Effects of rifampicin on the pharmacokinetics of alflutinib, a selective third-generation EGFR kinase inhibitor, and its metabolite AST5902 in healthy volunteers.

Background Alflutinib is a novel irreversible and highly selective third-generation EGFR inhibitor currently being developed for the treatment of non-small cell lung cancer patients with activating EGFR mutations and EGFR T790M drug-resistant mutation. Alflutinib is mainly metabolized via CYP3A4 to form its active metabolite AST5902. Both alflutinib and AST5902 contribute to the in vivo pharmacological activity. The aim of this study was to investigate the effects of rifampicin (a strong CYP3A4 inducer) on the pharmacokinetics of alflutinib and AST5902 in healthy volunteers, thus providing important information for drug-drug interaction evaluation and guiding clinical usage. Methods This study was designed as a single-center, open-label, and single-sequence trial over two periods. The volunteers received a single dose of 80 mg alflutinib on Day 1/22 and continuous doses of 0.6 g rifampicin on Day 15-30. Blood sampling was conducted on Day 1-10 and Day 22-31. The pharmacokinetics of alflutinib, AST5902, and the total active ingredients (alflutinib and AST5902) with or without rifampicin co-administration were respectively analyzed. Results Co-administration with rifampicin led to 86% and 60% decreases in alflutinib AUC0-∞ and Cmax, respectively, as well as 17% decrease in AST5902 AUC0-∞ and 1.09-fold increase in AST5902 Cmax. The total active ingredients (alflutinib and AST5902) exhibited 62% and 39% decreases in AUC0-∞ and Cmax, respectively. Conclusions As a strong CYP3A4 inducer, rifampicin exerted significant effects on the pharmacokinetics of alflutinib and the total active ingredients (alflutinib and AST5902). The results suggested that concomitant strong CYP3A4 inducers should be avoided during alflutinib treatment. This trial was registered at http://www.chinadrugtrials.org.cn . The registration No. is CTR20191562, and the date of registration is 2019-09-12.

N,N'-1,10-Bis(Naringin) Triethylenetetraamine, Synthesis and as a Cu(II) Chelator for Alzheimer's Disease Therapy.

The bis-Schiff base of N,N'-1,10-bis(naringin) triethylenetetraamine (1) was prepared, as a copper(II) ion chelator, compound 1 was used for Alzheimer's disease therapy in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of compound 1 showed that this Schiff base could promote PC12 cells proliferation, and also, compound 1 could inhibit Cu2+-amyloid-ß (Aß)1-42 mediated cytotoxicity on PC12 cells. The thioflavine T (ThT) assay showed that 1 can effectively attenuate Cu2+-induced Aß1-42 aggregation. In addition, compound 1 is determined to be potent antioxidants on the basis of in vitro antioxidant assay, it can effectively decease the level of reactive oxygen species (ROS) in Cu2+-Aß1-42-treated PC12 cells and elevate the superoxide dismutase (SOD) activity in Cu2+-Aß1-42-treated PC12 cells. The results show that N,N'-1,10-bis(naringin) triethylenetetraamine is a potential agent for therapy of Alzheimer's disease.

Genetic dereplication driven discovery of a tricinoloniol acid biosynthetic pathway in Trichoderma hypoxylon.

A genetic dereplication approach in combination with differential gene expression led to the discovery of three new sesquiterpenes, tricinoloniol acids (TRAs) A-C (1-3) and the known fusidilactone A (4) from T. hypoxylon. Comparative transcriptomic analysis and targeted deletion identified the biosynthetic route for TRAs. Our results demonstrate an alternative application of the genetic dereplication method for exploring the biosynthesis of cryptic secondary metabolites (SMs), which utilizes the coordinated expression of trichothecene (tri) and tra cluster genes.

Development of a cell-based high-throughput peroxisome proliferator-activated receptors (PPARs) screening model and its application for evaluation of the extracts from Rhizoma Coptis.

To date, peroxisome proliferator-activated receptors (PPARs) are becoming the new therapeutic targets for the treatment of metabolic diseases, such as Type 2 diabetes, obesity, and cardiovascular disease. In this study, a cell-based high-throughput PPARs (PPARα/ß/γ) model was developed for the screening of PPARs agonists. The screening conditions were evaluated through analyzing the expression value of luciferase. Finally, 24 h of drug acting time, 5 times of the dilution factor of luciferase zymolyte, and about 2 × 10(4) cells/ well on HeLa cells in 96-well plates were used, respectively. Furthermore, the quality of high-throughput screening (HTS) in stability and reliability was evaluated by the Z'-factor. Additionally, different extracts of Rhizoma Coptis and berberine were tested by the developed method. The results suggested that both the EtOAc extract and berberine were able to activate PPARα/ß/γ, and Rhizoma Coptis contains potential natural agonists of PPARs besides berberine. In conclusion, the developed HTS assay is a simple, rapid, stable, and specific method for the screening of PPARs natural agonists.

Glucagon-like peptide 1 receptor plays a critical role in geniposide-regulated insulin secretion in INS-1 cells.

AIM: To explore the role of the glucagon-like peptide 1 receptor (GLP-1R) in geniposide regulated insulin secretion in rat INS-1 insulinoma cells. METHODS: Rat INS-1 insulinoma cells were cultured. The content of insulin in the culture medium was measured with ELISA assay. GLP-1R gene in INS-1 cells was knocked down with shRNA interference. The level of GLP-1R protein in INS-1 cells was measured with Western blotting. RESULTS: Geniposide (0.01-100 µmol/L) increased insulin secretion from INS-1 cells in a concentration-dependent manner. Geniposide (10 µmol/L) enhanced acute insulin secretion in response to both the low (5.5 mmol/L) and moderately high levels (11 mmol/L) of glucose. Blockade of GLP-1R with the GLP-1R antagonist exendin (9-39) (200 nmol/L) or knock-down of GLP-1R with shRNA interference in INS-1 cells decreased the effect of geniposide (10 µmol/L) on insulin secretion stimulated by glucose (5.5 mmol/L). CONCLUSION: Geniposide increases insulin secretion through glucagon-like peptide 1 receptors in rat INS-1 insulinoma cells.

[Pharmacokinetics and bioequivalence of atorvastatin calcium tablets in healthy male Chinese volunteers].

OBJECTIVE: To compare the bioequivalence and pharmacokinetics of national made and imported atorvastatin in healthy male Chinese volunteers after single oral administration. METHODS: This randomized sequence, open-label, two-period crossover study with a one-week washout period between doses was performed in 24 fasting healthy Chinese males. They were randomly assigned to receive 20 mg of either the test (national made) or reference (imported) formulation orally. The blood samples were collected over a 72-hour period. Plasma concentrations of parent atorvastatin (AT), ortho-hydroxy-atorvastatin (o-OAT) and para-hydroxy-atorvastatin (p-OAT) were simultaneously determined using the validated liquid chromatography-tandem mass spectrometry method, the bioequivalence was also evaluated throughout the study. RESULTS: The main pharmacokinetic parameters of test and reference formulations were as follows: the values of C(max) for AT were (10.6 ± 11.9) µg/L and (10.6 ± 9.8) µg/L, t(1/2z) were (11.4 ± 3.9) h and (11.4 ± 5.3) h, AUC(0-t) were (54.2 ± 37.4) µg×h(-1)×L(-1) and (51.7 ± 34.1) µg×h(-1)×L(-1), respectively. The values of C(max) for o-OAT were (7.8 ± 4.5) µg/L and (7.6 ± 4.3) µg/L, t(1/2z) were (12.3 ± 4.2) h and (11.9 ± 3.4) h, AUC(0-t) were (96.8 ± 48.2) µg×h(-1)×L(-1) and (92.3 ± 44.4) µg×h(-1)×L(-1), respectively. The values of C(max) for p-OAT were (0.5 ± 0.4) µg/L and (0.4 ± 0.3) µg/L, t(1/2z) were (18.4 ± 12.4) h and (23.3 ± 17.8) h, AUC(0-t) were (15.9 ± 12.3) µg×h(-1)×L(-1) and (13.8 ± 8.11) µg×h(-1)×L(-1), respectively. The relative bioavailability of AT and o-OAT in test formulation were (105.3 ± 20.7)% and (107.8 ± 23.2)%, respectively. The 90% confidence interval of the test/reference geometric mean ratios of AUC(0-t) for AT and o-OAT were (97.7 - 110.5)% and (98.3 - 111.3)%, C(max) for AT and o-OAT were (75.8 - 114.0)% and (90.6 - 122.9)%, they were all located within the bioequivalence criteria range (80% - 125% for AUC, and 70% - 143% for C(max)). CONCLUSION: The result demonstrated that two formulations were bioequivalent.

[Activating effect of citrus flavonoids on neuromedin U2 receptor and analysis on siRNA interference].

OBJECTIVE: To screen out active substances on Neuromedin U2 receptor (NMU2R) by using stable NMU2R cell lines and negative cell lines and analyzing siRNA interference. METHOD: NMU2R cells were used to observe the activating effect of nine nine citrus flavonoids on NMU2R cell. Afterwards, false-positive interference of citrus flavonoids that showed higher activating effect was eliminated by using negative cells and analyzing the efficiency of siRNA interference. RESULT: Hesperidin and nobiletin contained in citrus flavonoids were found to effectively activate NMU2R. The efficacy, EC50 and potency values of hesperidin were 4.688, 318.970 micromol x L(-1) and 200.933 micromol x L(-1), while the efficacy, EC50 and potency values of nobiletin were 4.758, 5.832 micromol x L(-1) and 3.124 micromol x L(-). CONCLUSION: Hesperidin and nobiletin contained in citrus flavonoids can activate NMU2R. Nobiletin shows such a low EC50 that it has medicinal value.

Quantification of Cross-Contamination of Campylobacter jejuni during Food Preparation in a Model Kitchen in China.

ABSTRACT: Numerous outbreak investigations and case-control studies of campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a high risk factor for infection and illness. In this study, the cross-contamination and transfer rates of Campylobacter jejuni from chicken to ready-to-eat food were determined in various food handling scenarios. Skinless raw chicken breasts were artificially contaminated with C. jejuni and diced on cutting boards of three different materials. Whether cold water, cold water with detergent, or hot water was used, statistically significant differences were found between the transfer rates of C. jejuni to unwashed and washed cutting boards or hands, respectively. When both kitchen knife and cutting board were reused after dicing the artificially contaminated chicken, the transfer rates of C. jejuni to cucumber cut on bamboo, wooden, and plastic cutting boards were 16.28, 12.82, and 5.32%, respectively. The transfer rates from chicken to bread, a large lift-up water faucet handle, and a small twist faucet handle via unwashed hands were 0.49, 4.64, and 3.14%, respectively. This research provides scientific evidence that various types of contaminated kitchenware and cook's hands are vital potential vehicles for the cross-contamination of Campylobacter from raw chicken to ready-to-eat food and emphasizes the importance of timely and proper cleaning to prevent cross-contamination during food handling; therefore, high-quality consumer education to reduce the risk of foodborne infection is urgent and necessary.

GLP-1 receptor plays a critical role in geniposide-induced expression of heme oxygenase-1 in PC12 cells.

AIM: To explore the role of activation of glucagon-like peptide 1 receptor (GLP-1R) and its relative cell signaling pathway in the cytoprotection of geniposide. METHODS: Cell viability was determined by MTT assay. Knockdown of the Glp-1r gene was carried out with shRNA. The levels of HO-1 protein and cAMP response element binding protein (CREB) phosphorylation were measured by Western blotting. RESULTS: Geniposide protected PC12 cells from oxidative damage induced by 3-morpholinosydnonimine hydrochloride (SIN-1) by enhancing the expression of heme oxygenase 1 (HO-1) via the cAMP-PKA-CREB signal pathway. After transfecting PC12 cells with the AB1 enhancer from the HO-1 gene, luciferase activity induced by geniposide increased in a dose-dependent manner, but not in the PC12 cells whose Glp-1r gene was disrupted. Additionally, inhibition of HO-1 activity by Sn-protoporphyrin IX (SnPP) or shRNA-mediated knockdown of Glp-1r decreased the neuroprotection of geniposide in PC12 cells. CONCLUSION: GLP-1R plays a critical role in geniposide-induced HO-1 expression to attenuate oxidative insults in PC12 cells.

Effects of Apatinib on the Pharmacokinetics of Nifedipine and Warfarin in Patients with Advanced Solid Tumors.

BACKGROUND AND PURPOSE: Apatinib is a small-molecule tyrosine kinase inhibitor for the treatment of recurrent or progressive advanced-stage gastric adenocarcinoma or gastroesophageal junction cancer. The in vitro inhibition studies suggested that apatinib exerted potent inhibition on CYP3A4 and CYP2C9. To evaluate the potential of apatinib as a perpetrator in CYP450-based drug-drug interactions in vivo, nifedipine and warfarin were, respectively, selected in the present study as the probe substrates of CYP3A4 and CYP2C9 for clinical drug-drug interaction studies. Since hypertension and thrombus are common adverse effects of vascular targeting anticancer agents, nifedipine and warfarin are usually coadministered with apatinib in clinical practice. METHODS: A single-center, open-label, single-arm, and self-controlled trial was conducted in patients with advanced solid tumors. The patients received a single dose of 30 mg nifedipine on Day 1/14 and a single dose of 3 mg warfarin on Day 3/16. On Day 9-21, the subjects received a daily dose of 750 mg apatinib, respectively. The pharmacokinetics of nifedipine and warfarin in the absence or presence of apatinib was, respectively, investigated. RESULTS: Compared with the single oral administration, coadministration with apatinib contributed to the significant increases of AUC0-48h and Cmax of nifedipine by 83% (90% confidence interval [CI] 1.46-2.31) and 64% (90% CI 1.34-2.01), respectively. Similarly, coadministration with apatinib contributed to the significant increases of AUC0-t and Cmax of S-warfarin by 92% (90% CI 1.68-2.18) and 24% (90% CI 1.10-1.39), respectively. CONCLUSION: Concomitant apatinib administration resulted in significant increases in systemic exposure to nifedipine and S-warfarin. Owing to the risk of pharmacokinetic drug-drug interactions based on CYP3A4/CYP2C9 inhibition by apatinib, caution is advised in the concurrent use of apatinib with either CYP2C9 or CYP3A4 substrates.

Neuroprotection of geniposide against hydrogen peroxide induced PC12 cells injury: involvement of PI3 kinase signal pathway.

AIM: Oxidative stress plays a critical role in the pathogenic cascade leading to neuronal degeneration in AD. Consequently, the induction of endogenous antioxidative proteins by antioxidants seems to be a very reasonable strategy for delaying the disease's progression. In previous work, we identified the neurotrophic and neuroprotective effects of geniposide, which result from the activation of glucagon-like peptide 1 receptor (GLP-1R). In this study, we explore the role of PI3 kinase signaling pathway in the neuroprotection of geniposide in PC12 cells. METHODS: Cell viability was determined by MTT assay. Apoptosis was detected by Hoechst and PI double staining. The protein expression of Bcl-2 and phosphorylation of Akt308, Akt473, GSK-3beta, and PDK1 was measured by Western blot. RESULTS: Geniposide induced the expression of the antiapoptotic protein Bcl-2, which inhibited apoptosis in PC12 cells induced by H(2)O(2), and this effect could be inhibited by preincubation with LY294002, a selective inhibitor of PI3K. Furthermore, geniposide enhanced the phosphorylation of Akt308, Akt473, GSK-3beta and PDK1 under conditions of oxidative stress. CONCLUSION: These results demonstrate that the PI3K signaling pathway is involved in the neuroprotection of geniposide in PC12 cells against the oxidative damage induced by H(2)O(2) in PC12 cells.

p-Synephrine exhibits anti-adipogenic activity by activating the Akt/GSK3ß signaling pathway in 3T3-L1 adipocytes.

This work aimed to investigate the effects of p-synephrine on the differentiation of adipocyte and explore the underlying mechanism. We found that p-synephrine suppressed the 3T3-L1 cell adipogenesis by reducing the expression level of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), which subsequently led to a reduction in the fatty acid-binding protein 4 (aP2) expression. p-Synephrine treatment markedly activated the protein kinase B (PKB/Akt) pathway and sequentially inhibited glycogen synthase kinase 3ß (GSK3ß) activity. Inhibition of GSK3ß activity by LiCl was found to partially ameliorate the above-mentioned effects. All these data suggested that p-synephrine exhibited the anti-adipogenic effects via the regulation of Akt signaling pathway and the suppression of adipogenesis-related proteins. PRACTICAL APPLICATIONS: Citrus aurantium often uses as herbal or dietary supplement in various countries around the world, including in Seville, Spain and South Africa. In traditional Chinese herbs, it is referred to as "Fructus aurantii immaturus," "Zhi shi," or "Zhi ke," and has been used for hundreds of years for various digestive problems. Its primary protoalkaloid, p-synephrine, exhibited lipolytic effects and energy expenditure, which has rapidly replaced ephedrine as an "ephedra-free" alternative dietary supplement. The current study firstly demonstrated the anti-adipogenic effects of p-synephrine in 3T3-L1 preadipocytes, which was due to the regulation of Akt signaling pathway and the subsequent suppression of adipogenesis-related proteins. The present study may offer invaluable opinions into the mechanisms of body weight/fat-losing activities of p-synephrine in theory, and scientific experimental evidence on dietary supplement in practice. p-Synephrine could be utilized for the preventive and therapeutic uses against metabolic syndrome.

[Effect of total glucosides of paeony on nuclear factor-kappaB activation in rat peritoneal macrophages].

OBJECTIVE: To study the effect of total glucosides of paeony (TGP) on lipopolysaccharides (LPS)-induced nuclear factor-kappaB (NF-kappaB) activation in macrophages. METHOD: Rat peritoneal macrophages were pre-treated with TGP for 2 h and stimulated with LPS for 20 min or 0.5 h. Inhibitory kappaBalpha (IkappaBalpha) protein in the cytoplasm and NF-kappaB p65 protein in the nuclear were analyzed by western blot. Further, DNA binding activity of NF-kappaB complex was detected. RESULT: TGP enhanced the amounts of IkappaBalpha protein in the cytoplasm and decreased the amounts of NF-kappaB p65 protein in the nuclear of LPS-induced macrophages. TGP also inhibited the LPS-mediated DNA binding activity of NF-kappaB complex in macrophages. CONCLUSION: TGP can inhibit LPS-induced NF-kappaB activation in macrophages through arresting IKBalpha protein degradation, NF-kappaB p65 protein nuclear translocation and DNA binding activity of NF-kappaB complex.

Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines.

RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments. Reference gene expression stability was analysed using three programs (geNorm, NormFinder and BestKeeper), and the recommended comprehensive ranking is provided. Our results reveal that ACTB and GAPDH, and miR-16 and miR-1228-3p are the most suitable mRNA and miRNA reference genes across all cell lines, respectively. Considering prostate cancer cell types, ACTB/GAPDH and ACTB/HPRT1 are the most suitable reference gene combinations for mRNA analysis, and miR-16/miR-1228-3p and RNU6-2/RNU43 for miRNA analysis in AR+, and AR- and normal cell lines, respectively. Comparison of relative target gene (PCA3 and miR-141) expression reveals different patterns depending on reference genes used for normalisation. To our knowledge, this is the first report on validation of reference genes under different DHT treatments in prostate cancer cells. This study provides insights for discovery of reliable DHT-regulated genes in prostate cells.

A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction.

Most RNA positive controls currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentiable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of Nipah virus (NiV). A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5' terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.

A Population-Based Assessment of 24-Hour Ocular Perfusion Pressure Among Patients With Primary Open Angle Glaucoma: The Handan Eye Study.

PURPOSE: To compare the 24-hour ocular perfusion pressure (OPP) among patients with primary open angle glaucoma (POAG) and those with suspected POAG identified in a population-based study in China. METHODS: Forty-seven patients with POAG and 35 with suspected POAG attended the 24-hour OPP study. Intraocular pressure (IOP) and blood pressure (BP) were measured at 2, 6, and 10 AM and 2, 6, and 10 PM. Subjects were not taking any medications to lower IOP, which was measured with Goldmann applanation in an upright sitting position. Blood pressure was measured in a supine position using a digital automatic BP monitor (OMRON, model HEM-907). Mean arterial pressure was calculated as diastolic BP + 1[Fraction Slash]3 × (systolic BP - diastolic BP). Mean OPP (MOPP) was defined as 2[Fraction Slash]3 × mean arterial pressure - IOP, systolic OPP (SOPP) was defined as 2[Fraction Slash]3 × systolic BP - IOP, and diastolic OPP (DOPP) was defined as 2[Fraction Slash]3 × diastolic BP - IOP. RESULTS: After adjustment for age, sex, and IOP, the maximum, mean, and minimum SOPP, DOPP, and MOPP were statistically significantly lower in subjects with POAG than in those with suspected POAG (P < 0.05). The minimum MOPP, SOPP, and DOPP occurred from 10 AM to 2 PM in approximately 60% of eyes with POAG and between 20% and 30% of minimum MOPP, SOPP, and DOPP occurred around 10 PM. CONCLUSIONS: Systolic OPP, DOPP, and MOPP were consistently lower in eyes with POAG than in those with suspected POAG, providing further evidence that OPP plays a role in the development of glaucoma.

Differential phosphorylation of p38 induced by apoptotic and anti-apoptotic stimuli in murine hepatocytes.

AIM: To investigate the differential phosphorylation and activation of p38 in hepatocytes by pro-apoptotic Transforming Growth Factor-beta1 (TGF-beta1), pro-survival factors Epidermal Growth Factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) and the potential mechanisms. METHODS: The phosphorylation and activation of p38 were determined by immunoblotting. Apoptosis was analyzed by morphological staining and observation, FACS analysis of sub-G1 content and DNA fragmentation assay. To quantitatively determine caspase activation, caspase activity assay was performed in vitro. RESULTS: TGF-beta1-induced apoptosis was associated with the phosphorylation of p38, and SB202190, a specific inhibitor of p38, which was able to inhibit TGF-beta1-induced caspase activation and apoptosis. TPA and EGF also blocked apoptosis induced by TGF-beta1. Both of them induced the phosphorylation of p38. The results showed SB202190 had no effect on TGF-beta1-induced phosphorylation of p38, but effectively inhibited both EGF and TPA-induced phosphorylation of p38. CONCLUSION: Pro-apoptotic TGF-beta1, anti-apoptotic TPA and EGF induce the phosphorylation of p38 through different mechanisms that can be distinguished by SB202190. The data suggest that TPA and EGF-induced p38 phosphorylation is through an autophosphorylation-dependent mechanism. Since p38 phosphorylation induced by TGF-beta1 plays an important role in caspase activation and apoptosis, TPA and EGF-induced p38 phosphorylation may not be requisite for their anti-apoptotic function.

Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype.

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901) and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism, and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells. METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay. RESULTS: Telomerase activity was detected in well, moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively). Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 mumol/L, and was entirely inhibited at 10 mumol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901 gastric cancer cell lines (the inhibition ratio was 40.89% and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0.05). Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells. CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.

Telomerase inhibition and telomere loss in BEL-7404 human hepatoma cells treated with doxorubicin.

AIM: To study the effects of doxorubicin on telomerase activity and telomere length in hepatocellular carcinoma. METHODS: Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplification protocol-based method. The effect of doxorubicin (DOX) on the growth of BEL-7404 human hepatoma cells was determined by microculture tetrazolium assay. Mean telomere length (terminal restriction fragment) was detected by Southern blot method. The expression of telomerase subunits genes was investigated by RT-PCR. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. RESULTS: Telomerase activity was inhibited in a dose and time-dependent manner in BEL-7404 human hepatoma cells treated with DOX for 24, 48 or 72 h in concentrations from 0.156 to 2.5 microM which was correlated with the inhibition of cell growth. No changes were found in the mRNA expression of three telomerase subunits (hTERT, hTR and TP1) after drug exposure for 72 h with indicated concentrations. The cells treated with DOX showed shortened mean telomere length and accumulated at the G(2)/M phase. However, there was almost no effects on cell apoptosis by DOX. CONCLUSION: The telomerase inhibition and the telomere shortening by DOX may contribute to its efficiency in the treatment in hepatocellular carcinoma.