| Objective:Now,lung cancer remains one of the most common malignancies and is the leading cause of all cancer-related deaths in the world.Non-small cell lung cancer(NSCLC)accounts for approximately 85% of lung cancers.Gracillin is an atural steroidal saponin compound widely existed in a variety of plants.Numerous studies have demonstrated that Gracillin has great anti-tumor potential,with broad-spectrum anti-proliferative effects on a variety of tumor cells.However,there are relatively few studies on its anti-tumor mechanism,and its anti-tumor mechanism is not yet clear enough.The objective of this project is to verify the anti-tumor effect of Gracillin on NSCLC and to clarify its possible mechanism of action against NSCLC.Methods:In this project,A549 cells were used as tumor models in vitro,and immunodeficient mice carrying A549 cells were used as tumor models in vivo.First,an immunodeficient mouse tumor model was established,and the antitumor effect of Gracillin on NSCLC was clarified by tumor volume,weight and Ki67 staining.Then,the inhibitory effect of Gracillin on the proliferation of A549 cells and BEAS-2B cells was verified by CCK-8;through RNA-Seq high-throughput transcriptome sequencing screening,bioinformatics analysis and literature research,autophagy was identified as the research direction,and a significantly overexpressed gene WIPI1 attracted our attention;therefore,3-MA and Rapamycin were used to investigate the inhibitory or inducing effect of Gracillin on autophagy;then,the ultrastructure of autophagy was observed by transmission electron microscopy;moreover,the punctate aggregation of LC3 protein on autophagosomes was observed by laser confocal after transfection of p EGFP-LC3 plasmid;At the same time,autophagy-related proteins(Beclin-1,P62,WIPI1 and LC3)and mTOR signaling pathway-related proteins(PI3K,p-PI3K,Akt,p-Akt,AMPK,p-AMPK,mTOR and p-mTOR)were detected.expression changes: in addition,to study the role of WIPI1 in autophagy by Gracillin regulated in A549 cells,lentivirus was used to silence WIPI1 in A549 cells,and then the protein expression changes of Beclin-1,P62 and LC3 were detected.Results:1.In animal models,the growth of tumors was significantly inhibited by Gracillin.Compared with the solvent group,the tumor weight and volume in the Gracillin-treated groups were significantly reduced.The results of Ki67 immunohistochemical staining showed that the proliferation of tumor cells in the Gracillin-treated mice was significantly reduced.At the same time,H&E staining results of mouse heart,liver,lung,and kidney showed that Gracillin had no obvious physiological toxicity.2.In the cell models,the proliferation of A549 cells was significantly inhibited by Gracillin and the proliferation of BEAS-2B cells was not significantly affected.The results of qRT-PCR and Western blot showed that the expression of WIPI1 in Gracillintreated A549 cells were significantly higher at both gene and protein levels,which was consistent with the sequencing results;Gracillin-induced autophagic death was found in Gracillin-treated A549 cells after inhibiting autophagy with 3-MA or activating autophagy with Rapamycin;and a typical double-layer membrane structure and autophagic vacuoles were observed in A549 cells,and LC3 protein was significantly aggregated on the autophagosome membrane;the expression of autophagy-related protein Beclin-1 was up-regulated,the expression of P62 was down-regulated,and the transformation of LC3-II was increased.At the same time,the inhibition of autophagy by 3-MA was reversed by Gracillin,manifested as re-up-regulation of Beclin-1,downregulation of P62,and promotion of LC3-II transformation.3.Molecular mechanism study found that the anti-tumor mechanism of Gracillin may be related to the inhibition of mTOR signaling pathway.Its upstream positive regulators PI3K and Akt phosphorylated proteins p-PI3K and p-Akt were significantly inhibited,and its upstream negative regulator AMPK phosphorylated protein p-AMPK was significantly activated,resulting in the expression of p-mTOR significantly reduced;after WIPI1 was silenced,it was found that there was no significant effect on the expression of P62 and Beclin-1,but the transformation of LC3-II was significantly decreased,indicating that autophagy was inhibited.Based on the literature and the research of this topic,WIPI1 was involved in the formation of autophagosomes and regulates autophagy as the downstream of mTOR in the process of Gracillin-induced autophagy.Conclusion:Gracillin showed obvious anti-tumor effect in non-small cell lung cancer in vitro and in vivo.Further anti-tumor mechanism studies showed that Gracillin may induce autophagic death by affecting autophagy-related proteins and regulating autophagyrelated mTOR signaling pathway. |
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