PO.ET02.10 · 实验与分子治疗
奈必洛尔抑制细胞生长、抑制自噬流，并在前列腺癌细胞中与比卡鲁胺联合增强生长抑制作用
Nebivolol inhibits cell growth, inhibits autophagic flux, and enhances growth inhibition in combination with bicalutamide in prostate cancer cells
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摘要 Abstract
中文摘要
引言：奈必洛尔是一种beta1-肾上腺素受体阻滞剂，正被探索作为乳腺癌的重新利用疗法，显示出对细胞生长和自噬流的抑制作用。本研究评估了奈必洛尔在前列腺癌（PCa）细胞中的抗肿瘤作用，并确定其是否增强雄激素受体拮抗剂比卡鲁胺的作用。
方法：将PCa细胞系LNCap和22Rv1用赋形剂或奈必洛尔（0.01-30 μM）处理7天，使用EnSight™多模式酶标仪评估细胞生长。用赋形剂或奈必洛尔（0.1-10 μM）处理14-15天后测量克隆形成生长。在用赋形剂、奈必洛尔（1或10 μM）或氯喹（50 μM）处理24小时后，使用Hoechst 33342、Cyto-ID® Green和Lyso-ID® Red染色并随后进行共聚焦成像，评估自噬和溶酶体形成。使用浓度反应曲线在7天内进行奈必洛尔与比卡鲁胺（0-30 μM）的联合研究，以评估生长抑制和相互作用效应。通过非线性回归曲线分析计算IC50值。在用赋形剂或奈必洛尔（10 μM）处理22Rv1细胞24、48和72小时后，通过免疫印迹测量LC3B蛋白表达。酌情使用配对student's t检验或单因素方差分析（one-way ANOVA）及随后的Dunnett's多重比较检验，使用GraphPad Prism v.10确定统计学显著性。使用CompuSyn软件采用Chou-Talalay法检验奈必洛尔与比卡鲁胺的药物协同作用。
结果：奈必洛尔以浓度依赖的方式抑制细胞生长（IC50：LNCap中37.1 μM；22Rv1中9.8 μM）并抑制克隆形成潜能（IC50：LNCap中1.1 μM；22Rv1中2.5 μM）。与赋形剂相比，奈必洛尔（10 μM）显著增加了自噬体和溶酶体的积累（p<0.05）。奈必洛尔以时间依赖的方式显著上调了22Rv1细胞中的LC3B蛋白表达（p<0.05），提示自噬流受到抑制。Chou-Talalay分析表明，1至30 μM比卡鲁胺与10至30 μM奈必洛尔联合时存在潜在协同作用，联合指数<1。
结论：奈必洛尔抑制了PCa细胞生长和克隆形成潜能，并抑制了自噬流同时增加LC3B表达。此外，奈必洛尔与比卡鲁胺联合时增强了生长抑制作用。这些发现支持进一步研究奈必洛尔（联合或不联合比卡鲁胺）在PCa中的应用。
查看英文原文 English abstract
Introduction: Nebivolol, a beta1-adrenergic receptor blocker, is being explored as a repurposed therapeutic for breast cancer, showing inhibition of cell growth and autophagic flux. This study evaluated nebivolol's anti-tumor effects in prostate cancer (PCa) cells and determined whether it enhances the effect of the androgen receptor antagonist bicalutamide.
Methods: PCa cell lines, LNCap and 22Rv1, were treated with vehicle or nebivolol (0.01-30μM) for 7 days to assess cell growth using the EnSight™ Multimode Plate Reader. Clonogenic growth was measured after 14-15 days of vehicle or nebivolol (0.1-10μM). Autophagy and lysosome formation were evaluated after 24-hour treatment with vehicle, nebivolol (1 or 10μM), or chloroquine (50μM) using Hoechst 33342, Cyto-ID® Green, and Lyso-ID® Red staining followed by confocal imaging. Combination studies with nebivolol and bicalutamide (0-30μM) were performed over 7-day using concentration-response curves to access growth inhibition and interaction effects. IC 50 values were calculated by non-linear regression curve analysis. LC3B protein expression was measured in 22Rv1 cells by immunoblotting following vehicle or nebivolol (10μM) treatment for 24, 48, and 72 hours. Statistical significance was determined using paired student's t-test or one-way ANOVA followed by Dunnett's multiple comparison test, as appropriate, using GraphPad Prism v.10. Drug synergism of nebivolol and bicalutamide was tested by CompuSyn software using the Chou-Talalay method.
Results: Nebivolol inhibited cell growth (IC 50 : 37.1μM in LNCap; 9.8μM in 22Rv1) and suppressed clonogenic potential (IC 50 : 1.1μM in LNCap; 2.5μM in 22Rv1) in a concentration-dependent manner. Nebivolol (10μM) significantly increased autophagosome and lysosome accumulation compared with vehicle (p<0.05). Nebivolol significantly upregulated LC3B protein expression in 22Rv1 cells in a time-dependent manner (p<0.05), suggesting inhibition of autophagic flux. Chou-Talalay analysis indicated a potential synergism at 1 to 30μM bicalutamide in combination with nebivolol 10 to 30μM, with combination index<1.
Conclusion: Nebivolol inhibited PCa cell growth and clonogenic potential, and inhibited autophagic flux with increased LC3B expression. Furthermore, nebivolol enhanced growth inhibition when combined with bicalutamide. These findings support further investigation of nebivolol, with/without bicalutamide, in PCa.
利益披露 Disclosure
S. Mandal, None..
C. Wu, None..
S. S. Kanna, None..
W. Cao, None..
M. V. Trivedi, None.