[Pseudomonas Aeruginosa Affects the Function of Pulmonary Vascular Endothelial Cells].

Objective To investigate the impact of Pseudomonas aeruginosa(PA) infection on the function of pulmonary vascular endothelial cells,and explore the mechanism of this bacterium in exacerbating lung inflammation in mice. Methods Two hours after human lung microvascular endothelial cell(HULEC-5a) were infected with the PA strain PAO1,the mRNA levels of autophagy-related gene 5(ATG5),6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3),and calcium adhesion protein 5(CDH5) were determined by reverse transcription real-time fluorescent quantitative PCR(RT-qPCR).The protein levels of ATG5,PFKFB3,and vascular endothelial calcium adhesion protein(VE-cadherin) were detected by immunofluorescence.After the expression of ATG5 and PFKFB3 was respectively knocked down by small interfering RNA(siRNA),RT-qPCR was employed to measure the mRNA levels of ATG5,PFKFB3,and CDH5,and immunofluorescence to detect the protein levels of PFKFB3 and VE-cadherin.In addition,the lactate assay kit was used to determine the level of lactate in the cells.After mice were infected with PAO1,lung inflammation was assessed through histopathological section staining.Confocal microscopy was employed to capture and analyze fluorescence-labeled PFKFB3 and VE-cadherin in endothelial cells. Results Compared with the control group,the HULEC-5a cells infected with PAO1 showed up-regulated mRNA and protein levels of PFKFB3(all P<0.05),down-regulated mRNA level of CDH5(P=0.023),disrupted continuity and down-regulated protein level of VE-cadherin(P<0.001),and elevated lactate level(P=0.017).Compared with PAO1-infected HULEC-5a cells,knocking down PFKFB3 led to the up-regulated mRNA level of CDH5(P=0.043),lowered lactate level(P=0.047),and restored continuity of VE-cadherin;knocking down ATG5 led to up-regulated mRNA and protein levels of PFKFB3(P=0.013 and P=0.003),elevated lactate level(P=0.015),and down-regulated mRNA level of CDH5(P=0.020) and protein level of VE-cadherin(P=0.001).The HE staining results showed obvious red blood cell leakage,inflammatory cell infiltration,alveolar septal widening,and partial detachment of vascular endothelial cells in the alveoli of PA-infected mice.Immunofluorescence staining showed up-regulated expression of PFKFB3 and decreased fluorescence signal of VE-cadherin in endothelial cells of infected mice compared with normal mice. Conclusion PA may regulate the PFKFB3 pathway via AGT5 to disrupt the function of pulmonary vascular endothelial cells,thereby exacerbating the inflammation in the lungs of mice.

A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance / 药物分析学报

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50％of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine le-sions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glio-blastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell char-acteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

Inhibition of the CDK9-cyclin T1 protein-protein interaction as a new approach against triple-negative breast cancer

Cyclin-dependent kinase 9 (CDK9) activity is correlated with worse outcomes of triple-negative breast cancer (TNBC) patients. The heterodimer between CDK9 with cyclin T1 is essential for maintaining the active state of the kinase and targeting this protein-protein interaction (PPI) may offer promising avenues for selective CDK9 inhibition. Herein, we designed and generated a library of metal complexes bearing the 7-chloro-2-phenylquinoline CˆN ligand and tested their activity against the CDK9-cyclin T1 PPI. Complex 1 bound to CDK9 via an enthalpically-driven binding mode, leading to disruption of the CDK9-cyclin T1 interaction in vitro and in cellulo. Importantly, complex 1 showed promising anti-metastatic activity against TNBC allografts in mice and was comparably active compared to cisplatin. To our knowledge, 1 is the first CDK9-cyclin T1 PPI inhibitor with anti-metastatic activity against TNBC. Complex 1 could serve as a new platform for the future design of more efficacious kinase inhibitors against cancer, including TNBC.

Aurone derivatives as Vps34 inhibitors that modulate autophagy

We report in this study the identification of a natural product-like antagonist () of Vps34 as a potent autophagy modulator structure-based virtual screening. Aurone derivative strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, prevents autophagy in human cells induced either by starvation or by an mTOR inhibitor. modeling and kinetic data revealed that could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy and without inducing heart or liver damage in mice. could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.

Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells / 中国天然药物

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.