[Characteristics and Source Apportionment of VOCs Initial Mixing Ratio in Beijing During Summer].

To clarify the characteristics and source apportionment of the VOCs initial mixing ratio in Beijing in summer, continuous monitoring of VOCs was conducted in the Beijing urban area from May to August 2022, and the initial mixing ratio was calculated using the photochemical ratio method. The results showed that:① during the study period, initial φ(TVOCs) in the Beijing urban area were (30.0 ±11.5)×10-9, in which the proportion of VOCs and alkanes containing oxygen reached 34.2% and 33.2%, respectively. The species with high volume fractions were low carbon substances such as acetone, ethane, acetaldehyde, and propane. ② The initial TVOCs mixing ratio in Beijing showed a slightly unimodal trend, reaching the peak at 11:00 and slightly decreasing in the afternoon. ③ Isoprene, acetaldehyde, n-butanal, and ethylene were the major contributors to the generation of O3, whereas toluene, isoprene, m-paraxylene, and ethylbenzene were the major contributors to the generation of secondary organic aerosols. ④ Based on the initial mixing ratio of PMF analysis, it was found that aging background and secondary sources (30%) contributed the most to VOCs in Beijing, and motor vehicle sources (25%) were the main primary human sources. In addition, solvent and fuel volatile sources contributed 16%, combustion sources contributed 11%, industrial process sources contributed 9%, and natural sources contributed 9%. ⑤ The anthropogenic sources of Beijing were mainly from the eastern and southern regions, whereas the natural sources were from the western and northwestern regions. This research showed that vehicle emissions should be further reduced, and regional joint prevention and control to reduce VOCs in the whole region is an effective means to control VOCs in Beijing.

Pharmacokinetics and Bioequivalence of Abiraterone Acetate Tablets in Healthy Chinese Volunteers: An Open, Randomized, Single-Dose, Three-Period, Three-Sequence Crossover Study.

BACKGROUND AND OBJECTIVE: Abiraterone acetate tablet is an inhibitor of androgen synthesis, primarily for the treatment of metastatic castration-resistant prostate cancer (mCRPC). This study evaluated the bioequivalence and pharmacokinetics of the reference and test formulations of abiraterone acetate tablets in healthy Chinese volunteers. METHODS: A single-center, open, single-dose, randomized, three-period, three-sequence, semi-repeat (only repeated reference formulations), and reference formulation-corrected fasting reference-scaled average bioequivalence test was conducted in 36 healthy volunteers included in this study. Volunteers were randomly assigned to one of three groups in a 1:1:1 ratio. There was a minimum 7-day washout period between each dose. Blood samples were collected at prescribed time intervals, the plasma concentration of abiraterone acetate tablets was determined by liquid chromatography-tandem mass spectrometry, and adverse events were recorded. RESULTS: Under fasting conditions, the maximum plasma concentration (Cmax) was 27.02 ± 14.21 ng/mL, area under the concentration-time curve from time zero to time t (AUCt) was 125.30 ± 82.41 h·ng/mL, and AUC from time zero to infinity (AUC∞) was 133.70 ± 83.99 h·ng/mL. The 90% confidence intervals (CIs) of the geometric mean ratio (GMR) of AUCt and AUC∞ were in the range of 0.8000-1.2500, and the coefficient of variation (CVWR) of Cmax was more than 30%. The Critbound result was - 0.0522, and the GMR was between 0.8000 and 1.2500. CONCLUSION: Both test and reference formulations of abiraterone acetate tablets were bioequivalent in healthy Chinese subjects under fasting conditions. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04863105, registered 26 April 2021-retrospectively registered ( https://register. CLINICALTRIALS: gov/prs/app/action/SelectProtocol?sid=S000ARAA&selectaction=Edit&uid=U00050YQ&ts=2&cx=-vbtjri.

[Characteristics and Sources of PM2.5 and Its Light-scattering Properties with Different Chemical Compositions in Urban Area of Beijing].

In recent years, the concentration of PM2.5 in the Beijing urban area has decreased with the increase in the proportion of secondary inorganic ions. In order to explore the characteristics and sources of the light scattering of PM2.5 with different chemical compositions, PM2.5 with its chemical components and scattering coefficient were continuously measured at hourly resolution in the Beijing urban area from December 2020 to November 2021. The components, scattering characteristics, and sources of PM2.5 were analyzed. The results showed that NO3- was the major component of PM2.5 in the Beijing urban area, and the ω(NO3-) and ω(SNA) were 24% and 46% in PM2.5, respectively. PM2.5 could be divided into six types according to mass concentration and component proportion. The occurrence frequency of the good-type was the highest during the study with a similar duration in the four seasons, and the ω(SNA), ω(OM), and ω(FS) were 32%, 32%, and 28% in PM2.5, respectively. The dust(D)-type and the OM(O)-type appeared mainly in spring and summer with the lowest frequency during the study. FS and OM were their major components, and the ω(FS) and ω(OM) were 66% and 46% in PM2.5, respectively. The OM+SO42-(OS)-type, OM+NO3-(ON)-type, and NO3-(N)-type appeared mainly in the afternoon in summer, in the early morning and morning in winter, and at approximately 07:00 every day in spring. Under the condition of low humidity[relative humidity (RH)<40%], the MSE of N-type PM2.5 was the highest (4.3 m2·g-1), and that of D-type PM2.5 was the lowest (2.1 m2·g-1), reflecting the high scattering ability of SNA. The MSE increased with relative humidity. Under the condition of high humidity (RH>80%), the MSE of all types of PM2.5 rose to 1.5 to 1.8 times the values under low humidity. The variation trends of SAE showed that particle size increased with the rising of RH level. Under non-high humidity conditions, the scattering coefficients reconstructed by the revised IMPROVE formula fitted well with the measured values at hourly resolution, the correlation coefficients were between 0.81 and 0.97, and the slopes were between 1.00 and 1.21 except for that of D-type. The N-type fitting result was the best. Under high-humidity conditions, the R and the slopes were from 0.82 to 0.84 and from 0.48 to 0.53, respectively. The annual Bsca was 203.8 Mm-1, and N-type PM2.5 contributed the most, accounting for 53%, in which the large particles of NH4NO3 were the major contributor. Bsca of good-type PM2.5 was 67.2 Mm-1, in which small particles of OM were the major contributor. Bsca was 1.5 times the annual Bsca(dry), whereas the Bsca values of SNA were 1.8 to 2.1 times the Bsca(dry). The peak value of NO3- and RH simultaneously appeared around 07:00, resulting in the maximum Bsca of NH4NO3 at this time. The peak value of SO42- and the Bsca of (NH4)2SO4 mainly appeared at 16:00 and at 04:00, respectively. The diurnal variation curves of OM concentration and Bsca were consistent, and the bimodal peaks appeared at 13:00 and 20:00, respectively. In spring and winter, NO3-, SO42- and OM mainly came from the plains east of the Taihang Mountains, and their potential source regions were not in any particular place in summer and autumn; the main potential source regions of FS were the northwest areas of Beijing in spring and autumn. The flow with high RH across the south and southeast of the north China plain and the eastern rim of Bohai Sea was likely to increase the weighted potential source contribution factor values of Bsca of SNA in this region.

Evaluation of the Effect of Food on the Pharmacokinetics of SHR6390, An Oral CDK4/6 Inhibitor, in Healthy Volunteers.

BACKGROUND AND INTRODUCTION: SHR6390 is a new developed highly effective and selective small-molecule oral CDK4/6 inhibitor. We aimed to evaluate the effect of food on the pharmacokinetics of SHR6390 tablets. METHODS: In an open-label two-way crossover study, 24 healthy Chinese volunteers were randomly divided into Group A and Group B, and 12 volunteers in each group received a single oral dose of a SHR6390 150-mg tablet under fasting and high-fat conditions. Blood samples were collected and determined for pharmacokinetic analyses. A liquid chromatography-tandem mass spectrometry method was developed and validated for determining the SHR6390 concentration. RESULTS: The time to maximum plasma concentration was not significantly affected by a high-fat diet. Compared with the fasting group, maximum plasma concentration, i.e., the area under the concentration-time curve (AUC0-t and AUC0-∞) was altered significantly, as evidenced by an increase of 56.9%, 38.6%, and 37.5% respectively. We identified seven metabolites of SHR6390 from the plasma samples, and we found no sex differences in metabolic pathways. All treatment-emergent adverse events were Grade 1 or 2. CONCLUSIONS: Food intake increased the maximum plasma concentration, AUC0-t, and AUC0-∞ significantly compared with the fasting condition. Meanwhile, single-dose SHR6390 for two treatment cycles is safe. SHR6390 was administered in a fasting status in the pivotal phase III study (NCT03927456) and chosen for the final drug label.

eIF3a R803K mutation mediates chemotherapy resistance by inducing cellular senescence in small cell lung cancer.

Drug resistance in small cell lung cancer (SCLC) significantly affects the efficacy of chemotherapy treatment. However, due to the lack of tumor tissue samples, especially serial tumor samples during chemotherapy, the mechanism of chemotherapy resistance has not been fully studied. Circulating tumor DNA, which can be obtained in a noninvasive manner, can complement tumor sampling approaches for research in this field. We identified an SCLC patient with acquired drug resistance from 52 SCLC patients for whom follow-up data were available. By comparing somatic mutations in circulating tumor DNA before and after chemotherapy, for the first time, we found that the somatic mutation eIF3A R803K may be related to acquired chemotherapy resistance. Then, the association between the eIF3A R803K mutation and chemotherapy resistance was confirmed by samples from 254 lung cancer patients receiving chemotherapy. We found that the eIF3a R803K mutation weakened the proliferation ability of tumor cells but increased their resistance to chemotherapy. Further studies revealed that the eIF3A R803K mutation promotes cellular senescence. In addition, fisetin showed a synergistic effect with chemotherapy in eIF3A R803K mutant cells. These results suggest that the eIF3A R803K somatic mutation has the potential to predict chemotherapy resistance in SCLC. Moreover, the eIF3A R803K mutation promotes chemotherapy resistance by inducing senescence. Furthermore, a senolytic drug, fisetin, can reverse chemotherapy resistance mediated by the eIF3A R803K mutation.

Magnesium demethylcantharidate inhibits hepatocellular carcinoma cell invasion and metastasis via activation transcription factor FOXO1.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, develops rapidly and has a high mortality rate. Relapsed metastasis is the most important factor affecting prognosis and is also the main cause of death for patients with HCC. Cantharidin is a kind of folk medicine for malignant tumors in China. Because of its cytotoxicity, the application of cantharidin is very limited. Magnesium demethylcantharidate (MDC) is a derivative of cantharidin independently developed by our laboratory. Our results show that MDC has anticancer activity and exhibited lower toxicity than cantharidin. However, whether MDC affects the invasion and metastasis of HCC cells and the underlying molecular mechanisms remain obscure. Transwell and Matrigel assays showed that MDC could effectively inhibit the invasion and metastasis of the HCC cell lines SMMC-7721 and SK-Hep1 in a dose-dependent manner. Moreover, MDC significantly inhibited the expression of invasion and metastasis related proteins MMP-2 and MMP-9. In addition, our study found that MDC inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 by activating transcription factor FOXO1. Interestingly, the combination of MDC and sorafenib significantly inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 compared with the single drug treatment via the activated transcription factor FOXO1. Our work revealed that MDC obviously inhibited the invasion and metastasis of HCC cells, and suggested that MDC could be a potential candidate molecule against the invasion and metastasis of HCC.

Pharmacokinetic and Safety Comparison of Two Capecitabine Tablets in Patients with Colorectal or Breast Cancer Under Fed Conditions: A Multicenter, Randomized, Open-Label, Three-Period, and Reference-Replicated Crossover Study.

INTRODUCTION: In this study, we assessed the pharmacokinetics (PK), bioequivalence, and safety of 150 mg capecitabine compared to the branded reference formulation in colorectal or breast cancer patients receiving a high-fat diet. METHODS: This was a multicenter, open, random, balanced, three-period, three-sequence and semi-repetitive cross study with 48 subjects. In each study period, the eligible subject received the test or reference formulation, followed by a 1-day washout period. Serial blood samples for pharmacokinetic assessment were collected at predose up to 8 h postdose. The plasma concentrations of capecitabine were analyzed by LC/MS-MS. Pharmacokinetic parameters (non-compartmental model) were assessed with WinNonlin software. The pharmacokinetic parameters assessed were the area under the plasma concentration-time curve from time 0 to the time of last measurable concentration (AUC0-t), the AUC from time zero to infinity (AUC0-∞), the peak plasma concentration of the drug (Cmax), the time needed to reach maximum concentration (Tmax), the elimination half-life (t1/2), and the terminal elimination rate (λz). All were analyzed using an analysis of variance (ANOVA) model after logarithmic transformation of the data. To establish the bioequivalence (BE) for capecitabine, reference-scaled average bioequivalence (RSABE) acceptance criteria and average bioequivalence (ABE) acceptance criteria were used. Safety and tolerability were assessed during the entire study period. RESULTS: Reference scaled maximum plasma concentration (Cmax) was higher than 0.294, permitting use of RSABE. The within-subject SDs of the reference intervention (SWR) for AUC0-t and AUC0-∞ were < 0.294, meeting ABE criteria. The point estimate for the geometric least squares mean (GLSM) ratio for the point estimate of Cmax was 0.962, within the range of 0.80-1.25. The 90% upper confidence boundary for the test/reference of GLSM ratios was 97.84-105.40% for AUC0-t and 97.33-103.51% for AUC0-∞, all of which were within the prespecified limits. The 90% confidence intervals for AUC0-t and AUC0-∞ and 95% upper confidence limit for Cmax indicated bioequivalence. No serious adverse events were found among the subjects. CONCLUSIONS: According to the criteria for bioequivalence, the test formulation was bioequivalent to the reference formulation in terms of the rate and extent of absorption under fed conditions by measurement of total capecitabine and was safe and well tolerated. TRIAL REGISTRATION: NCT04420871.

Pharmacokinetics and Bioequivalence of Cefprozil for Suspension and Granule Formulation in Healthy Chinese Volunteers: Two Single-Dose Crossover Studies.

INTRODUCTION: Cefprozil, an oral second-generation semi-synthetic cephalosporin, possesses a broad spectrum of antimicrobial activity. A granule formulation has been developed to improve medication adherence of the patients. This study was conducted to assess the bioequivalence of the granule formulation to a dry suspension in healthy Chinese volunteers and estimate the pharmacokinetic (PK) profiles of cefprozil. METHODS: An open-label, randomized, single-dose, two-period, two-group, crossover study was conducted in 60 healthy Chinese volunteers under fasted or fed conditions (30 volunteers for each condition) to assess the bioequivalence between two formulations of cefprozil. Blood samples were collected at specified time intervals, and the plasma concentrations of cis- and trans-cefprozil were determined by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. PK and bioavailability parameters were estimated via non-compartmental methods. Adverse events (AEs) were also recorded. RESULTS: Under fasted conditions, the mean Cmax was (3534.70 ± 634.67) ng/ml, Tmax was (0.98 ± 0.25) h, t1/2 was (1.37 ± 0.13) h and AUC0-t was (9302.86 ± 1618.39) ng·h/ml, respectively, after a single dose of 125 mg cefprozil for suspension. Under fed conditions, the mean Cmax was (2438.80 ± 493.78) ng/ml, Tmax was (1.66 ± 0.76) h, t1/2 was (1.36 ± 0.24) h and AUC0-t was (9332.36 ± 1373.61) ng·h/ml, respectively. The PK parameters of the granule formulation of cefprozil were similar to those of the suspension. The 90% CI values of the GMRs of Cmax, AUC0-t and AUC0-∞ under both fasted and fed conditions were within the prespecified bioequivalence range (80.00-125.00%). CONCLUSIONS: According to the criteria for bioequivalence, the test granule formulations of cefprozil and "Cefprozil for Suspension®" were determined to be bioequivalent whether under fasted or fed conditions by measurement of cis-, trans- and total cefprozil. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT04414254.

Holliday junction recognition protein promotes pancreatic cancer growth and metastasis via modulation of the MDM2/p53 signaling.

Holliday junction recognition protein (HJURP) refers to a histone H3 chaperone that has been implicated in different kinds of malignancies. Yet, its character in pancreatic cancer remains unclear. The expression of HJURP was assessed in PDAC tissues by RT-qPCR, immunoblotting, and immunohistochemistry. HJURP-deficient or overexpressed PDAC cell lines were constructed, using shRNA or plasmids with HJURP insert. MTT, sphere formation assay, migration, and invasion assays were performed to evaluate the viability, proliferation, migration, and invasion of PDAC cells. We used xenograft mice models to assess the tumor growth and metastasis in vivo. RNA-seq was applicated in search of the potential downstream target of HJURP in PDAC and subsequent verification were fulfilled via multiple assays, including immunofluorescence. Additionally, chromatin immunoprecipitation and luciferase reporter assay were conducted to explore the potential regulation of MDM2 expression by HJURP through H3K4me2. In this current research, we found that the expression of HJURP in PDAC cells and tissue was significantly higher than those of adjacent normal tissue, and high HJURP expression predicted poor survival. HJURP significantly promoted the viability, sphere formation, migration, and invasion of PDAC cells in vitro, HJURP also facilitated tumor growth and metastasis in vivo. Mechanically, MDM2/p53 axis is critical for HJURP-mediated malignant behaviors in PDAC, and HJURP regulates MDM2 expression through H3K4me2. HJURP could serve as a promising biomarker, and target for PDAC prognosis and treatment.

Icariin treatment reduces blood glucose levels in type 2 diabetic rats and protects pancreatic function.

Icariin, a flavonoid isolated from traditional oriental herbal medicines, has been demonstrated to exhibit several health benefits in animal models and in humans. The aim of the present study was to investigate the effect of Icariin on hyperglycemia in type 2 diabetes mellitus (T2DM) in rats. A model of diabetes was established in 50 Sprague Dawley rats using a high-sugar and high-fat diet and peritoneal injection of streptozotocin. Diabetic rats were divided into five groups: Diabetic control; metformin; and rats treated with three different doses of Icariin, 5, 10 and 20 mg/kg. Body weight and blood glucose levels were measured, and serum adiponectin levels, expression of phospho-AMP mediated protein kinase (p-AMPK) and glucose transporter isoform 4 (GLUT-4) were measured using ELISA, Realtime PCR and western blotting, respectively. Diabetic rats without drug treatment exhibited reduced body weight, increased blood glucose levels and decreased the number of islets. In T2DM rats treated with 10 or 20 mg/kg Icariin, the blood glucose levels were reduced, whereas serum adiponectin levels were not affected. Additionally, the mRNA and protein expression levels of p-AMPK and GLUT-4 protein were increased in the T2DM rats treated with Icariin. In conclusion, in the diabetes rat model, Icariin alleviated the severity of diabetes, and the effects may be associated with reduction of hyperglycemia by activating an AMPK/GLUT-4 pathway.

Circular RNA screening from EIF3a in lung cancer.

Eukaryotic initiation factor 3 (EIF3) is one of the largest and most complex translation initiation factors, which consists of 13 subunits named eukaryotic translation initiation factor 3 subunit A (EIF3a) to EIF3m. EIF3a is the largest subunit of EIF3. Previous studies suggested that EIF3a is a housekeeping gene, recent results have found that EIF3a is closely related to the tumorigenesis and drug resistance. Circular RNAs (circRNAs) derived from biologically important gene can play an important role in gene regulation. However, the mechanism underlying circRNAs' biological functions is not well understood yet. In this work, we screened 31 EIF3a-derived circRNAs, in which two circEIF3as were identified to be correlated with cisplatin drug sensitivity in lung cancer. Two circEIF3as were found involved in RNA-binding proteins-mediated biological processes and may be related to translational regulation according to bioinformatics analyses. CircEIF3as, the transcriptional initiation factor EIF3a transcribed circRNAs, are associated with both drug sensitivity and translation regulation. These findings mean that they may have a functional synergy effect with EIF3a or be valuable therapeutic targets for treatment like EIF3a. This is the first study that exploits circRNAs screening from EIF3a in lung cancer, our findings provide a novel perspective on the function of EIF3a and circEIF3as in lung cancer.

Prognostic and predictive values of CDK1 and MAD2L1 in lung adenocarcinoma.

Lung cancer remains as the leading cause of cancer-related death worldwide, and lung adenocarcinoma (LUAD) is the most common histological subtype. This study aims to investigate biomarkers associated with cancer progression and prognosis of LUAD. We integrated expression profiles of 668 lung cancer patients in five datasets from the Gene Expression Omnibus (GEO) and identified a panel of differentially expressed genes (DEGs). Function enrichment analysis highlighted that these genes were closely associated with the carcinogenesis of LUAD, such as cell cycle, ECM-receptor interaction and p53 signaling pathway. Cyclin-dependent kinase 1 (CDK1) and MAD2 mitotic arrest deficient-like 1 (MAD2L1), two critical mitotic checkpoint genes, were selected for further study. Elevated expression of CDK1 and MAD2L1 was validated in an independent LUAD cohort. Kaplan-Meier analysis revealed that CDK1 and MAD2L1 expression was negatively correlated with both overall survival (OS) and relapse-free survival (RFS). In conclusion, CDK1 and MAD2L1 were adverse prognostic biomarkers for LUAD whose increased expression could render patients with LUAD a high risk of cancer recurrence and poor survival, suggesting that they might be applied as potential targets for LUAD treatment.

Targeting DNA Damage Response in the Radio(Chemo)therapy of Non-Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer death worldwide due to its high incidence and mortality. As the most common lung cancer, non-small cell lung cancer (NSCLC) is a terrible threat to human health. Despite improvements in diagnosis and combined treatments including surgical resection, radiotherapy and chemotherapy, the overall survival for NSCLC patients still remains poor. DNA damage is considered to be the primary cause of lung cancer development and is normally recognized and repaired by the intrinsic DNA damage response machinery. The role of DNA repair pathways in radio(chemo)therapy-resistant cancers has become an area of significant interest in the clinical setting. Meanwhile, some studies have proved that genetic and epigenetic factors can alter the DNA damage response and repair, which results in changes of the radiation and chemotherapy curative effect in NSCLC. In this review, we focus on the effect of genetic polymorphisms and epigenetic factors such as miRNA regulation and lncRNA regulation participating in DNA damage repair in response to radio(chemo)therapy in NSCLC. These may provide novel information on the radio(chemo)therapy of NSCLC based on the individual DNA damage response.

Role of NF-E2-related factor 2 in neuroprotective effect of l-carnitine against high glucose-induced oxidative stress in the retinal ganglion cells.

l-Carnitine (LC) has protective effects on high glucose-induced oxidative stress in the retinal ganglion cells (RGCs). The aim of this study was to investigate the role of NF-E2-related factor 2 (Nrf2), Kelch like-ECH-associated protein 1 (Keap1), haemoxygenase-1 (HO-1) and γ-glutamyl cysteine synthetase (γ-GCS) in the protective effect of LC on RGCs. RGCs were first processed with high concentrations of glucose. LC treatment at three concentrations (50µM, 100µM and 200µM) was applied to high glucose stimulated RGCs. The expression of Nrf2, Keap1, haemoxygenase-1 (HO-1) and γ-glutamyl cysteine synthetase (γ-GCS) was quantified by Western blot in the treatment and control (high glucose stimulation) groups. In the three LC groups (50µM, 100µM and 200µM), Nrf-2 (0.71±0.04, 0.89±0.05, 1.24±0.05 vs 0.56±0.03, p<0.05), HO-1 (0.58±0.04, 0.76±0.06, 0.89±0.07 vs 0.25±0.03, p<0.01), and γ-GCS protein expression (0.66±0.03, 0.79±0.05, 0.84±0.08 vs 0.84±0.08, p<0.01) was higher than in the control group. The levels of Keap1 protein were in the LC groups were lower than in the control group (0.50±0.03, 0.45±0.02, 0.53±0.03 vs 0.86±0.05, p<0.01). In conclusion, in high glucose stimulated RGCs, LC treatment was associated with an increased level of Nrf2, HO-1and γ-GCS. LC treatment was also associated with a reduced expression of Keap1 protein. These results suggest that the protective effect of LC treatment on RGCs may be related to Nrf2-Keap1 pathway.

Induction of H2AX phosphorylation in tumor cells by gossypol acetic acid is mediated by phosphatidylinositol 3-kinase (PI3K) family.

BACKGROUND: H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including Ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. Our study shows that gossypol acetic acid (GAA) alone can induce γH2AX in Human mucoepidermoid carcinoma cell line (MEC-1) in vitro. Thus, we further examined the possible mechanisms of GAA to induce γH2AX in tumor cells. MATERIALS AND METHODS: The PI3K inhibitors caffeine and wortmannin were used in an effort to identify the kinase(s) responsible for GAA -induced γH2AX in MEC-1 cells. DNA dependent protein kinase (DNA-PK) - proficient and -deficient cells, human glioma cell lines M059K and M059J, were also used to evaluate the kinases responsible for GAA induced H2AX phosphorylation. γH2AX expression was detected by immunofluorescent microscopy. Flow cytometry assay was used to assay γH2AX and cell cycle. RESULTS: GAA induced H2AX phosphorylation in a cell cycle-dependent manner and a significant G0/G1 phase arrest in MEC-1 cells was shown. Caffeine and wortmannin significantly inhibited GAA-induced H2AX phosphorylation in MEC-1 cells. GAA induced H2AX phosphorylation in M059K, but not in M059J. Taken together, these data suggested that GAA treatment alone could induce H2AX phosphorylation in a cell cycle dependent manner in MEC-1 and M059K, but not in M059J cells. A significant G0/G1 phase arrest was shown in MEC-1. CONCLUSIONS: The member of PI3K family, DNA-PK, ATM and ATR are involved in the H2AX phosphorylation of MEC-1 cells.

Urinary excretion of L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine and their antioxidant activities after single dose administration of L-carnitine in healthy subjects

The urine excretion of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-Lcarnitine (PLC) and their relations with the antioxidant activities are presently unknown. Liquid L-carnitine (2.0 g) was administered orally as a single dose in 12 healthy subjects. Urine concentrations of LC, ALC and PLC were detected by HPLC. Superoxide dismutase (SOD), total antioxidative capacity (T-AOC), malondialdehyde (MDA) and nitrogen monoxidum (NO) activities were measured by spectrophotometric methods. The 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excretion of LC was 53.13±31.36 µmol, 166.93±76.87 µmol, 219.92±76.30 µmol, 100.48±23.89 µmol, 72.07±25.77 µmol, respectively. The excretion of ALC was 29.70±14.43 µmol, 80.59±32.70 µmol, 109.85±49.21 µmol, 58.65±18.55 µmol, and 80.43±35.44 µmol, respectively. The urine concentration of PLC was 6.63±4.50 µmol, 15.33±12.59 µmol, 15.46±6.26 µmol, 13.41±11.66 µmol and 9.67±7.92 µmol, respectively. The accumulated excretion rate of LC was 6.1% within 24h after its administration. There was also an increase in urine concentrations of SOD and T-AOC, and a decrease in NO and MDA. A positive correlation was found between urine concentrations of LC and SOD (r = 0.8277) or T-AOC (r = 0.9547), and a negative correlation was found between urine LC excretions and NO (r = -0.8575) or MDA (r = 0.7085). In conclusion, a single oral LC administration let to a gradual increase in urine L-carnitine excretion which was associated with an increase in urine antioxidant enzymes and the total antioxidant capacities. These data may be useful in designing therapeutic regimens of LC or its analogues in the future.

A excreção urinária de L-carnitina (LC), acetil-L-carnitina (ALC) e propionil-L-carnitine (PLC) e as suas relações com as atividades antioxidantes são presentemente desconhecidos. Líquido de L-carnitina (2,0 g) foi administrada por via oral como uma dose única em 12 indivíduos saudáveis. As concentrações urinárias de LC, PLC e ALC foram detectados por HPLC. Atividades superóxido dismutase (SOD), a capacidade antioxidante total (T-AOC), malondialdeído (MDA) e óxido nítrico (NO) foram medidas por métodos espectrofotométricos. O 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excreção de LC foi 53,13±31.36 µmol, 166,93±76.87 µmol, 219,92±76.30 µmol, 100,48±23.89 µmol, 72,07±25.77 µmol, respectivamente. A excreηão de ALC foi 29,70±14.43 µmol, 80,59±32.70 µmol, 109,85±49.21 µmol, 58,65±18.55 µmol, e 80,43±35.44 µmol, respectivamente. A concentraηão de urina de PLC foi 6,63±4.50 µmol, 15,33±12.59 µmol, 15,46±6.26 µmol, 13,41±11.66 µmol e 9,67±7.92 µmol, respectivamente. A taxa de excreηão acumulada de LC foi de 6,1% 24 horas após sua administração. Houve também um aumento nas concentrações de urina de SOD e T-COA e diminuição de NO e de MDA. Correlação positiva foi encontrada entre as concentrações de urina de LC e SOD (r = 0,8277) ou T-AOC (r = 0,9547) e correlação negativa entre a excreção de LC e NO (r = -0,8575) ou MDA (r = 0,7085). Em conclusão, a administração oral única de LC leva ao aumento gradual na excreção urinária de L-carnitina, que foi associada com o aumento das enzimas antioxidantes na urina e as capacidades antioxidantes totais. Estes dados podem ser úteis no futuro para o planejamento de esquemas terapêuticos de LC ou os seus análogos, no futuro.

Pravastatin inhibits C-reactive protein generation induced by fibrinogen, fibrin and FDP in isolated rat vascular smooth muscle cells.

OBJECTIVE: The available evidence indicates that C-reactive protein (CRP) participates directly in atherosclerosis formation as an inflammatory molecule. Our previous investigation suggested that fibrinogen, fibrin and fibrinogen degradation products (FDP) produce a pro-inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing CRP generation. In the present study, we observed the effect of pravastatin on CRP generation induced by fibrinogen, fibrin and FDP in rat VSMCs. METHODS: VSMCs from Sprague-Dawley rats were cultured. Fibrinogen, fibrin and FDP were used as stimulants for CRP generation. VSMCs were preincubated with pravastatin at 10, 30, 100 µM for 30 min prior to stimulation. CRP mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). CRP levels in the supernatant of VSMCs were measured by enzyme-linked immunosorbent assay (ELISA). CRP expression in VSMCs was examined with immunocytochemical staining. RESULTS: ELISA analysis showed that the pravastatin concentration-dependently reduced fibrinogen-, fibrin- and FDP-stimulated generation of CRP in VSMCs, with maximal inhibition of 56.6, 55.7 and 62.3%, respectively. Immunocytochemical staining and RT-PCR revealed that pravastatin inhibited protein and mRNA expression of CRP in VSMCs significantly. CONCLUSIONS: Pravastatin at the concentrations used in the present experiment has ability to relieve vascular inflammation and to restrain atherosclerotic processes via inhibiting the CRP production induced by fibrinogen, fibrin and FDP in VSMCs, which helps explain the beneficial effects of pravastatin on atherosclerosis.

[Gossypol acetic acid induces DNA double-strand breaks in human mucoepidermoid carcinoma cell MEC-1].

The present study was conducted to investigate the effects of gossypol acetic acid (GAA) on the proliferation of human mucoepidermoid carcinoma cell line MEC-1 in vitro and its possible molecular mechanisms of DNA double-strand breaks (DSB). MTT assay was performed to test the inhibition of proliferation of MEC-1 cells by GAA. DSB and γH2AX foci formation induced by GAA were detected by neutral comet assay and immunostaining. GAA (5-40 µmol/L) inhibited the growth of MEC-1 cells in a dose- and time-dependent manner. One of the indexes of comet assay, percentage of head DNA was decreased, however other indexes, including tail length, percentage of tail DNA, tail moment (TM) and Olive tail moment (OTM) were increased when treated with 2.5- 40 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with those in control. The percentage of γH2AX-positive cells was also increased when MEC-1 was treated with 2.5-20 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with that in control. All these results show that GAA inhibits the proliferation of MEC-1, and DSB maybe one of the mechanisms of inhibitory effect of GAA on the growth of tumor cells.

Calcitonin gene-related peptide inhibits angiotensin II-induced endothelial progenitor cells senescence through up-regulation of klotho expression.

BACKGROUND: It has been shown that angiotensin II (Ang II) is able to accelerate endothelial progenitor cells (EPCs) senescence through induction of oxidative stress. Calcitonin gene-related peptide (CGRP), a major neurotransmitter of the capsaicin-sensitive sensory nerves, protects endothelial function. Whether CGRP protects against EPCs senescence is unknown. METHODS AND RESULTS: In cord-derived EPCs, the effects of CGRP on Ang II-induced cell senescence were evaluated by exogenous application of CGRP and rutaecarpine (to stimulate the endogenous CGRP production) or by over-expression of CGRP. The anti-senescence mechanisms of CGRP on EPCs were investigated either by applying CGRP antagonist or by silence of klotho, an anti-aging protein. The results showed that both CGRP and klotho mRNA expression were reduced in Ang II-induced senescent EPCs. Exogenous application of CGRP inhibited Ang II-induced EPCs senescence by down-regulating the expression of NADPH oxidase and reactive oxygen species production. Similarly, rutaecarpine or CGRP I over-expression also inhibited Ang II-induced EPCs senescence. The effects of CGRP and rutaecarpine were reversed by CGRP(8-37), a select antagonist of CGRP receptor and capsazepine, a selective antagonist of transient receptor potential vanilloid 1, respectively. Furthermore, gene silence of klotho markedly attenuated the anti-senescence effect of CGRP on EPCs. CONCLUSIONS: The results suggest that CGRP can counteract Ang II-induced EPCs senescence through down-regulating the expression of NADPH oxidase and reactive oxygen species production and increasing the production of klotho.

Accelerated senescence of endothelial progenitor cells in hypertension is related to the reduction of calcitonin gene-related peptide.

OBJECTIVES: To explore whether the accelerated senescence of endothelial progenitor cells (EPCs) is related to the reduction of calcitonin gene-related peptide (CGRP) in hypertension. METHODS AND RESULTS: In-vivo studies, plasma levels of CGRP and the number of senescent EPCs were measured in hypertensive humans and animals, from which the EPCs were isolated to examine the production of CGRP. Moreover, rutaecarpine, as an agent or tool to stimulate CGRP production, was used in hypertensive animals. The effects of rutaecarpine on angiotensin II-induced EPCs senescence were evaluated in vitro. The results showed that the number of circulating senescent EPCs was significantly increased in hypertension concomitantly with the decreased plasma level of CGRP and the decreased CGRP mRNA expression in EPCs. Administration of rutaecarpine reversed EPC senescence along with an elevation in CGRP production in spontaneously hypertensive rats. In the angiotensin II-induced EPCs senescence, the CGRP mRNA expression was reduced, which was reversed by rutaecarpine. The effect of rutaecarpine on EPCs was canceled in the presence of capsazepine, a selective antagonist of transient receptor potential vanilloid 1. CONCLUSION: The results suggest that CGRP may work as an endogenous protective substance to counteract EPCs senescence in hypertension and the accelerated EPCs senescence in hypertension was related to the reduction of CGRP.