Molecular Mechanism Of Camptothecin Derivative RY-1-11 Exerts Anti-lung Cancer Activity By Inhibiting EZH2

Lung cancer is one of the malignant tumors with high mortality.Despite new progress has been made in the understanding of the mechanism of lung cancer and the development of anticancer drugs,the clinical treatment effect is still unsatisfactory.Therefore,the discovery of effective anti-lung cancer drugs has become a hot spot for researchers.The compound RY-1-11 is a new type of camptothecin derivative,and previous studies have shown that RY-1-11 significantly inhibits the proliferation and migration of lung cancer cells by affecting the Wnt/β-catenin signaling pathway,with good antitumor activity.Meanwhile,previous studies have also shown that RY-1-11 can reduce the expression of histone methyltransferase EZH2.EZH2 is a well-known and potential anti-tumor drug target,which plays a key role in the development of lung cancer.Therefore,this study aims to explore the exact molecular mechanism of RY-1-11’s anti-lung cancer effect,and further investigate whether RY-1-11 acts as a novel EZH2 inhibitor.The results of the study are as follows:1.RY-1-11 interacts with EZH2,and EZH2 can be a potential target of RY-1-11Molecular docking and MD simulation of RY-1-11 and EZH2 was performed by using Auto Dock Vina and Desmond software,the results showed that compound RY-1-11 interacts with EZH2 protein with high matching degree,the affinity value is-8.74 kcal/mol,and it can form strong hydrophobic interactions with multiple hydrophobic amino acids ILE-109/TRP-624/PHE-686/PHE-665/TYR-658/TYR-111,etc,especially the benzene ring and pyridine ring of this compound can form π-πconjugated interaction with PHE-665.At the same time,the active groups of RY-1-11 can form hydrogen bonding interactions with ARG-685 and MET-110,and the hydrogen bonding distances are 2.2 ? and 1.9 ?,respectively,which is of great significance for anchoring molecules.In addition,RY-1-11 is driven to competitively bind to the catalytically active site of EZH2 by hydrophobic interactions,and form a stable complex.Western blot results showed that RY-1-11 could significantly inhibit EZH2 protein expression and its downstream H3K27 methylation level.Taken together,EZH2 is a potential target of RY-1-11.2.RY-1-11 inactivates the Wnt pathway by regulating the EZH2/mi R-29c-3p/Dvl2 signaling axisFirst,the effect of Wnt/β-catenin signaling pathway was detected by knocking down EZH2.The results showed that interference with EZH2 could significantly up-regulate the expression of tumor suppressor mi R-29c-3p.Correspondingly,RT-q PCR and Western blot results showed that overexpression of mi R-29c-3p could downregulate the expression of Dvl2;however,inhibition of mi R-29c-3p could significantly upregulate the expression of Dvl2.The above results show that knocking down EZH2 can inactivate the Wnt pathway by upregulating mi R-29c-3p and then transcriptionally inhibiting the Dvl2 expression.Next,the effect of RY-1-11 on the above-mentioned mediator mi R-29c-3p and Dvl2 was detected.The results showed that RY-1-11 could significantly up-regulate the expression of mi R-29c-3p and down-regulate the expression of Dvl2 and its downstream related genes.At the same time,the results of interference with EZH2 expression combined with RY-1-11 treatment showed that the effect of RY-1-11 on the expression of mi R-29c-3p and Dvl2 could be significantly reversed by interfering with EZH2 expression.Taken together,the results show that RY-1-11 can inactivate the Wnt pathway by targeting EZH2 and upregulating the expression of tumor suppressor mi R-29c-3p,which in turn transcriptionally inhibits the expression of Dvl2.3.RY-1-11 inhibits the expression of EZH2 by down-regulating the expression of β-catenin,a key regulator of the Wnt pathwayThe results of Wnt pathway activation combined with RY-1-11 treatment showed that RY-1-11 can significantly reduce the protein expression of β-catenin and EZH2,and the effect of RY-1-11 on the expression of β-catenin and EZH2 can be reversed by adding the activator Li CL.Taken together,the results show that EZH2 also acts as a target gene of the Wnt pathway and RY-1-11 can inhibit the expression of EZH2 at the transcriptional level by down-regulating the expression of the key proteinβ-catenin.Collectively,EZH2 as an antitumor drug target and RY-1-11 can competitively bind to the catalytic domain of EZH2 to inhibit the enzymatic activity of EZH2.Meanwhile,RY-1-11 can inactivate the Wnt/β-catenin pathway by regulating the EZH2/mi R-29c/3p/Dvl2 signaling axis;it can also inhibit the expression of the downstream gene EZH2 by down-regulating the expression of β-catenin.It can be seen that there may be some feedback regulation between the EZH2 and Wnt pathways in the anti-lung cancer effect of RY-1-11.Therefore,this study can provide valuable research ideas in the prevention and targeted therapy of lung cancer in the future,and also provide a solid theoretical basis for the further development of RY-1-11,which acts as a new anti-lung cancer targeted drug.