ABSTRACT
Drug resistance in cells is a major impedance to successful treatment of lung cancer. Taxus chinensis var. inhibits the growth of tumor cells and promotes the synthesis of interleukins 1 and 2 and tumor necrosis factor, enhancing immune function. In this study, T. chinensis var.-induced cell death was analyzed in lung cancer cells (H460) enriched for stem cell growth in a defined serum-free medium. Taxus-treated stem cells were also analyzed for Rhodamine 123 (Rh-123) expression by flow cytometry, and used as a standard functional indicator of MDR. The molecular basis of T. chinensis var.-mediated drug resistance was established by real-time PCR analysis of ABCC1, ABCB1, and lung resistance-related protein (LRP) mRNA, and western blot analysis of MRP1, MDR1, and LRP. Our results revealed that stem cells treated with higher doses of T. chinensis var. showed significantly lower growth inhibition rates than did H460 cells (P < 0.05). The growth of stem and H460 cells treated with a combination of T. chinensis var. and cisplatin was also significantly inhibited (P < 0.05). Rh-123 was significantly accumulated in the intracellular region and showed delayed efflux in stem cells treated with T. chinensis var. (P < 0.05), compared to those treated with verapamil. T. chinensis var.-treated stem cells showed significant downregulation of the ABCC1, ABCB1, and LRP mRNA and MRP1, MDR1, and LRP (P < 0.05) compared to H460 cells. Thus, T. chinensis var.-mediated downregulation of MRP1, MDR1, and LRP might contribute to the reversal of drug resistance in non-small cell lung cancer stem cells.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Neoplastic Stem Cells/drug effects , Plant Extracts/pharmacology , Taxus/chemistry , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cisplatin/pharmacology , Drug Combinations , Drug Resistance, Neoplasm/genetics , Drugs, Chinese Herbal , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Plant Extracts/chemistry , Rhodamine 123/metabolism , Signal Transduction , Vault Ribonucleoprotein Particles/antagonists & inhibitors , Vault Ribonucleoprotein Particles/genetics , Vault Ribonucleoprotein Particles/metabolismABSTRACT
Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0.94 ± 0.03 and 0.92 ± 0.05 vs 1.0 ± 0.02, 0.98 ± 0.05, and 0.98 ± 0.09, respectively; P < 0.05). Quantitative polymerase chain reaction revealed that DNMT1 gene expression of fibroblasts administered 10, 20, and 100 mM RG108 was significantly lower than those in the 0 and 5 mM groups (0.2 ± 0.05, 0.18 ± 0.07, and 0.3 ± 0.09 vs 1.0 ± 0.12 and 1.4 ± 0.12, respectively; P < 0.05). Treatment with 20 mM RG108 resulted in the lowest expression levels. Fibroblasts incubated with 20 mM RG108 for 72 h were used as donor cells to generate SCNT embryos. A greater number of such embryos developed into blastocysts compared to the non-treated group (28.9 ± 3.9 vs 15.3 ± 3.4%; P < 0.05). RG108 treatment can modify DNA methylation in buffalo adult fibroblasts and promote development of subsequent SCNT embryos.
Subject(s)
Buffaloes/genetics , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic , Fibroblasts/drug effects , Nuclear Transfer Techniques , Phthalimides/pharmacology , Tryptophan/analogs & derivatives , Animals , Blastocyst/cytology , Blastocyst/enzymology , Breeding , Buffaloes/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/drug effects , Dose-Response Relationship, Drug , Embryonic Development , Female , Fertilization in Vitro , Fibroblasts/cytology , Fibroblasts/enzymology , Male , Oocytes/cytology , Oocytes/metabolism , Pregnancy , Spermatozoa/cytology , Spermatozoa/metabolism , Tryptophan/pharmacologyABSTRACT
In this case-control study, we assessed the influence of IL-10 -1082A/G and -819T/C on the development of preeclampsia. The IL-10 -1082A/G and -819T/C polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism. The genotype distributions of the IL-10 -1082A/G and -819T/C polymorphisms in the control subjects were in conformance with Hardy-Weinberg equilibrium (HWE; P = 0.46 and 0.17). Unconditional logistic regression analyses revealed that individuals carrying the CC genotype of IL-10 -819T/C were associated with an increased risk of preeclampsia compared to the TT genotype. The odds ratio (95% confidence interval) for the CC genotype of IL-10 -819T/C was 1.71 (1.07-3.27) compared to the TT genotype. In conclusion, the results of our study indicated that the IL-10 -819T/C polymorphism was associated with an increased risk of preeclampsia in a Chinese population.
Subject(s)
Genetic Predisposition to Disease , Interleukin-10/genetics , Polymorphism, Single Nucleotide , Pre-Eclampsia/genetics , Promoter Regions, Genetic , Adult , Alleles , Asian People , Early Diagnosis , Female , Gene Expression , Gene Frequency , Genetic Linkage , Humans , Logistic Models , Odds Ratio , Pre-Eclampsia/diagnosis , Pre-Eclampsia/ethnology , Pre-Eclampsia/pathology , Pregnancy , RiskABSTRACT
Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.