Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Dietary Patterns and Intrinsic Capacity in Community-Dwelling Older Adults: A Cross-Sectional Study.

OBJECTIVES: Few studies have investigated the link between diet and intrinsic capacity (IC), and the potential sex difference in such association. This study examined the association between dietary patterns and IC and its sub-domains in Chinese community-dwelling older adults. DESIGN: Cross-sectional analysis using baseline data from the MrOs and MsOs study. SETTING: Community. PARTICIPANTS: Chinese community-dwelling older adults aged ≥65 years in Hong Kong. MEASUREMENTS: Dietary intake was assessed using a validated food frequency questionnaire and priori and posteriori dietary pattern scores were generated. IC including measures of cognitive, locomotor, vitality, sensory and psychological domains was assessed. Multiple logistic regression was performed to examine the associations between dietary pattern scores and the likelihood of greater IC and sub-domain scores with adjustment for sociodemographic and lifestyle factors. RESULTS: Data of 3730 participants (aged 72.2±5.0 years, 50.4% men) was available. In men, higher Diet Quality Index-International (DQI-I) and Okinawan diet scores, and lower "meat-fish" pattern scores were associated with greater IC. A higher DQI-I score was associated with greater locomotion, whereas higher "snacks-drinks-milk products" pattern score was associated with a greater sensory function. In women, none of the dietary pattern scores was associated with IC. Higher DQI-I score, Mediterranean-DASH Intervention for Neurodegenerative Delay Diet (MIND) score and "vegetables-fruits" pattern score were associated with greater psychological function. CONCLUSION: Various dietary patterns were associated with greater IC and its sub-domains in Chinese community-dwelling older adults, and more associations were observed in men than women. Strategies to improve diet and IC should take sex differences into account.

Statin use and lung cancer risk in chronic obstructive pulmonary disease patients: a population-based cohort study.

BACKGROUND: Patients living with chronic obstructive pulmonary disease (COPD) are at an increased risk of lung cancer. A common comorbidity of COPD is cardiovascular disease; as such, COPD patients often receive statins. This study sought to understand the association between statin exposure and lung cancer risk in a population-based cohort of COPD patients. METHODS: We identified a population-based cohort of COPD patients based on having filled at least three prescriptions for an anticholinergic or short-acting beta-agonist (SABA). We used an array of methods of defining medication exposure including three conventional methods (ever statin exposure, cumulative duration of use, and cumulative dose) and two novel methods (recency-weighted cumulative duration of use and recency-weighted cumulative dose). To assess residual confounding, a negative control exposure was used to test the validity of our results. All exposure variables were time-dependent. RESULTS: The population-based cohort of COPD had 39,879 patients with mean age of 70.6 (SD: 11.2) years and, of which, 53.5% were female. There were 12,469 patients who received at least one statin prescription. Results from the reference case multivariable analysis indicated a reduced risk from statin exposure (HR: 0.85 (95% CI: 0.73-1.00) in COPD patients, but this result not statistically significant. Using the two recency-weighted modelling approaches, statin exposure was associated with a statistically significant reduction in lung cancer risk (recency-weighted cumulative dose, HR: 0.85 (95% CI: 0.77-0.93) and recency-weighted cumulative duration of use, HR: 0.97 (95% CI: 0.96-0.99). Multivariable analysis incorporating the negative control exposure was not statistically significant (HR: 0.89 (95% CI: 0.75-1.10). CONCLUSIONS: The results of this population-based analysis indicate that statin use in COPD patients may reduce the risk of lung cancer. While the effect was not statistically significantly across all exposure definitions, the overall results support the hypothesis that COPD patients might benefit from statin therapy.

Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

Novel genes and insights in complete asthma remission: A genome-wide association study on clinical and complete asthma remission.

BACKGROUND: Asthma is a chronic respiratory disease without a cure, although there exists spontaneous remission. Genome-wide association (GWA) studies have pinpointed genes associated with asthma development, but did not investigate asthma remission. OBJECTIVE: We performed a GWA study to develop insights in asthma remission. METHODS: Clinical remission (ClinR) was defined by the absence of asthma treatment and wheezing in the last year and asthma attacks in the last 3 years and complete remission (ComR) similarly but additionally with normal lung function and absence of bronchial hyperresponsiveness (BHR). A GWA study on both ClinR and ComR was performed in 790 asthmatics with initial doctor diagnosis of asthma and BHR and long-term follow-up. We assessed replication of the 25 top single nucleotide polymorphisms (SNPs) in 2 independent cohorts (total n = 456), followed by expression quantitative loci (eQTL) analyses of the 4 replicated SNPs in lung tissue and epithelium. RESULTS: Of the 790 asthmatics, 178 (23%) had ClinR and 55 ComR (7%) after median follow-up of 15.5 (range 3.3-47.8) years. In ClinR, 1 of the 25 SNPs, rs2740102, replicated in a meta-analysis of the replication cohorts, which was an eQTL for POLI in lung tissue. In ComR, 3 SNPs replicated in a meta-analysis of the replication cohorts. The top-hit, rs6581895, almost reached genome-wide significance (P-value 4.68 × 10-7 ) and was an eQTL for FRS2 and CCT in lung tissue. Rs1420101 was a cis-eQTL in lung tissue for IL1RL1 and IL18R1 and a trans-eQTL for IL13. CONCLUSIONS AND CLINICAL RELEVANCE: By defining a strict remission phenotype, we identified 3 SNPs to be associated with complete asthma remission, where 2 SNPs have plausible biological relevance in FRS2, CCT, IL1RL1, IL18R1 and IL13.

PTTG1IP and MAML3, novel genomewide association study genes for severity of hyperresponsiveness in adult asthma.

BACKGROUND: The severity of bronchial hyperresponsiveness (BHR) is a fundamental feature of asthma. The severity of BHR varies between asthmatics and is associated with lack of asthma control. The mechanisms underlying this trait are still unclear. This study aimed to identify genes associated with BHR severity, using a genomewide association study (GWAS) on the slope of BHR in adult asthmatics. METHODS: We performed a GWAS on BHR severity in adult asthmatics from the Dutch Asthma GWAS cohort (n = 650), adjusting for smoking and inhaled corticosteroid use, and verified results in three other cohorts. Furthermore, we performed eQTL and co-expression analyses in lung tissue. RESULTS: In the discovery cohort, one genomewide significant hit located in phosphodiesterase 4D, cAMP-specif (PDE4D) and 26 SNPs with P-values < 1*10-5 were found. None of our findings replicated in adult and childhood replication cohorts jointly. In adult cohorts separately, rs1344110 in pituitary tumour-transforming 1 interacting protein (PTTG1IP) and rs345983 in Mastermind-like 3 (MAML3) replicated nominally; minor alleles of rs345983 and rs1344110 were associated with less severe BHR and higher lung tissue gene expression. PTTG1IP showed significant co-expression with pituitary tumour-transforming 1, the binding factor of PTTG1lP, and with vimentin and E-cadherin1. MAML3 co-expressed significantly with Mastermind-like 2 (MAML2), both involved in Notch signalling. CONCLUSIONS: PTTG1IP and MAML3 are associated with BHR severity in adult asthma. The relevance of these genes is supported by the eQTL analyses and co-expression of PTTG1lP with vimentin and E-cadherin1, and MAML3 with MAML2.

Effect of controlled human exposure to diesel exhaust and allergen on airway surfactant protein D, myeloperoxidase and club (Clara) cell secretory protein 16.

BACKGROUND: Air pollution is a major cause of global morbidity and mortality. Air pollution and aeroallergens aggravate respiratory illness, but the variable effects of air pollutants and allergens in the lung are poorly understood. OBJECTIVE: To determine the effects of diesel exhaust (DE) and bronchial allergen challenge as single and dual exposures on aspects of innate immunity in the airway as reflected by surfactant protein D (SPD), myeloperoxidase (MPO) and club (Clara) cell secretory protein 16 (CC16) in 18 atopic individuals. METHODS: In this double-blind, randomized crossover study, atopic individuals were exposed to DE or filtered air, followed by endobronchial allergen or saline 1 hour after inhalational exposure. Bronchoalveolar lavage, bronchial washings, nasal lavage and blood samples were obtained 48 hours after exposures and assayed for CC16, MPO and SPD by ELISA. RESULTS: In bronchial samples, the concentration of SPD increased from 53.3 to 91.8 ng/mL after endobronchial allergen, with no additional contribution from DE. MPO also increased significantly in response to allergen (6.8 to 14.7 ng/mL), and there was a small additional contribution from exposure to DE. The concentration of CC16 decreased from 340.7 to 151.0 ng/mL in response to DE, with minor contribution from allergen. These changes were not reflected in nasal lavage fluid or plasma samples. CONCLUSIONS AND CLINICAL RELEVANCE: These findings suggest that allergen and DE variably influence different aspects of the innate immune response of the lung. SPD and MPO, known markers of allergic inflammation in the lung, are strongly increased by allergen while DE has a minor effect therein. DE induces a loss of CC16, a protective protein, while allergen has a minor effect therein. Results support site- and exposure-specific responses in the human lung upon multiple exposures.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

Independent association of subclinical coronary artery disease and emphysema in HIV-infected patients.

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) and coronary artery disease are inflammatory states with a significant clinical impact. The relationship between them has not been investigated in patients with HIV infection. We assessed the presence of subclinical emphysema and coronary artery disease using chest computed tomography (CT) imaging in a cohort of HIV-infected patients receiving antiretroviral therapy. METHODS: Gated chest CT scans were performed in 1446 consecutive patients to assess the presence and severity of coronary artery calcium (CAC) (classified as a score of 0, 1-100 or > 100) and emphysema (classified using a visual semiquantitative scale: 0, absent; 1-4, mild to moderate; > 4, severe). Univariable and multivariable logistic regression analyses were performed to identify factors independently associated with CAC and emphysema. RESULTS: The emphysema score was significantly higher in patients with CAC scores of 1-100 and > 100 compared with those with a CAC score of 0. After adjustments for age, sex, smoking status, pack-years of smoking, visceral adiposity and duration of HIV infection, the presence of any emphysema was significantly associated with a CAC score > 0 [odds ratio (OR) 1.43; 95% confidence interval (CI) 1.08-1.88; P = 0.012]. The association persisted after adjustment for the Framingham risk score (OR 1.52; 95% CI 1.16-1.99; P = 0.002). There was a dose-dependent effect in the association between emphysema score and CAC score. CONCLUSIONS: In this cross-sectional study of HIV-infected patients, there was an independent association between emphysema and CAC, after adjustment for traditional cardiovascular risk factors, suggesting a common pathogenesis of these chronic inflammatory conditions in a chronic inflammatory disease such as HIV infection.

Deficiency of FHL2 attenuates airway inflammation in mice and genetic variation associates with human bronchial hyper-responsiveness.

BACKGROUND: Asthma is an inflammatory disease that involves airway hyper-responsiveness and mucus hypersecretion. The LIM-only protein FHL2 is a crucial modulator of multiple signal transduction pathways and functions as a scaffold in specific protein-protein interactions. OBJECTIVE: We sought to investigate the role of FHL2 in airway inflammation. METHODS: Allergic airway inflammation was induced in WT and FHL2-knock out (FHL2-KO) mice with ovalbumin (OVA). Lung tissue, bronchoalveolar lavage fluid (BALF) and draining lymph node cells were analysed for inflammation. FHL2 loss and gain of function studies were performed in lung epithelial cells. RESULTS: FHL2-deficient mice challenged with OVA show significantly reduced airway inflammation as evidenced by reduced infiltration of inflammatory cells including eosinophils, dendritic cells, B cells and T cells. Furthermore, mucus production was decreased in FHL2-KO mice. In BALF, the levels of IL-5, IL-13, eotaxin-1 and eotaxin-2 were significantly lower in FHL2-KO mice. In addition, draining lymph node cells from FHL2-KO mice show reduced levels of IL-5 and IL-13. Consistent with this, OVA-specific serum IgG and IgE levels were reduced in FHL2-KO mice. We also found that phosphorylation of ERK1/2 is markedly attenuated in FHL2-KO lung. Knock-down of FHL2 in human lung epithelial cells resulted in a striking decrease in ERK1/2 phosphorylation and mRNA levels of inflammatory cytokines and MUC5AC, whereas FHL2 overexpression exhibited opposite effects. Finally, the SNP rs4851765 shows an association with the severity of bronchial hyper-responsiveness. CONCLUSION: These results highlight functional involvement of FHL2 in airway inflammation and identify FHL2 as a novel gene associated with asthma severity in human.

Characteristics of COPD in never-smokers and ever-smokers in the general population: results from the CanCOLD study.

BACKGROUND: There is limited data on the risk factors and phenotypical characteristics associated with spirometrically confirmed COPD in never-smokers in the general population. AIMS: To compare the characteristics associated with COPD by gender and by severity of airway obstruction in never-smokers and in ever-smokers. METHOD: We analysed the data from 5176 adults aged 40 years and older who participated in the initial cross-sectional phase of the population-based, prospective, multisite Canadian Cohort of Obstructive Lung Disease study. Never-smokers were defined as those with a lifetime exposure of <1/20 pack year. Logistic regressions were constructed to evaluate associations for 'mild' and 'moderate-severe' COPD defined by FEV1/FVC <5th centile (lower limits of normal). Analyses were performed using SAS V.9.1 (SAS Institute, Cary, North Carolina, USA). RESULTS: The prevalence of COPD (FEV1/FVC

Exacerbation-like respiratory symptoms in individuals without chronic obstructive pulmonary disease: results from a population-based study.

RATIONALE: Exacerbations of COPD are defined clinically by worsening of chronic respiratory symptoms. Chronic respiratory symptoms are common in the general population. There are no data on the frequency of exacerbation-like events in individuals without spirometric evidence of COPD. AIMS: To determine the occurrence of 'exacerbation-like' events in individuals without airflow limitation, their associated risk factors, healthcare utilisation and social impacts. METHOD: We analysed the cross-sectional data from 5176 people aged 40 years and older who participated in a multisite, population-based study on lung health. The study cohort was stratified into spirometrically defined COPD (post-bronchodilator FEV1/FVC < 0.7) and non-COPD (post bronchodilator FEV1/FVC ≥ 0.7 and without self-reported doctor diagnosis of airway diseases) subgroups and then into those with and without respiratory 'exacerbation-like' events in the past year. RESULTS: Individuals without COPD had half the frequency of 'exacerbation-like' events compared with those with COPD. In the non-COPD group, the independent associations with 'exacerbations' included female gender, presence of wheezing, the use of respiratory medications and self-perceived poor health. In the non-COPD group, those with exacerbations were more likely than those without exacerbations to have poorer health-related quality of life (12-item Short-Form Health Survey), miss social activities (58.5% vs 18.8%), miss work for income (41.5% vs 17.3%) and miss housework (55.6% vs 16.5%), p<0.01 to <0.0001. CONCLUSIONS: Events similar to exacerbations of COPD can occur in individuals without COPD or asthma and are associated with significant health and socioeconomic outcomes. They increase the respiratory burden in the community and may contribute to the false-positive diagnosis of asthma or COPD.

Risk of new onset diabetes mellitus in patients with asthma or COPD taking inhaled corticosteroids.

BACKGROUND: A recent case-controlled study reported an increased risk of diabetes mellitus in patients treated with inhaled corticosteroids for asthma or COPD, versus age-matched controls. OBJECTIVE: The purpose of the current study was to evaluate whether there was an increased risk of new onset diabetes mellitus or hyperglycaemia among patients with asthma or COPD treated with inhaled corticosteroids. METHODS: A retrospective analysis evaluated all double-blind, placebo-controlled, trials in patients ≥4 years of age involving budesonide or budesonide/formoterol in asthma (26 trials; budesonide: n = 9067; placebo: n = 5926), and in COPD (8 trials; budesonide: n = 4616; non-ICS: n = 3643). A secondary dataset evaluated all double-blind, controlled trials in asthma involving the use of inhaled corticosteroids (60 trials; budesonide: n = 33,496; fluticasone: n = 2773). RESULTS: In the primary asthma dataset, the occurrence of diabetes mellitus/hyperglycaemia adverse events (AEs) was 0.13% for budesonide and 0.13% for placebo (HR 0.98 [95% CI: 0.38-2.50], p = 0.96) and serious adverse events (SAEs) was 0% for budesonide and 0.05% for placebo. In the secondary dataset, the occurrence of diabetes/hyperglycaemia as AE and SAE was 0.19% and 0.03%, respectively. In the COPD dataset, the occurrence of diabetes mellitus/hyperglycaemia AEs was 1.3% for budesonide and 1.2% for non-ICS (HR 0.99 [95% CI: 0.67-1.46], p = 0.96) and SAEs was 0.1% for budesonide and 0.03% for non-ICS. CONCLUSION AND CLINICAL RELEVANCE: Treatment with inhaled corticosteroids in patients with asthma or COPD was not associated with increased risk of new onset diabetes mellitus or hyperglycaemia.

Canadian prediction equations of spirometric lung function for Caucasian adults 20 to 90 years of age: results from the Canadian Obstructive Lung Disease (COLD) study and the Lung Health Canadian Environment (LHCE) study.

BACKGROUND: Currently, no reference or normative values for spirometry based on a randomly selected Canadian population exist. OBJECTIVE: The aim of the present analysis was to construct spirometric reference values for Canadian adults 20 to 90 years of age by combining data collected from healthy lifelong nonsmokers in two population-based studies. METHOD: Both studies similarly used random population sampling, conducted using validated epidemiological protocols in the Canadian Obstructive Lung Disease study, and the Lung Health Canadian Environment study. Spirometric lung function data were available from 3042 subjects in the COLD study, which was completed in 2009, and from 2571 subjects in the LHCE study completed in 1995. A total of 844 subjects 40 to 90 years of age, and 812 subjects 20 to 44 years of age, were identified as healthy, asymptomatic, lifelong nonsmokers, and provided normative reference values for spirometry. Multiple regression models were constructed separately for Caucasian men and women for the following spirometric parameters: forced expiratory volume in 1 s (FEV(1)), forced vital capacity (FVC) and FEV(1)/FVC ratio, with covariates of height, sex and age. Comparison with published regression equations showed that the best agreement was obtained from data derived from random populations. RESULTS: The best-fitting regression models for healthy, never-smoking, asymptomatic European-Canadian men and women 20 to 90 years of age were constructed. When age- and height-corrected FEV(1), FVC and FEV(1)/FVC ratio were compared with other spirometry reference studies, mean values were similar, with the closest being derived from population-based studies. CONCLUSION: These spirometry reference equations, derived from randomly selected population-based cohorts with stringently monitored lung function measurements, provide data currently lacking in Canada.

Can age and sex explain the variation in COPD rates across large urban cities? A population study in Canada.

OBJECTIVE: To measure the prevalence of chronic obstructive pulmonary disease (COPD) and determine the effect of age and sex on the variation in prevalence across major cities within the same country and health care system. METHOD: We used the Burden of Obstructive Lung Disease (BOLD) methodology to estimate the prevalence of COPD in adults aged ≥ 40 years in different Canadian cities. The study used interviewer-administered questionnaires on respiratory, smoking and occupational history, medication use and comorbidities. Post-bronchodilator spirometry was used to classify subjects. We determined the prevalence and severity of COPD with and without adjustments for age and sex distribution across different cities. RESULTS: The study population was 3042. Overall, 16.7% (95%CI 14.8-18.7) of study subjects met the criteria for Global Initiative for Chronic Obstructive Lung Disease (GOLD) severity Stage 1 or higher. The prevalence according to the criteria for the lower limits of normal of the ratio forced expiratory volume in 1 second/forced vital capacity was 11.6% (95%CI 9.9-13.3). COPD prevalence varied by severity across site (P = 0.0025). After age-sex adjustment, the variation disappeared (P> 0.16). CONCLUSION: Age and sex differences account for most of the heterogeneity in COPD estimates across large cities within the same country. Adjustments for age and sex are essential in comparing COPD rates across the country.

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest.

OBJECTIVES: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti-cancer treatment have been sought from natural resources. Here, we have investigated anti-proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. MATERIAL AND METHODS: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate levels were analysed by HPLC. RESULTS: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G(2) /M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21(waf1/cip1) was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate in myriocin-treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. CONCLUSIONS: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21(waf1/cip1) , followed by inhibition of cyclin B1 and cdc2, resulting in G(2) /M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism-based therapy for this type of skin cancer.