一种新型喜树碱衍生物 3j 通过拓扑异构酶 I 介导的 DNA 损伤诱导细胞周期阻滞抑制 NSCLC 增殖。
A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage.
机构信息
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu, China.
Department of Gynaecology and Obstetrics, Southeast University Affiliated Zhongda Hospital, Nanjing, Jiangsu, China.
出版信息
Anticancer Agents Med Chem. 2019;19(3):365-374. doi: 10.2174/1871520619666181207102037.
OBJECTIVE
The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms.
BACKGROUND
Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression.
METHODS
Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage.
RESULTS
Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells.
CONCLUSION
Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.
目的
本研究旨在探讨喜树碱衍生物 3j 对非小细胞肺癌（NSCLC）细胞的抑制作用及其潜在的抗肿瘤机制。
背景
喜树碱类化合物被认为是世界范围内广泛研究的第三大天然药物，但由于严重的毒性，如出血性膀胱炎和骨髓抑制，其应用受到限制。
方法
采用细胞增殖试验和 S180 肿瘤小鼠模型，筛选并评价了一系列 20(S)-O-取代苯甲酰基 7-乙基喜树碱化合物的体外和体内抗肿瘤活性。选用喜树碱衍生物 3j 进一步研究，采用流式细胞术检测 NSCLC 细胞。采用 Western Blot 检测细胞周期相关蛋白 cyclin A2、CDK2、cyclin D 和 cyclin E。然后，通过计算机分子对接确认 3j 与 Topo I 的相互作用。此外，还通过 DNA 松弛实验和碱性彗星实验研究了 3j 对 DNA 损伤的作用机制。
结果
我们的研究结果表明，喜树碱衍生物 3j 在十一组 20(S)-O-取代苯甲酰基 7-乙基喜树碱化合物中具有更强的抗肿瘤作用，无论是在体外还是体内实验中。3j 的 IC50 值为 1.54±0.41 μM，低于喜树碱衍生物 irinotecan 的 IC50 值（13.86±0.80 μM），降低了 8 倍。在 NCI-H1975 细胞中，3j 的 IC50 值为 1.87±0.23 μM，低于喜树碱衍生物 irinotecan（IC50±SD，5.35±0.38 μM），降低了 1.8 倍。流式细胞术分析显示，3j 诱导的细胞积累呈剂量依赖性。用 10 μM 的 3j 处理 24 小时后，NCI-H460 细胞在 S 期的比例显著增加（达到 93.54±4.4%），与对照组（31.67±3.4%）相比。同样，用 10 μM 的 3j 处理 NCI-H1975 细胞后，S 期的比例也显著增加（达到 83.99±2.4%），与对照组（34.45±3.9%）相比。此外，cyclin A2、CDK2 水平升高，cyclin D、cyclin E 水平降低，进一步证实了细胞周期阻滞是由 3j 诱导的。此外，分子对接研究表明，3j 与 Topo I-DNA 相互作用，DNA 松弛实验同时证实，3j 抑制了 Topo I 的活性。对作用机制的研究表明，3j 通过激活 DNA 损伤反应通路和抑制 NSCLC 细胞的修复通路发挥抗肿瘤活性。
结论
新型喜树碱衍生物 3j 已被证明是一种有前途的抗肿瘤药物，有待进一步研究。
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