养肺控瘤方通过Beclin-1促进自噬并抑制肺癌细胞A549转移

Effect of Yangfeikongliu Formula in Promoting Autophagy and Inhibiting Metastasis of Lung Tumor Cell A549 via Beclin-1

目的研究养肺控瘤方(yangfeikongliu formula,YKF)在肺癌A549细胞自噬和转移中的作用。方法大鼠的YKF的给药剂量将通过对人体临床的剂量换算后得出,给大鼠灌服人体等效剂量的5倍、10倍和15倍的YKF,并收集它们的血液以制备5×、10×和15×的含药血清。使用含有10%YKF含药血清的培养基对A549细胞进行培养。通过电子显微镜、LC3Ⅱ/LC3Ⅰ的比例和自噬流串联mRFP-GFP-LC3来评估A549细胞自噬情况。通过Transwell测定法评估细胞迁移和侵袭,并使用CCK-8测定细胞活性。结果3种剂量YKF含药血清均抑制了肿瘤细胞活性,增加了A549细胞的LC3Ⅱ/LC3Ⅰ比值和p62及Beclin-1表达水平,表明YKF可促进A549细胞自噬死亡。支持本结果的指标为,5×YKF含药血清诱导的LC3点状点的增加在3-MA抑制自噬后被消除。此外,研究观察到沉默Beclin-1可以废除YKF诱导的LC3Ⅱ/Ⅰ比值和p62表达的增加,以及细胞活性、迁移和侵袭的减少。结论养肺控瘤方具有促进肺癌细胞A549自噬和抑制转移的抗肿瘤活性,且Beclin-1是其抗肿瘤活性的关键分子。

Objective Traditional Chinese medicine has been used for serval centuries and proved to be an alternative treatment method for cancer.This study aims to investigate the role of traditional Chinese medicine Yangfei-kongliu Formula(YKF)in autophagy and metastasis of lung tumor A549 cells.Methods The YKF dose for rats was converted according to the human clinical dose.Rats were given YKF by gavage of 5-,10-,and 15-fold of the equivalent human dose,and their blood was collected to prepare 5×,10×,and 15×YKF-contained serum.A549 cells were cultured with a medium containing 10%YKF-contained serum.Autophagy was evaluated by electron microscopy,LC3Ⅱ/LC3Ⅰratio,and autophagic flux tandem mRFP-GFP-LC3.Cell migration and invasion were evaluated by Transwell assay.Cell activity was determined using CCK-8.Results Three doses of YKF-contained serum inhibited cell activity and increased LC3/LC3Ⅰratio and p62 and Beclin-1 expression level in A549 cells indicating YKF promotes autophagic death.The result in support of this indication is that the increase of LC3 puncta induced by 5×YKF-contained serum was abolished after autophagy inhibition by 3-MA.Besides,the research observed that silencing Beclin-1 abolished YKF-induced the increase in LC3Ⅱ/Ⅰratio and p62 expression,and the decrease in cell activity,migration,and invasion.Conclusion Traditional Chinese medicine YKF exerts antitumor activity in promoting autophagy and inhibiting metastasis of lung tumor cell A549 and Beclin-1 is the key molecule of its antitumor activity.