sensitivity through SMAD7.

BACKGROUND/AIMS: SMAD7 is a key inhibitor of transforming growth factor ß (TGFß) receptor signaling, which regulates the alteration of cancer cell invasiveness through epithelial-mesenchymal cell conversion. Carboplatin is a commonly used drug in the chemotherapy for non-small cell lung cancer (NSCLC). Nevertheless, the molecular mechanisms underlying its suppressive effects on the NSCLC cell invasion are not completely understood. In the current study, we addressed this question by analyzing the effects of Carboplatin on microRNA-regulated SMAD7. METHODS: We used Carboplatin to treat NSCLC cell lines. We performed bioinformatics analyses on the binding of microRNA-21 (miR-21) to the 3'-UTR of SMAD7 mRNA, and verified the biological effects of this binding using promoter luciferase reporter assay. The effects of Carboplatin or miR-21-modification on NSCLC cell invasion were evaluated in either a transwell cell invasion assay, or a scratch wound healing assay. RESULTS: We found that Carboplatin inhibited the NSCLC cell invasion, in either a transwell cell invasion assay, or a scratch wound healing assay. Moreover, Carboplatin increased the levels of SMAD7 protein, but not mRNA, in NSCLC cells, suggesting presence of post-transcriptional control of SMAD7 by Carboplatin. Furthermore, expression of miR-21 was found to be inhibited by Carboplatin, and bioinformatics analyses showed that miR-21 targeted the 3'-UTR of SMAD7 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. CONCLUSION: Carboplatin may upregulate SMAD7 through suppression of miR-21 to inhibit TGFß receptor signaling mediated NSCLC cell invasion.

Cigarette Smoke-Induced Hypermethylation of the GCLC Gene Is Associated With COPD.

BACKGROUND: Cigarette smoking is a major environmental contributor to COPD, but understanding its epigenetic regulation of oxidative genes involved in the pathogenesis of COPD remains elusive. METHODS: We analyzed DNA methylation on glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase M1 (GSTM1), glutathione S-transferase P1 (GSTP1), and superoxide dismutase 3 (SOD3) promoters in clinical samples from patients with COPD (current-smoker [CS-COPD]; ex-smoker [ES-COPD]) and subjects with normal pulmonary function (current-smoker [CS-NS]; ex-smoker [ES-NS]; never-smoker [NC]). Expression of GCLC messenger RNA (mRNA) and glutathione (GSH) synthesis in these clinical samples and human bronchial epithelial (BEAS-2B) cells stimulated by cigarette-smoke extract (CSE) was evaluated. GCLC mRNA and protein levels were measured to determine effects of demethylation and deacetylation agents on CSE-treated BEAS-2B cells. RESULTS: The DNA methylation level of the GCLC promoter was significantly increased in CS-COPD, CS-NS, and ES-COPD groups compared with ES-NS and NC groups. However, there were no significant differences in DNA methylation values of GSTM1, GSTP1, and SOD3 promoters among these groups. Expression of GCLC mRNA was downregulated in the lungs, and GSH levels decreased in plasma as a consequence of hypermethylation of the GCLC promoter. Similarly, CSE-treated BEAS-2B cells had hypermethylation of the GCLC gene, mRNA downregulation, and a decreased intracellular GSH level. GCLC expression in CSE-treated BEAS-2B cells was restored by the methylation inhibitor, 5-aza-2'-deoxycytidine, but not by the deacetylation agent, trichostatin A. CONCLUSIONS: Cigarette smoke-induced hypermethylation of the GCLC promoter is related to the initiation and progression of COPD. Our finding may provide a new strategy for COPD intervention by developing demethylation agents targeting GCLC hypermethylation.

Ascorbic acid kills Epstein-Barr virus positive Burkitt lymphoma cells and Epstein-Barr virus transformed B-cells in vitro, but not in vivo.

Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.

A novel mutation of estrogen receptor gene detected in girls with precocious puberty.

Female precocious puberty is caused by premature activation of the hypothalamic-pituitary-gonadal axis, exposure to exogenous sex steroid hormones, and the presence of endogenous sex steroids caused by various factors. Estrogen is the final key factor to start onset of puberty. However,in some cases of precocious puberty in girls estrogen elevation could not be detected. The raised sensitivity of estrogen receptor, which may caused by ESR1 mutation or polymorphism, has been frequently mentioned for interpreting the etiology of sporadic low estrogen type cases. But no case evidence has been found in clinical practice. For the purpose of screening possible mutations in estrogen receptor gene, leukocyte genomic DNA were collected from 16 girls with precocious puberty of sporadic low estrogen,and exons of ESR1 were amplified and analysized using PCR-SSCP/silver staining method. A single strand conformation change in exon 8 was found in one of the patients (No. 14). The suspected fragment were cloned to a T vector and sequenced for analysis. Sequencing of these clones revealed that this conformation change is caused by a C to T transition. This mutation results in the replacement of arginine by cystine at position 548 of ESR1 protein. The mutation created an extra Btsl digest site and made it can be readily identified by PCR-PFLP method. Further detection using this method, and sequencing of cloned exon8 colonies from patients proved that the patient No. 14 is Arg548/Cys548 heterozagous in genotype. This mutation increased hydrophobility of the area dramatically. The position and the conservative of this residue in vertebrates suggested Arg548 may play an important role in ESR1 function. For study the role of this mutation in the onset of precocious puberty, a firefly luciferase reporter plasmid pGL3-promoter-ERE was constructed,and a pCR3. 1-hermut pisimid expressing Cys548 ER was constructed based on wild type pCR3. 1her. Co-transfection of reporter and pCR3. 1 -hermut in CMF-7 cell strain proved that Cys548 mutant can significantly increase the transcription activity over the Arg548 wild type.

Identification of an E-box motif as a transcriptional repressor element in the proximal promoter region of the GCLC gene in rat lung epithelial L2 cells.

Glutathione (GSH) is a critical antioxidant for protecting the airway epithelium from oxidant injury and its levels are mainly controlled by glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in GSH synthesis. A full understanding of the gene regulation mechanism of this important enzyme may disclose the role it plays in respiratory diseases. GCL is made up of two differentially regulated subunits, a catalytic or heavy subunit (GCLC) and a modifier or light subunit (GCLM). Many studies in this field led to the findings of important positive regulatory regions of the GCLC promoter. For a detailed analysis of this gene regulation in the respiratory system, we cloned a 1.76-kb 5'-flanking region of the rat GCLC gene, inserted into a luciferase reporter vector. Exonuclease III was used to cut the 5'-flanking region of the rat GCLC gene unidirectionally into deletion mutants of different lengths. Sequential deletion analysis revealed that regions from -403 to -111 and from -705 to -613 are involved in positive regulation and the region from -745 to -705 is involved in negative regulation of the GCL gene in rat lung epithelial L2 cells. Specific proteins binding to these regions were confirmed by electrophoretic mobility-shift assays (EMSAs) and antibody supershift assays. An E-box motif was found in the negative regulatory region -745 to -705. Site-directed mutagenesis proved that the functional element in this negative regulatory region was a putative E-box element. EMSA and supershift assays showed that USF1 and USF2 can specifically bind to the E-box element. Overexpression of USFs in L2 cells led to a decreased activity of the GCLC promoter. Western blotting demonstrated that the expression of GCLC protein was decreased in the retroviral USFs-expressing cells than in nontransfected (no DNA added) cells, suggesting that USF binding to the E-box at -729/-724 serves to trans-repress GCLC gene expression. These findings indicate that the E-box is an important transcriptional suppressor element in the GCLC promoter in rat lung epithelial L2 cells. Inhibition of interaction between the E-box and the USF may provide an effective means of ameliorating oxidant injury of the lung.

[Preliminary analysis of the 5'-flanking region of rat gamma-glutamylcysteine synthetase catalytic subunit gene].

OBJECTIVE: To analyze the characteristic of regulatory elements and corresponding transcriptional factors in the 5'-flanking region of rat gamma-glutamylcysteine synthetase (gamma-GCS) catalytic subunit (GCLC) gene. METHODS: A 1 760 bp 5'-flanking region of the rat GCLC was cloned and constructed into pGL-3 enhancer vector which includes luciferase reporter gene. Exonuclease III was used to cut the 5'-flanking region of rat GCLC gene unidirectionally into deletion mutants of different length, GCLC-Luc and its deletion mutants were used to transfect rat alveolar epithelium cells CCL-149, then the regulatory region of the gene was determined by luciferase activity assay of the transfected cells. Analysis of the transcription-factor-binding site was done using Transcription Factor Search software to indicate possible transcriptional factors. Electrophoresis mobility shift assay (EMSA) was used to determine the cis-acting elements and transcriptional factors in these regulatory regions. RESULTS: The experiment cloned the upstream regulatory sequence of rat GCLC gene and its reporter vector GCLC-Luc, as well as 11 deletion mutants of GCLC-Luc. Luciferase activity assay of the cells transfected by GCLC-Luc (-1 758/+2-Luc), mutant 1 (-1 231/+2-Luc), mutant 2 (-1 108/+2-Luc), mutant 3 (-1 087/+2-Luc), mutant 4 (-876/+2-Luc), mutant 5 (-745/+2-Luc), mutant 6 (-705/+2-Luc), mutant 8 (-613/+2-Luc), mutant 9 (-595/+2-Luc), mutant 10 (-403/+2-Luc) and mutant 11(-111/+2-Luc) were (90 012 +/- 2 445), (77 652 +/- 840), (149 927 +/- 4 915), (71 588 +/- 1 108), (99 283 +/- 2 612), (75 443 +/- 1 438), (282 772 +/- 7 046), (96 891 +/- 2 275), (148 917 +/- 5 966), (258 991 +/- 5 015) and (895 +/- 49) U, respectively. EMSA proved that activated protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) can bind to the region of -403 to -111 bp; nuclear factor-1 (NF-1) and CCAAT/enhancer binding protein (C/EBP) can bind to the region of -705 to -613 bp; and upstream stimulatory factor (USF) can bind to the region of -745 to -705 bp. CONCLUSIONS: Two DNA regions -403 to -111 bp and -705 to -613 bp of GCLC gene, which can be bound by transcriptional factors such as NF-1, C/EBP, AP-1, and NF-kappaB on EMSA, are involved in positive gene regulation. A newly identified region -745 to -705 bp of GCLC gene, which can be bound by USF, is involved in negative gene regulation, suggesting that the interaction between E-box and USF can inhibit the expression of gamma-GCS.

Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea.

Src I is the first reported acidic actinoporin from sea anemone Sagartia rosea with a pI value of 4.8 and comprises 13.9% alpha-helix, 65.1% beta-sheet, and 18.2% random coil. For structure-function studies, Src I was expressed in Escherichia coli as a cleavable fusion protein. Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic alpha-helix for its functional activity. The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells. Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID(50) 2.8 microg/ml and BGC-823 was the least sensitive cells with ID(50) 7.4 microg/ml. After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent. When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein. According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3',5'-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.