Proteomic Study Of Anlotinib Inhibiting Proliferation Of H1299 Lung Cancer Cells

Lung cancer is a malignant tumor of the respiratory system with high morbidity and mortality all over the world.According to the cancer burden data compiled by the International Agency for Research on Cancer,the number of new cases and deaths of lung cancer in the world in 2020 ranked second and first among all types of malignant tumors.In China,lung cancer ranked first in both incidence and death in 2022.Non-small cell lung cancer(NSCLC)is the most common histopathological type of lung cancer,and most patients are found in advanced stages due to its inconspicuous early symptoms.In recent years,the emergence of new molecular targeted drugs has brought remarkable hope for patients with advanced NSCLC with mutations of different gene types(such as EGFR,ALK and BRAF),but there are no molecular targeted drugs for the treatment of TP53 mutant NSCLC.Anlotinib is a novel oral small-molecule,multi-target tyrosine kinase inhibitor independently developed in China.It can not only inhibit neoangiogenesis by acting on vascular endothelial growth factor receptor(VEGFR),but also inhibit tumor growth by acting on stem cell factor receptor(KIT)and platelet-derived growth factor receptor(PDGFR).Anlotinib has shown good efficacy in several clinical studies targeting solid tumors,and it has been approved for the treatment of advanced NSCLC,soft tissue sarcoma,small cell lung cancer(SCLC),medullary thyroid cancer and other types of tumors.In recent years,some scholars have found that anlotinib can bring relatively good survival benefits for patients with TP53 mutant NSCLC who have failed previous treatment.However,the mechanism of anlotinib in the treatment of TP53 mutant NSCLC is still unclear,which is the main content of this study.Objective:To study the inhibitory effect and mechanism of anlotinib on H1299 lung cancer cells(TP53 mutant type).The difference of protein expression profile of H1299 lung cancer cells before and after treatment with anlotinib was compared by proteomic technology,in order to explore the mechanism of anlotinib inhibiting the proliferation of lung cancer cells.Methods:(1)The effect of anlotinib on the proliferation of A549(TP53 wild type)and H1299(TP53 mutant type)lung cancer cells was detected by CCK-8 assay.(2)Non-labeled quantitative proteomic based on liquid chromatography-tandem mass spectrometry was used to identify and screen differentially expressed proteins in H1299 lung cancer cells before and after treatment with anlotinib.(3)The differentially expressed proteins were submitted to the bioinformatics website(DAVID,PANTHER,STRING)for analysis using bioinformatics techniques,aiming to find that anlotinib may inhibit the proliferation of H1299 lung cancer cells through p53-related signaling pathways.(4)Western Blot assay was used to detect the expression of related differential proteins in A549 and H1299 lung cancer cells treated with anlotinib.The differential proteins ELAVL1 and HDAC2 were used to verify the results of proteomic results,and the differential proteins CDK1 and MAP2K3 associated with p53 signaling pathway were used to verify the possibility of the hypothesis of Map2K3-p53-CDK1 signaling pathway.Results:(1)Anlotinib inhibited the proliferation of A549 and H1299 lung cancer cells in a dose-dependent and time-dependent manner within the concentration range of 1-64 μM.Compared with A549 lung cancer cells,anlotinib showed stronger inhibitory effect on the proliferation of H1299 lung cancer cells within the concentration range of 8-64 μM.(2)In the proteomic study of H1299 lung cancer cells,3200 trusted proteins weridentified.A total of 126 differentially expressed proteins were screened according tthe differential expression multiple and significance difference analysis,among whic37 were up-regulated and 89 were down-regulated.(3)The main differential proteins related to p53 signaling pathway were CDK1 and MAP2K3 by bioinformatics analysis.CDK1 can form Cyclin B/CDK1 complex with Cyclin B,which plays an important role in regulating the initiation,progression and termination of cell cycle.MAP2K3 can specifically activate p38,and p38 affects the expression of its downstream transcription factor p53,thus regulating cell apoptosis.(4)Western Blot results showed that anlotinib could up-regulate the expression of HDAC2 and down-regulate the expression of ELAVL1,which was consistent with the results of proteomic studies on H1299 lung cancer cells.Compared with A549 lung cancer cells,anlotinib significantly down-regulated the p53 signaling pathway-related differential proteins CDK1 and MAP2K3 in H1299 lung cancer cells.Conclusion:Compared with A549(TP53 wild type)lung cancer cells,anlotinib showed stronger inhibitory effect on the proliferation of H1299(TP53 mutant type)lung cancer cells.Proteomics and bioinformatics techniques showed that CDK1 and MAP2K3 were the important target proteins related to p53 signaling pathway that mediated the above effects of anlotinib.Western Blot results also proved that anlotinib could significantly down-regulate the expressions of CDK1(the downstream protein of p53 pathway)and MAP2K3(the upstream protein of p53 pathway).Based on the results of this study,we speculated that the inhibition of MAP2K3-p53-CDK1 signaling pathway by anlotinib may be an important mechanism in the treatment of TP53 mutant NSCLC.