Glioma is the most devastating cancer in the brain and has a poor prognosis in adults. Therefore, there is a critical need for novel therapeutic strategies for the management of glioma patients. Isogambogenic acid, an active compound extracted from the Chinese herb Garcinia hanburyi, induces autophagic cell death.Cell viability was detected with MTT assays. Cell proliferation was assessed using the colony formation assay. Morphological changes associated with autophagy and apoptosis were tested by TEM and Hoechst staining, respectively. The apoptosis rate was measured by flow cytometry. Western blot, immunofluorescence and immunohistochemical analyses were used to detect protein expression. U87-derived xenografts were established for the examination of the effect of isogambogenic acid on glioma growth in vivo.Isogambogenic acid induced autophagic death in U87 and U251 cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activities of isogambogenic acid. Moreover, we observed the activation of AMPK-mTOR signalling in isogambogenic acid-treated glioma cells. Furthermore, the activation of AMPK or the inhibition of mTOR augmented isogambogenic acid-induced autophagy. Inhibition of autophagy attenuated apoptosis in isogambogenic acid-treated glioma cells. Finally, isogambogenic acid inhibited the growth of U87 glioma in vivo.Isogambogenic acid inhibits the growth of glioma via activation of the AMPK-mTOR signalling pathway, which may provide evidence for future clinical applications in glioma therapy.
Rapid advancement in genomic technologies has greatly enhanced the potential of clinical recognition and application of molecular targets. Specifically, gene mutation detection technologies are of great significance in the early diagnosis, customized drug delivery guidance, treatment progression, and monitoring of tumor’s drug resistance. Gene mutation detection or genetic typing of patients is a prerequisite for molecular targeted therapies. Most kinds of targeted therapies treat cancers by interfering with specific proteins that are involved in tumorigenesis. Compared with traditional chemotherapies, molecular targeted therapies have many advantages, despite that they still have risks of resistance, side effects and leak of genetic information.
Green fluorescent protein(GFP)as a reporter protein is widely used in signal conduction and gene expression and regulation in microbiology. In this research,GFP genes were successfully expressed in Gluconacetobacter xylinus with shuttle plasmid p MV24. GFP genes were cloned from p MUTIN-gfp+ plasmid and connected to p MV24 to build prokaryotic expression plasmid p MV24-gfp+. Electrotransformation was applied to introduce p MV24-gfp+ into G. xylinus. The transformant exhibits bright green fluorescence when exposed to blue light in the ultraviolet range of fluorescence microscope. It can provide very important theoretical basis for the observation of cell movement and the analysis of kinesin of G. xylinus under chemotaxis.
In this paper, puerarin (PUE) was nanocrystallized by emulsion solvent evaporation (ESE) method, followed by freeze-drying. The solubility, dissolution rate and oral bioavailability of PUENs were significantly improved compared with raw PUE.
Abstract The TGF-β signaling pathway plays a dual role in epithelial malignancies. While attenuated function of TGF-β signaling enables the development of epithelial cancers, activation of alternative TGF-β signaling in established cancer promotes a more invasive and metastatic phenotype. While the loss of its tumor suppressor function may be due to a variety of defects in the TGF-β Smad-dependent signal transduction pathway, the mechanisms responsible for the pro-oncogenic effects are less well characterized. Since aberrant activation of transcription factor Nuclear Factor-kappaB (NF-κB) promotes the malignant phenotype in head and neck squamous cell carcinomas (HNSCC), we examined whether TGF-β cross-activates NF-κB. Here, we show that TGFβ-1 treatment induced NF-κB activation and gene expression in HNSCC lines. TGFβ-1 and TNFα-induced NF-κB activation was mediated through TGF-β-activated kinase 1 (TAK1), since knocking down of TAK1 using siRNA decreased both TGFβ-1 and TNFα induced NF-κB-dependent reporter gene activity. Furthermore, TAK1 knockdown decreased degradation of IκBα, the inhibitor of NF-κB, and promoted nuclear translocation of the transactivating NF-κB subunit p65 (RELA). Consequently, p65 DNA binding activity and transcription of NF-κB downstream genes was attenuated. As TGFβ-1 treatment of HNSCC cells with mutated TGF-β Receptor II (TβRII) did not affect NF-κB-dependent reporter gene expression, functional TβRII signaling was required for activation of TAK1. Transient transfection and expression of the inhibitor SMAD7 was able to attenuate NF-κB activation as shown by NF-κB-dependent reporter gene activity, however SMAD7 preferentially decreased TGFβ-1 over TNFα induced NF-κB activation. Conversely, knocking down of SMAD7 using siRNA increased TGFβ-1 induced NF-κB activation to a greater extent than that induced by TNFα. Furthermore, TGFβ-1 treatment increased SMAD7 mRNA and overexpression of SMAD7 nearly abrogated TGF-β-induced reporter gene activity, indicating SMAD7 provides a negative feedback loop preferentially suppressing canonical TGF-β signaling. Knockdown of the NF-κB subunit p65 using siRNA decreased SMAD7 mRNA, indicating that NF-κB activation further contributes to the expression and greater inhibitory effect of SMAD7 upon TGF-β signaling. In conclusion, we show that both TGFβ-1 and TNFα can induce NF-κB activation through TAK1. Although SMAD7 was able to attenuate this TAK1/NF-κB activation, the inhibitory effect on canonical TGF-β signaling was more potent, favoring increased NF-κB-mediated cell growth and survival signaling in HNSCC. This study helps explain the dual role of TGF-β signaling, which involves activation of NF-κB and suppression of canonical TGF-β signaling in HNSCC.(Supported by NIDCD intramural project ZIADC-000016) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5052.
Cipangopaludina chinensis is an important economic value snail species with high medicinal value. The gut microbes of aquatic animals plays a vital role in food digestion and nutrient absorption. Herein, we aimed at high-throughput sequencing of 16S rRNA to further investigate whether there were differences in the composition and function of gut microbes of adult and juvenile C. chinensis snails, as well as sediments. This study found that the microbial diversity of the sediment was significantly higher than that of the snails gut ( P < 0.001), but there was no significant difference between the gut flora of adult and juvenile snails ( P > 0.05). A total of 47 phyla and 644 genera were identified from all samples. Proteobacteria and Verrucomicrobia were the two dominant phyla in all samples, and overall relative abundances was 48.2% and 14.2%, respectively. Moreover, the relative abundances of Aeromonas and Luteolibacter in the gut of juvenile snails (30.8%, 11.8%) were higher than those of adults (27.7%, 10.6%) at the genus level ( P > 0.05). Then, four indicator genera were found, namely Flavobacterium , Silanimonas , Geobacter and Zavarzinella , and they abundance in the gut of juvenile snails was significantly higher than that of adults ( P < 0.05). This results imply the potential development of Silanimonas as a bait for juvenile snail openings. We observed that Aeromonas was the primary biomarker of the snail gut and sediments ( P < 0.001), and it may be a cellulose-degrading bacteria. Function prediction revealed significantly better biochemical function in the snail gut than sediments ( P < 0.001), but no significant differences in adult and juvenile snail ( P > 0.05). In conclusion, studies show that the snail gut and sediment microbial composition differ, but the two were very similar. The microbial composition of the snail gut was relatively stable and has similar biological functions. These findings provide valuable information for in-depth understanding of the relationship between snails and environmental microorganisms.
Adrenomedullin is a secreted peptide hormone with multiple activities. Several reports have indicated that adrenomedullin may be involved in tumor survival, but this has not been directly shown. Here we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast cancer cells.The human breast cancer cell lines T47D and MCF7, both of which express low basal levels of adrenomedullin, were stably transfected with an expression construct that contained the coding region of the human adrenomedullin gene or with empty expression vector. Properties of the transfected cells were assessed by proliferation and apoptosis assays, in vitro and in vivo angiogenesis assays, cell migration experiments, and xenograft implants. The effect of synthetic adrenomedullin on human ovarian (ECV) cancer cell motility was also tested. Western blot analysis was used to compare expression levels of several genes whose products are associated with cell growth and regulation of apoptosis.T47D and MCF7 cells transfected with the adrenomedullin construct both expressed high levels of adrenomedullin mRNA and protein. Compared with cells transfected with empty vector, cells that overexpressed adrenomedullin displayed a more pleiotropic morphology, an increased angiogenic potential both in vitro and in vivo, and less apoptosis after serum deprivation. T47D and MCF7 cells did not display measurable motility, but ECV ovarian cancer cells treated with synthetic adrenomedullin were more motile than saline-treated ECV cells. Adrenomedullin-overexpressing T47D cells had higher levels of proteins involved in oncogenic signal transduction pathways (such as Ras, Raf, PKC, and MAPKp49) and lower levels of pro-apoptotic proteins (such as Bax, Bid, and caspase 8) than T47D cells transfected with empty vector. In a preliminary in vivo experiment, three of 10 nude mice injected with adrenomedullin-overexpressing T47D cells developed xenograft tumors, whereas none of the 10 nude mice injected with cells carrying the empty plasmid developed tumors.These results further support the role of adrenomedullin as a survival factor for tumors. Development of physiologically efficient inhibitors of adrenomedullin may prove useful in the clinical management of cancer.
Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.
Abstract Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by l-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.
The main goal is to investigate the role of P120-catenin (P120ctn) in cadherin switching, as well as migration and invasion, of oral squamous cell cancer (OSCC) cells.The plasmid pGFP-V-RS-P120ctn shRNA was used to transfect TSCCA cells and significantly reduce the expression of P120ctn in these cells. Real-time fluorescent quantitative polymerase chain reaction and Western blot were conducted to determine the mRNA and protein expression levels of P120ctn, E-cadherin (E-cad), and N-cadherin (N-cad). By contrast, the Transwell cell invasion and cell migration assay was used to determine the invasion and migration capacities before and after the transfection.After the plasmid pGFP-V-RS-P120ctn shRNA was transfected into the TSCCA cells, we found that as the P120ctn expression significantly decreased, E-cad mRNA and protein expression decreased significantly. Moreover, N-cad mRNA and protein expression increased significantly (P<0.05). Lastly, the cell migration and invasion capacities were augmented significantly (P<0.05).In OSCC cells, P120ctn may be involved in cadherin switching and promote metastasis and invasion.目的 探索P120-连环蛋白（P120ctn）在口腔鳞状细胞癌（OSCC）细胞钙黏蛋白转换中的作用及其对肿瘤细胞迁移和侵袭的影响。方法 采用质粒pGFP-V-RS-P120ctn shRNA转染OSCC细胞株TSCCA，采用实时荧光定量聚合酶链反应、Western blot检测细胞中P120ctn、E-钙黏蛋白（E-cad）和N-钙黏蛋白（N-cad）的mRNA和蛋白表达的变化，通过Transwell细胞侵袭及细胞迁移实验检测转染前后细胞迁移和侵袭能力的变化。结果 质粒pGFP-V-RS-P120ctn shRNA转染TSCCA细胞后，P120ctn表达明显降低，E-cad的mRNA和蛋白表达水平也明显降低，而N-cad的mRNA和蛋白表达水平明显提高，细胞迁移和侵袭能力也明显提高，差异均有统计学意义（P<0.05）。结论 在OSCC中P120ctn可能通过介导钙黏蛋白转换来调节肿瘤的浸润和转移过程。.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
