Objectives: Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer, with highmorbidity and mortality rate. Nove drug development for NSCLC is urgently needed.This study aims to investigate the activity of lathyrol derivatives and the mechanism for its inhibitory effect on the growth of NSCLC cells.
Methods: Three lathyrol derivatives were synthesized from lathyrol and their structures were verified by nuclear magnetic resonance. MTT assay was used to detect the effects of the lathyrol derivatives on the proliferation activity of NSCLC cells (A549 and H1299 cells), and the compound with the best activity was selected for subsequent experiments. Colony forming assay, wound-healing assay, and transwell assay were applied to detect in vitro cell proliferation, migration and invasion ability in A549 and H1299 cells, respectively. Quantitative real-time RT-PCR and Western blotting were performed to detect mRNA and protein levels of E-cadherin, N-cadherin, β-catenin, and MMP2 in A549 cells, respectively.
Results: Three lathyrol derivatives inhibited the growth of A549 and H1299 cells in a dose-dependent manner, and they showed a weak inhibitory effect on normal cells Beas-2B and 16HBE, indicating that they possessed certain selective toxic effects. Therefore, C-5 benzoylated lathyrol with the best activity was selected as the ideal drug for the subsequent experiments. Compared with the control group, the number and size of cell clusters in the treatment group of A549 and H1299 cells were significantly decreased, the relative mobility were significantly decreased, and the number of invaded cells were significantly decreased (all P<0.05), indicating that the in vitro cell proliferation, migration and invasion ability were decreased. The mRNA levels of integrin α2, integrin β1, MMP2, MMP9, β-catenin, and N-cadherin were decreased, while the expression of E-cadherin was increased (all P<0.05). The protein levels of N-cadherin, β-catenin, MMP2, and integrin αV were decreased, while the expression of E-cadherin was increased (all P<0.05).
Conclusions: The lathyrol derivatives synthesized in this study possess good inhibitory activity against NSCLC. Among them, C-5 benzoylated lathyrol significantly inhibits the proliferation, migration, and invasion ability of NSCLC cells in vitro through regulating the process of epithelial-mesenchymal transition.
目的: 非小细胞肺癌占所有肺癌的85%，具有较高的发病率和病死率。非小细胞肺癌的治疗研究和新药开发迫在眉睫。本研究主要探讨千金子二萜醇衍生物的活性及其抑制非小细胞肺癌细胞生长的作用机制。方法: 以千金子二萜醇为原料合成3个千金子二萜醇衍生物，采用磁共振验证结构；采用MTT法检测上述衍生物对非小细胞肺癌细胞(A549和H1299细胞)增殖活力的影响，筛选出活性最佳的化合物进行后续实验。集落形成、划痕、Transwell实验分别用于检测A549和H1299细胞的体外增殖、迁移和侵袭能力，实时荧光定量PCR(real-time RT-PCR)和蛋白质印迹法用于检测A549细胞中E-cadherin、N-cadherin、β-catenin、MMP2等mRNA和蛋白质的表达水平。结果: 3个千金子二萜醇衍生物均呈剂量依赖性地抑制A549和H1299细胞的生长，且对正常细胞Beas-2B和16HBE细胞抑制作用较弱，具有一定的选择性杀伤作用。其中C-5苯甲酰化千金子二萜醇的活性最佳，作为后续实验的药物。与对照组相比，处理组的A549和H1299细胞克隆团明显变小且数目明显减少，相对迁移率明显降低，侵袭细胞数目明显减少(均P<0.05)。在mRNA水平上，integrin α2、integrin β1、MMP2、MMP9、β-catenin、N-cadherin表达均降低，E-cadherin表达增高(均P<0.05)；在蛋白质水平上，N-cadherin、β-catenin、MMP2和integrin αV表达均降低，E-cadherin表达增高(均P<0.05)。结论: 本研究合成的千金子二萜醇衍生物抑制非小细胞肺癌的活性较好，其中C-5苯甲酰化千金子二萜醇可能通过调控上皮间质转化过程显著抑制非小细胞肺癌细胞的体外增殖、迁移和侵袭能力。.
Keywords: cell proliferation; epithelial-mesenchymal transition; invasion; lathyrol derivatives; migration; non-small cell lung cancer.