Antitumor Mechanism Of Cudratricuxanthone A Against Lung Cancer Harboring EGFR Wild-type

Epidermal growth factor receptor(EGFR)is an important target in non-small cell lung cancer(NSCLC)targeted therapy.The emergence of EGFR tyrosine kinase inhibitors(TKIs)successfully alleviated the plight of lung cancer patients with EGFR mutation,but for EGFR wild-type lung cancer these drugs are less effective.Chemotherapy that makes toxic side effects large and does not have targeted effects is still the main treatment for these patients.Therefore,it is important to develop new therapeutic agents for EGFR wild-type lung cancer.Yunnan is rich in plant resources,which not only are processed foods rich in nutrients,but some of the natural compounds found in them are useful in cancer treatment.In the previous study,1200 natural compounds were screened by surface plasma resonance technology and cell proliferation experiment,found a natural compound Cudratricusxanthone A(CTXA)from Cudrania tricuspidata,which interacts with EGFR extracellular domains with strong affinity,inhibits proliferation of wild-type lung cancer cells at 10 μM level.Therefore,this study speculated that CTXA could show good antitumor activity against EGFR wild-type lung cancer by targeting EGFR.Based on the above research,the following results are obtained:(1)EGFR wild-type lung cancer cell A549 was selected as the study object,and the cell proliferation test showed that CTXA could significantly inhibit the growth of A549 cells,and the IC50 at 24 h and 48 h were 5.803 μM and 3.311 μM.The effects of CTXA on the cell state were observed and photographed under a microscope at different concentrations.It was found that the cell state changed at low concentration,and the cells died at high concentration.Subsequent cloning experiments showed that CTXA could significantly inhibit the formation of A549 cell colonies in a concentration-dependent manner.Cell scratch experiments showed that CTXA could significantly inhibit the invasion and migration of tumor cells.(2)In order to explore the antitumor mechanism of CTXA,the influence of CTXA on EGFR and its downstream pathway in A549 cells was detected by Western blot assay.The results showed that,after 1 h treatment,CTXA could significantly inhibit EGFR stimulated and activated by EGF and the phosphorylation of downstream signal proteins ERK and AKT.(3)In order to explore the antitumor mechanism of CTXA,the influence of CTXA on EGFR and its downstream pathway in A549 cells was detected by Western blot.The results showed that CTXA could affect the arrest of A549 cells in G1 phase.Western blot detection of cycle-related proteins revealed that CTXA could decrease the expressions of CDK4 and cyclin D1,which were key proteins in G1 phase,leading to cell cycle rearrangement of A549.(4)Flow cytometry showed that CTXA could induce apoptosis of A549 cells in a concentration dependent manner.In order to explore the mechanism of apoptosis,the key proteins of apoptosis pathway were detected by Western blot assay.The results showed that CTXA mainly affected the expression of Bcl-2 family proteins.When Bax is up-regulated,Bcl-2 is down-regulated,caspase-9 and caspase-3 are activated,and finally PARP1 is cut to induce apoptosis of A549 cells.(5)Molecular docking simulation was used to explore the binding mode of CTXA and EGFR.The results showed that CTXA could bind EGFR and form two hydrogen bonds with GLY-259 and TYR-261.Combined with the above experimental results,compound binding in this region may affect the dimerization of EGFR,resulting in the corresponding cytological effects through signaling.In conclusion,CTXA plays a crucial role in regulating the proliferation of wild-type non-small cell lung cancer cells,and its mechanism of action is mainly through targeting EGFR and inhibiting the phosphorylation of its downstream pathway proteins,thus affecting the expression of key proteins of cycle and apoptosis,and finally causing cell cycle rearrangement and inducing apoptosis.This study can provide scientific basis for developing new antitumor drugs from plants.