| Hippo signaling pathway is related to cell proliferation and organ size.Its downstream transcriptional co-activator YAP protein appears as an important oncoprotein in many cancers.Based on the results of previous preliminary experiments,it was found that quinazoline derivatives can regulate the Hippo signaling pathway.Moreover,many research results have also shown that quinazoline-containing structures or quinazoline compounds have significant antitumor activities.After reviewing and referring to a large number of literatures,a series of quinazoline compounds with low toxicity and high efficacy were synthesized through design based on the previous work.The synthesized compounds were screened for antitumor activity in vitro by MTT colorimetry,and it was found that compound 3333 exhibited significant biological activity on gastric cancer cells and could activate the Hippo signaling pathway.The antitumor mechanism is further explored and the antitumor activity verified in vivo.The specific methods and results are as follows:1.The anti-proliferative activities of the compounds on human gastric cancer cells MGC-803,human breast cancer cells MCF-7 and human colon cancer cells HCT-116 were detected by MTT assay.The results showed that inhibitory effect of compound 3333 on MGC-803 was the most significant,and it was identified as the target compound.2.The biological activity of compound 3333 on gastric cancer cells(MGC-803,SGC-7901,HGC-27)was detected by MTT assay.The results showed that compound 3333 significantly reduced the viabilities of the above three types of gastric cancer cells in a concentration-dependent manner,while it had no significant toxicity no human gastric epithelial cells 1.3.To explore whether compound 3333 can activate the Hippo signaling pathway,the expression of related proteins was detected by Western Blot.The results showed that compound 3333 gradually increased the protein levels of P-LATS and P-YAP in cells MGC-803 and SGC-7901 in a concentration-time-dependent mannner,while the level of the main downstream effector molecule protein YAP in this pathway gradually decreased.It showed that compound 3333 was able to activate the Hippo pathway.4.In order to further verify that compound 3333 can activate the Hippo signaling pathway and inhibit the downstream effector molecule YAP,the nuclear and cytoplasmic separation experiments and Western Blot were used to detect changes in the protein levels of YAP in the nuclear and cytoplasm of MGC-803 and SGC-7901 cells,respectively.The results showed that YAP was hardly expressed in the nucleus under the action of 3333.It shows that 3333 obviously blocked the transfer of YAP from cytoplasm to nucleus.5.In order to explore the effects of 3333 negative regulation of YAP on downstream target genes,the changes of anti-apoptotic factor Bcl-2 and pro-proliferative factor c-MYC were detected by Western Blot and RT-qPCR.The results showed that the regulation of compound 3333 on Bcl-2 and c-MYC at the mRNA levels were consistent with that at the protein levels,both of which could be downregulated,reflecting that compound 3333 has anti-proliferative and apoptosis-promoting activities against gastric cancer cells.6.In order to verify that compound 3333 can induce apoptosis in gastric cancer cells,flow cytometry(FCM)tests were performedl.The results showed that with the increase of the concentration,the apoptosis rate of cells MGC-803 and SGC-7901 increased significantly.It can be seen that the cell nucleus had undergone significant characterization changes after DAPI fluorescent staining.Using the living dead cell detection kit,it was known that the higher concentration of compound 3333,the greater the number of dead cells.According to the Western Blot test,in a time-concentration-dependent manner,the apoptosis execution protein Cleaved-caspase 7 increased significantly and its substrate protein PARP appeared to be cleaved.The apoptosis inhibitory proteins XIAP and c-IAP1 gradually decreased.These results shown that compound 3333 can up-regulate pro-apoptotic proteins and down-regulate anti-apoptosis proteins,induce apoptosis of gastric cancer cells and reduce survival rate.7.Apoptosis has endogenous and exogenous apoptosis.Changes in the anti-apoptotic protein Bcl-2 acting on the mitochondrial membrane have been detected.In order to verify whether compound 3333 has an effect on the mitochondrial pathway,the kit(JC-1)treated cells MGC-803 and SGC-7901,respectively.FCM results showed that as the concentration of compound 3333 increased,the mitochondrial membrane potential of the two cells gradually decreased.The cell mitochondrial isolation kit was used to treat the cells MGC-803 and SGC-7901.The results of Western Blot showed that 3333 down-regulated the anti-apoptotic proteins Bcl-2 and Mcl-1,induced the release of cytochrome c and activated downstream caspase 9,indicating that compound 3333 induced apoptosis in gastric cancer cells through the mitochondrial pathway.8.MTT assay,clone formation experiments and growth curve results showed that compound 3333 had significant anti-proliferative activity.Cells MGC-803 and SGC-7901 were treated with DNA content detection kit(cell cycle).FCM results showed that the cell cycle was arrested at G2/M phase.However,the results of Western Blot showed that the proteins CDK1,Cyclin B1,and Cyclin B2,which regulate the G2 phase were not downregulated,and the expression of phosphorylated protein Histone H3 that marked the M phase was significantly increased.It shows that compound 3333 has anti-proliferative effect by blocking gastric cancer cells in M phase.9.A nude mouse xenograft model was established for in vivo experiments.5-FU was used as the positive control group.Compared with the negative control group,the compound 3333 administration group significantly reduced tumor volume and weight,effectively inhibited the tumor growth rate,and had no significant effect on nude mouse body weight.Blood biochemical indicators results showed that there was no significant difference between the administration group and the negative control group.TUNEL method was used to detect obvious apoptosis in the tumor tissue of the administration group and Western Blot was used to detect tissue proteins YAP,Bcl-2,c-MYC were all down-regulated.These results further confirmed that compound 3333 can activate the Hippo signaling pathway and produced significant anti-tumor effects with minimal toxic and side effects by mediating apoptosis and cycle inhibition.In summary,in gastric cancer cells,in addition to activating the Hippo signaling pathway to inhibit the activity of the downstream effector molecule YAP,the new quinazoline derivative 3333 can also induce apoptosis through the mitochondrial pathway and effectively inhibit the development of the cell cycle.This thesis explores and validates the mechanism that this compound can activate the Hippo pathway,as well as its roles in promoting apoptosis and inhibiting proliferation of gastric cancer cells,with a view to providing effective research evidence and theoretical support for the development of anticancer drugs and combined treatment programs. |
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