Fatty acid synthase causes drug resistance by inhibiting TNF-α and ceramide production

Fatty acid synthase (FASN) is a key enzyme in the synthesis of palmitate, the precursor of major nutritional, energetic, and signaling lipids. FASN expression is upregulated in many human cancers and appears to be important for cancer cell survival. Overexpression of FASN has also been found to associate with poor prognosis and higher risk of recurrence of human cancers. Indeed, elevated FASN expression has been shown to contribute to drug resistance. However, the mechanism of FASN-mediated drug resistance is currently unknown. In this study, we show that FASN overexpression causes resistance to multiple anticancer drugs via inhibiting drug-induced ceramide production, caspase 8 activation, and apoptosis. We also show that FASN overexpression suppresses tumor necrosis factor-α production and nuclear factor-κB activation as well as drug-induced activation of neutral sphingomyelinase. Thus, TNF-α may play an important role in mediating FASN function in drug resistance.     

Liquid chromatography tandem mass spectrometry in study of the pharmacokinetics of six steroidal saponins in rats

A rapid, simple and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of six main steroidal saponins in Paris polyphylla in rat plasma. Ginsenoside Rg3 was selected as the internal standard (IS). Plasma samples were pretreated with protein precipitation, and the separation was achieved on a reverse phase Agilent poroshell120 EC-C18 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, dioscin, gracillin and IS were m/z 899.5 > 853.4, 1059.5 > 1013.5, 783.4 > 737.4, 1075.5 > 1029.5, 913.5 > 867.4, 929.5 > 883.4 and 819.5 > 783.4, respectively. The intra- and inter-day precisions (RSD%) were less than 13% and the average extraction recoveries ranged from 85% to 97.0% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The established method was employed for simultaneous quantification and successfully used for the first time for the pharmacokinetics evaluation of the six main compounds after intragastric administration of P. polyphylla extract in Sprague–Dawley rats.     

Specificity of miR-378a-5p targeting rodent fibronectin

One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3′UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3′UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3′UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.     

Profiling of dynamic changes in the microbial community during the soy sauce fermentation process

Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus, Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii, Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future.     

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

携带blaNDM-1的质粒pNDM-BJ01在鲁氏不动杆菌中的适应度代价

[目的]了解编码新德里金属-β-内酰胺酶-1(blaNDM-1)的质粒pNDM-BJ01在鲁氏不动杆菌10621菌株中的适应度代价,进而评估该质粒在鲁氏不动杆菌群体中存续和扩散的潜能.[方法]通过不含亚胺培南的液体培养基连续传代培养,获得抗性质粒丢失的10621衍生菌株,利用生长曲线、生物被膜、无营养生存时间等体外适应度测量实验,分析10621NDM-1(+)及质粒缺失的10621NDM-1(-)之间的适应度差异.[结果]pNDM-BJ01在10621菌株中不稳定,连续传代11次即在群体中完全丢失.在26℃和37℃下,10621NDM-1( -)与10621NDM-1(+)菌株的生长曲线无显著性差异.在26℃条件下,10621NDM-1( -)菌株形成生物被膜的能力明显强于10621NDM-1(+),而在37℃条件下,10621 NDM-1(+)强于10621NDM-1(-).在无营养的PBS缓冲液中,10621NDM-1(-)菌株生存能力明显高于10621NDM-1(+).[结论]编码blaNDM-1的质粒pNDM-BJ01在鲁氏不动杆菌中具有较大的适应度代价,缺失这一质粒的菌株在外界环境中的生存能力强于抗性菌株,pNDM-BJ01在鲁氏不动杆菌存续与扩散的能力有限.     

Bone morphogenetic protein-7 (BMP-7) mediates ischemic preconditioning-induced ischemic tolerance via attenuating apoptosis in rat brain

A mild cerebral ischemic insult, also known as ischemic preconditioning (IPC), confers transient tolerance to a subsequent ischemic challenge in the brain. This study was conducted to investigate whether bone morphogenetic protein-7 (BMP-7) is involved in neuroprotection elicited by IPC in a rat model of ischemia. Ischemic tolerance was induced in rats by IPC (15 min middle cerebral artery occlusion, MCAO) at 48 h before lethal ischemia (2 h MCAO). The present data showed that IPC increased BMP-7 mRNA and protein expression after 24 h reperfusion following ischemia in the brain. In rats of ischemia, IPC-induced reduction of cerebral infarct volume and improvement of neuronal morphology were attenuated when BMP-7 was inhibited either by antagonist noggin or short interfering RNA (siRNA) pre-treatment. Besides, cerebral IPC-induced up-regulation of B-cell lymphoma 2 (Bcl-2) and down-regulation of cleaved caspase-3 at 24 h after ischemia/reperfusion (I/R) injury were reversed via inhibition of BMP-7. These findings indicate that BMP-7 mediates IPC-induced tolerance to cerebral I/R, probably through inhibition of apoptosis.     

IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.