Cyclooxygenase 2-dependent regulation of antitumor immunity in lung cancer.
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Animals↗
Biomedical subjects
Min Huang
Publications and source records attributed to Min Huang.
Tumor cyclooxygenase 2-dependent suppression of dendritic cell function.
Dendritic cells (DCs) serve as professional antigen-presenting cells and are pivotal in the host immune response to tumor antigens. To define the pathways limiting DC function in the tumor microenvironment, we assessed the impact of tumor cyclooxygenase (COX)-2 expression on DC activities. Bone marrow-derived DCs were cultured in either tumor supernatant (TSN) or TSN from COX-2-inhibited tumors. After culture, DCs were pulsed with tumor-specific peptides, and their ability to generate antitumor immune responses was assessed following injection into established murine lung cancer. In vitro, DC phenotype, alloreactivity, antigen processing and presentation, and interleukin (IL)-10 and IL-12 secretion were evaluated. DCs cultured in TSN failed to generate antitumor immune responses and caused immunosuppressive effects that correlated with enhanced tumor growth. However, genetic or pharmacological inhibition of tumor COX-2 expression restored DC function and effective antitumor immune responses. Functional analyses indicated that TSN causes a decrement in DC capacity to (a) process and present antigens, (b) induce alloreactivity, and (c) secrete IL-12. Whereas TSN DCs showed a significant reduction in cell surface expression of CD11c, DEC-205, MHC class I antigen, MHC class II antigen, CD80, and CD86 as well as a reduction in the transporter-associated proteins, transporter associated with antigen processing 1 and 2, the changes in phenotype and function were not evident when DCs were cultured in supernatant from COX-2-inhibited tumors. We conclude that inhibition of tumor COX-2 expression or activity can prevent tumor-induced suppression of DC activities.
Animals↗
[Genetic polymorphism of the thiopurine S-methyltransferase of healthy Han Chinese].
BACKGROUND & OBJECTIVE: Several mutations in the thiopurine S-methyltransferase(TPMT) gene have been identified to correlate with a low activity or deficiency phenotype. The distribution of TPMT activity in Chinese and Caucasian populations is different, and the molecular basis has not yet to be determined. This study was designed to investigate the type and frequency of four kinds of mutant TPMT alleles of healthy Han Chinese. METHODS: TPMT genotypes were determined in 225 healthy Han Chinese. Allele-specific PCR (ASPCR) and PCR-restriction fragment length polymorphism (RFLP) were used for analysis of G238C (TPMT*2), G460A and A719G (TPMT*3A,*3B,*3C). RESULTS: TPMT*2, TPMT*3B, and TPMT*3A were not detected in 225 healthy Han Chinese, and only six TPMT*3C heterozygotes were identified. The average TPMT activity of those TPMT*3C heterozygotes was (9.62+/-3.58) U/ml RBC. The frequency of TPMT mutant alleles was 1.33%(6/450) in healthy Han Chinese. CONCLUSION: The frequency of the known mutant TPMT alleles in Han Chinese population is low, and TPMT*3C appears to be the most prevalent among the known mutant TPMT allele in this population.
Alleles↗
[Mutant thiopurine S-methyltransferase alleles among Jing Chinese in Guangxi province].
OBJECTIVE: To investigate the mutant alleles of thiopurine S-methyltransferase (TPMT) among Jing Chinese. METHODS: Polymerse chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques were developed for assaying exons 5, 7 and 10 of the TPMT gene respectively and were used to detect mutant TPMT alleles among Jing Chinese. RESULTS: Two cases of TPMT*3C (A719G) heterozygotes were identified in 103 Jing Chinese; other deleterious alleles such as TPMT*2 (G238C), TPMT*3A (G460A/A719G) and TPMT*3B (G460A) were not found; 27 cases of silent mutant allele TPMT*1S (T474C) were also identified (5 homozygotes and 22 heterozygotes). CONCLUSION: The PCR-SSCP assay established and adopted in this study was sensitive and reliable, which could be used to detect mutant TPMT alleles. Allele frequency of TPMT*3C is low among Jing Chinese (1.0%), and TPMT*3C appears to be the most prevalent deleterious allele in this population.
Adolescent↗
Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA.
OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.
Animals↗
Mechanism of the antiulcerogenic effect of Ganoderma lucidum polysaccharides on indomethacin-induced lesions in the rat.
Many cytokines, in particular tumor necrosis factor (TNF)-alpha have been known to play an important role in the pathogenesis of gastric mucosal lesions caused by various factors such as drugs and Helicobacter pylori infection. Our previous studies have shown that the polysaccharide fractions isolated from the fruiting bodies of Ganoderma lucidum (GLPS) prevented indomethacin- and acetic acid-induced gastric mucosal lesions in the rat. However, the mechanisms remain unclear. This study aimed to investigate whether GLPS had a direct mucosal healing effect in the indomethacin-treated rat, and to explore the possible mechanisms by determining the gastric mucosal mRNA and protein levels of TNF-alpha and ornithine decarboxylase (ODC) activity. In addition, the effects of GLPS on the cellular proliferation, ODC and c-Myc protein expression and mucus synthesis in the rat gastric cell culture (RGM-1) were examined. The present study demonstrated that GLPS at 250 and 500 mg/kg by intragastric input caused ulcer-healing effect in the rat; this was accompanied with a significant suppression of TNF-alpha gene expression, but with an increased ODC activity. In RGM-1 cells, GLPS at 0.05, 0.25 and 1.0 mg/ml significantly enhanced [3H]thymidine incorporation and ODC activity in a concentration-dependent manner. However, these effects were abrogated by the addition of the ODC inhibitor, DL-alpha-difluoromethyl-ornithine (DFMO). GLPS at 0.25-1.0 mg/ml also increased mucus synthesis, as indicated by the increased D-[6-3H]glucosamine incorporation in RGM-1 cells. Furthermore, GLPS at 0.05-1.0 mg/ml increased the c-Myc protein expression. These findings indicated that GLPS produced a mucosal healing effect in the rat model, perhaps due partly to the suppression of TNF-alpha and induction of c-myc and ODC gene.
Animals↗
Inhibition of nucleoside transport by p38 MAPK inhibitors.
While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.
AraC Transcription Factor↗
Uptake of FITC-chitosan nanoparticles by A549 cells.
PURPOSE: The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. METHODS: Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. RESULTS: Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having Km and Vmax of 3.84 microM and 58.14 microg/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K+ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37 degrees C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37 degrees C. CONCLUSIONS: Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.
Chitin↗
Caspase-dependent cleavage of carbamoyl phosphate synthetase II during apoptosis.
Carbamoyl phosphate synthetase II (CPSII) is part of carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Herein, we identify CAD as a substrate for caspase-3 degradation in both in vitro and in vivo models of apoptosis. Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD. The rapid decline in the CPSII activity paralleled the degradation of CAD and preceded the appearance of Annexin-V-stained apoptotic cells and DNA fragmentation. These events correlated closely with the activation of caspase-3 in these cells and were prevented by the cell-permeable caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone. Moreover, the incubation of purified CAD with recombinant caspase-3 in vitro generated CAD fragments that were similar to those obtained in vivo. Edman sequencing revealed that two of the major caspase-3 cleavage sites occurred at the sequences EAVD/G and VACD/G within the catalytic (B2) and allosteric (B3) domains of CAD, thus providing a potential mechanism for the rapid inactivation of CPSII during apoptosis. Consistent with this, an enhanced loss of the intracellular pyrimidines (UTP and CTP) was observed in response to STS or DOX-induced apoptosis. Therefore, these studies show that CAD is a novel target for caspase-dependent regulation during apoptosis and suggest that the selective inactivation of pyrimidine nucleotide synthesis accompanies the process of apoptosis.
Animals↗
A77 1726 induces differentiation of human myeloid leukemia K562 cells by depletion of intracellular CTP pools.
A77 1726 (LEF) is the active metabolite of leflunomide, a recently approved immunosuppressive agent. We examined the ability of LEF to induce differentiation of a human erythroleukemia (K562) cell line and show that LEF induces a dose- and time-dependent differentiation of these cells as characterized by growth inhibition, hemoglobin production, and erythroid membrane protein glycophorin A expression. This effect was dependent on depletion of the intracellular pyrimidine ribonucleotides (UTP and CTP), and preceded by a specific S-phase arrest of the cell cycle. Supplementation of the cultures with exogenous uridine restored intracellular UTP and CTP to normal levels and prevented the LEF-induced cell cycle block and differentiation of K562 cells. Interestingly, addition of cytidine alone blocked the LEF-induced differentiation of K562 cells but only restored the CTP pool. By contrast, neither deoxycytidine nor thymidine prevented the effects of LEF on these cells. Similarly, pyrimidine starvation of a cell line lacking the de novo pyrimidine pathway (G9c) resulted in an S-phase arrest that was reversed by the addition of cytidine. Thus these studies demonstrate an important role for CTP in regulating cell cycle progression and show that LEF is an effective inducer of tumor cell differentiation through depletion of this ribonucleotide.
Aniline Compounds↗
IL-7 inhibits fibroblast TGF-beta production and signaling in pulmonary fibrosis.
Based on studies by our group and others, we hypothesized that IL-7 may possess antifibrotic activities in an IFN-gamma-dependent and independent manner. Here, we have evaluated the antifibrotic therapeutic potential of IL-7 in both in vitro and in vivo pulmonary fibrosis models. IL-7 inhibited both TGF-beta production and signaling in fibroblasts and required an intact JAK1/STAT1 signal transduction pathway. IL-7-mediated inhibition of TGF-beta signaling was found to be associated with an increase in Smad7, a major inhibitory regulator in the SMAD family. In the presence of IL-7, Smad7 dominant negative fibroblasts restored TGF-beta-induced collagen synthesis, indicating that an IL-7-mediated increase in Smad7 suppressed TGF-beta signaling. Consistent with these in vitro findings, recombinant IL-7 decreased bleomycin-induced pulmonary fibrosis in vivo, independent of IFN-gamma. The antifibrotic activities of IL-7 merit further basic and clinical investigation for the treatment of pulmonary fibrosis.
Animals↗
Multi-charged oligonucleotide ion formation in sonic spray ionization.
An oligonucleotide tends to release hydrogen atoms from a phosphoric acid group and to form negative ions that can be detected by mass spectrometry. Usually, with a solution-spray based ionization technique, the negative ions are present in different charge states. Ion formation for the nucleotide is quite complicated and is easily influenced by matrix and other constituents in a sample solution, as well as by the operating parameters for a mass spectrometer. In this work, we studied oligonucleotide ion formation by using an ion trap mass spectrometer combined with a sonic spray ionization (SSI) source. An oligonucleotide with 20 bases was measured. Effects from contaminants and parameters affecting the ion production, such as a high voltage applied to the ionization source and sample solution-flow rate, were investigated. Our results showed that an ion with about one charge for every three bases was most abundant. However, the signal intensity and the mass spectrum pattern were sensitive to the matrix and operating parameters. One of the reasons for such sensitivity is that there are various ion states for an oligonucleotide. Any change in the matrix or an operating parameter may shift the balances between the ion states. Adding Tris, or (hydroxymethyl)aminomethane, enhanced the signal intensity of the oligonucleotide and promoted formation of the oligonucleotide ion with higher charges, while adding acetic acid favored the ions with lower charges, compared with that obtained in the medium without adding Tris and acetic acid. The effects on charged droplets and chemical enhancement were investigated. The mechanism for oligonucleotide ion formation is discussed.
Acetic Acid↗
Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel(7402).
AIM: To investigate the resistance mechanism of 5-fluorouracil (5-FU) in Bel(7402)/5-FU cells which was established in our lab by in vitro continuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel(7402) to 5-FU. METHODS: The expression of multidrug resistance-associated protein (MRP) and thymidylate synthase (TS) in Bel(7402) cells was detected by immonocytochemistry. The fluorescein (FLU) accumulation, an index of MRP functional activity, was determined by flow cytometry. The distribution of FLU was observed by confocal laser scanning microscope. The spectrofluorometry was used to show the intracelluar content of glutathione (GSH). Cell growth inhibition was determined by MTT assay. The activity of glutathione S-transferases (GSTs) was determined by spectrophotometry. RESULTS: A higher expression of MRP in the Bel(7402)/5-FU cells was observed by using monoclonal mouse anti-MRP antibody, MRPr-1, in comparison with Bel(7402) cells. Bel(7402)/5-FU cells also showed a significant decrease of FLU accumulation. FLU mainly accumulated in the nucleus with a high nuclear/cytoplasmic ratio in Bel(7402) cells, whereas there was no difference of FLU accumulation between the nucleus and cytoplasm in Bel(7402)/5-FU cells. The intracellular GSH content in Bel(7402)/5-FU cells was almost 3 folds higher than that in Bel(7402) cells. Addition of D, L-buthione-S, R-sulfoximine (BSO) dose-dependently reduced the GSH content in Bel(7402)/5-FU cells, however, only a weak enhancement on the cytotoxicity of 5-FU and doxorubicin (Dox) to Bel(7402)/5-FU cells was observed. Bel(7402)/5-FU cells also exhibited 29.1 % higher total GSTs activity than Bel(7402) cells. Immunocytochemical staining by using anti-TS monoclonal antibody TS 106 showed that the level of TS in Bel(7402)/5-FU cells elevated markedly as compared with Bel(7402) cells. CONCLUSION: The continuous exposure of Bel(7402) cells to 5-FU led to overexpression of TS and MRP, as well as increased intracellular GSH content and total GST activity.
Buthionine Sulfoximine↗
[Effect of MNNG on ornithine decarboxylase activity in cells from adult Schistosoma japonicum].
OBJECTIVE: To observe the change of ornithine decarboxylase(ODC) activity in cells from adult Schistosoma japonicum after the cells were treated with N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG). METHODS: The cells were treated with MNNG at a concentration of 3 micrograms/ml for 48 hours after the cells being incubated for one week. The cells were then cultured with RPMI-1640 containing 10% calf serum. ODC activity was detected with spectrophotography. RESULTS: ODC activity rose significantly in two to three weeks after the cells were treated with MNNG. CONCLUSION: There was ODC activity in cells from adult S. japonicum and MNNG has an effect to reinforce ODC activity in the cells.
Animals↗
[Protective effect of tripchlorolide on dopaminergic neurons in partially lesioned rat model of Parkinson's disease].
AIM: To study whether the immunosuppressant tripchlorolide (T4) exerts neuroprotective effect on dopaminergic neurons. METHODS: A rat model of Parkinson's disease (PD) was set up by transection of the medial forebrain bundle (MFB) with a wire knife. The rotational behavior, HPLC-ECD, tyrosine hydroxylase (TH) immunocytochemistry, ELISA methods were used to evaluate the influence on the dopaminergic neurons following T4 treatment. RESULTS: T4 treatment was shown to effectively attenuate the rotational behavior challenged by amphetamine (2.5 mg.kg-1, i.p.) in the PD rats. T4 markedly prevented the decrease of dopamine content in the striatum and the loss of dopaminergic neurons in the substantia nigra pars compacta. T4 was found to suppress the abnormal increase of TNF-alpha and IL-2 level in brain tissues of PD rats after MFB transection. CONCLUSION: The evidence that the immunosuppressive Chinese herb extract T4 possesses neuroprotective activities on the dopaminergic neurons in PD rats was presented. The underlying mechanism of T4 may be relevant to its immunosuppressive activity.
Animals↗
A comparison of glutathione S-transferase mutant frequencies in healthy Han and Uygur Chinese.
OBJECTIVE: This study was to compare the frequencies of genetic polymorphisms of GSTM1, GSTT1, and GSTP1 in Uygur Chinese with those in Han Chinese. METHODS: GSTM1 and GSTT1 polymorphisms were analyzed by a PCR-Multiplex procedure, whereas GSTP1 polymorphism was analyzed by PCR-RFLP. RESULTS: The frequency of GSTM1 null genotype in Han Chinese (56.1%) was similar to that in Uygur Chinese (53.2%) (P = 0.592), whilst the frequency of GSTT1 null genotype in Han Chinese (50.0%) was significantly (P < 0.05) higher than that of Uygur Chinese (26.6%). GSTP1 had a genotype distribution of 60.7% I/I, 35.2% I/V and 4.1% V/V in Han Chinese, and 51.3% I/I, 40.2% I/V and 8.4% V/V in Uygur Chinese. CONCLUSION: There is marked ethnic difference in the mutant frequencies of GSTT1 and GSTP1, but not GSTM1, between Uygur and Han Chinese.
Adult↗
Large-sample Bayesian posterior distributions for probabilistic sensitivity analysis.
In probabilistic sensitivity analyses, analysts assign probability distributions to uncertain model parameters and use Monte Carlo simulation to estimate the sensitivity of model results to parameter uncertainty. The authors present Bayesian methods for constructing large-sample approximate posterior distributions for probabilities, rates, and relative effect parameters, for both controlled and uncontrolled studies, and discuss how to use these posterior distributions in a probabilistic sensitivity analysis. These results draw on and extend procedures from the literature on large-sample Bayesian posterior distributions and Bayesian random effects meta-analysis. They improve on standard approaches to probabilistic sensitivity analysis by allowing a proper accounting for heterogeneity across studies as well as dependence between control and treatment parameters, while still being simple enough to be carried out on a spreadsheet. The authors apply these methods to conduct a probabilistic sensitivity analysis for a recently published analysis of zidovudine prophylaxis following rapid HIV testing in labor to prevent vertical HIV transmission in pregnant women.
Bayes Theorem↗