Research On The Mechanism Of Qingre Jiedu Method In Modulating Ezrin-SNO Dependent Cellular Tension And Inhibiting The Invasion And Metastasis Of NSCLC

Object:Pulmonary carcinoma is one of the most common malignant tumors and has become a big challenge for human beings due to its high morbidity and mortality Non-small cell lung cancer(NSCLC),accounts for the highest proportion of pulmonary carcinoma,keeps bewildering clinicians.Especially the invasion and metastasis of NSCLC are the most important factors that correlate with prognostic.Hence,studies and drug development against NSCLC invasion and metastasis become an urgent task of medical researches.The cell membrane-cytoskeleton linker protein,ezrin,is highly relevant with NSCLC invasion and metastasis and possibly participates in the mechanical transduction during these processes.Conveying two cysteine residues,ezrin can be easily nitrosylated in the inflammatory microenvironment of NSCLC,the relevant mechanism may be involved in mechanical signaling and a potential molecular target.The Qingre Jiedu method(means clearing away heat and toxic materials)is an important therapy for inflammatory diseases in Chinese medicine,and some drugs belong to Qingre Jiedu method are reported to exhibit the effect in restraining malignant behaviors.However,whether these Chinese drugs could affect ezrin-SNO in the inflammatory microenvironment of NSCLC and play a role in the mechanical modulation during invasion and metastasis warrant further research.The relevant mechanism may explain the anti-dissemination activity of Qingre Jiedu method and acts as an effective screening index for drugs.Methods:The level of nitrosylated ezrin in non-small cell lung cancer tissues and A549 cell line were evaluated by biotin-switch assay.A few cysteine mutated plasmids of ezrin were used to identify active site for SNO.Newly designed ezrin or mutated-ezrin tension probes according to Forster resonance energy transfer(FRET)theory were applied to visually observe real-time tension change.Cytoskeleton depolymerizing and motor molecular inhibiting experiments were performed to reveal the alternation of the mechanical property of ezrin after SNO.Transwell assays and xenograft mouse model were used to assess cell aggressiveness in different groups.Fluorescent staining was also applied to view cellular location and structures.Results:1.High inducible nitric oxide synthase(iNOS)levels in the inflammatory microenvironment of NSCLC were observed to induce ezrin-SNO.2.The up-regulated ezrin-SNO level promotes malignant behaviors of NSCLC cells.3.Cys1 7 was identified as the only active site for ezrin-SNO.4.Ezrin tension was increased after iNOS-induced SNO.5.Enhanced ezrin tension positively correlated with aggressiveness of NSCLC.6.Microfilament(MF)forces instead of microtubule(MT)forces were identified to play dominant roles in modulating ezrin tension,especially after ezrin nitrosylation.7.The monomers of Qingre Jiedu class,Baicalein and Andrographolide,could inhibit iNOS induced ezrin-SNO effectively.8.Baicalein and Andrographolide could affect NSCLC invasion and metastasis by restraining ezrin-SNO related up-regulation of tension.Conclusions:This study revealed a SNO-associated mechanism underlying the mechanical tension of ezrin.Ezrin-SNO promotes NSCLC cells invasion and metastasis through facilitating mechanical transduction from the cytoskeleton to the membrane.These discoveries implicate the potential of therapeutic target towards ezrin for inhibition of NSCLC invasion and metastasis.The Qingre Jiedu method in Chinese medicine could inhibit NSCLC invasion and metastasis by decreasing iNOS-mediated ezrin-SNO level and down-regulating ezrin tension.