肺岩宁方调控TGF-β介导的骨转移相关PTHrP/RANKL信号通路抗肺癌侵袭转移

Effect of Feiyanning Formula on Invasion and Metastasis of Lung Cancer via Inhibiting TGF-β Mediated Bone Metastasis Related PTHrP/RANKL Signaling Pathway

目的：探讨肺岩宁方通过调控转化生长因子-β（transforming growth factor-β,TGF-β）介导的骨转移相关甲状旁腺激素相关蛋白（parathyroid hormone-related protein,PTHrP）/NF-κB受体活化因子配体（receptor or activator of NF-κB ligand,RANKL）信号通路抗肺癌细胞侵袭转移的作用机制。方法：体外培养A549肺癌细胞,用不同终浓度肺岩宁方进行处理,未加肺岩宁方为空白组。分别采用cell counting kit-8（CCK-8）检测肺岩宁方对A549细胞增殖抑制作用,计算半数浓度抑制率（IC50）;采用转移小室（transwell）侵袭实验、划痕实验检测肺岩宁方对A549细胞体外侵袭及迁移的抑制作用;以及蛋白免疫印迹法（Western blot）检测TGF-β,PTHrP,RANKL蛋白表达。结果：肺岩宁方作用于A549肺癌细胞48 h后,与空白组相比,肺岩宁方（12.5,25,50 mg·L^-1）组穿膜细胞明显减少（P〈0.05,P〈0.01）;肺岩宁方（12.5,25,50 mg·L^-1）组的划痕距离较空白组明显降低（P〈0.05,P〈0.01）;且肺岩宁方（25,50 mg·L^-1）组作用于A549细胞后,TGF-β蛋白表达明显下调（P〈0.05）,其对应下游靶基因PTHrP及RANKL蛋白也发生明显表达下调（P〈0.05）。结论：肺岩宁方可抑制A549肺癌细胞的侵袭迁移能力,直接下调TGF-β基因及其下游靶基因PTHrP,RANKL蛋白表达,且具有浓度相关性。本研究证实了肺岩宁方通过调控TGF-β介导的骨转移相关PTHrP/RANKL信号通路,以达到抗肺癌侵袭转移的作用机制,这为肺癌临床靶向治疗提供了一定的实验依据,也显示了肺岩宁方作为抗肺癌侵袭转移药物广泛的应用前景。

Objective： To investigate the effect mechanism of Feiyanning formula on invasion and metastasis of lung cancer cells via inhibiting transforming growth factor-β（TGF-β） mediated bone metastasis related parathyroid hormone-related protein（PTHrP）/receptor or activator of NF-κB ligand（RANKL） signaling pathway. Method： A549 cells were cultured in vitro and treated with Feiyanning formula with various concentrations,with no Feiyanning formula as blank group. The anti-proliferation effects and the 50% inhibitory concentration（IC50） of Feiyanning formula was measured by cell counting kit-8（CCK-8） method. The inhibiting effect against invasion and migration ability of A549 cells was detected by transwell assay and scratch test. Western blot was used to detect the protein expression of TGF-β,parathyroid hormone-related protein（PTHrP） and receptor or activator of NF-κB ligand（RANKL）. Result： After Feiyanning formula effected on A549 lung cancer cells for48 h,as compared with blank control group,the transmembrane cells in the low,medium and high dose Feiyanning formula（12. 5,25,50 mg·L^-1） groups were obviously reduced（P〈0. 05,P〈0. 01）,and the scratch distance in Feiyanning formula groups was also significantly shortened（P〈0. 05, P〈0. 01）.Furthermore,medium and high dose Feiyanning formula group significantly down-regulated the expression of TGF-β（P〈0. 05） and its corresponding downstream target genes of PTHrP and RANKL（P〈0. 05）. Conclusion：Feiyanning formula can directly down-regulate the protein expression of TGF-β and its downstream target genes PTHrP and RANKL,and inhibit the invasion and migration ability of A549 lung cancer cells in a concentrationdependent manner. This study confirmed the mechanism of Feiyanning formula on inhibiting the invasion and migration of lung cancer by regulating TGF-β mediated bone metastasis related PTHrP/RANKL signaling pathway,providing a certain experimental basis for the clinical target therapy of lung cancer,suggesting that Feiyanning formula could be a potential candidate in the treatment of lung cancer cell bone metastasis.