Background

ARN-509 (Apalutamide) is a unique androgen receptor (AR) antagonist for the treatment of castration-resistant (CR) prostate cancer (PC). It inhibits AR nuclear translocation, DNA binding and transcription of AR gene targets. As dysregulation of autophagy has been detected in PC, the targeting of autophagy is a potential approach to overcome early therapeutic resistance. Therefore, we investigated the characteristics of autophagic response to ARN-509 treatment and evaluated the potential effect of a combination with autophagy inhibition.

Methods

Human prostate cancer cells (LNCaP) were cultivated in a steroid-free medium. Cells were treated with ARN-509 (50 µM) alone or in combination with the autophagy inhibitors 3-methyladenine (3MA, 5 mM) or chloroquine (Chl, 20 µM) or with ATG5 siRNA knock-down. Cell viability and apoptosis were measured by flow cytometry and fluorescence microscopy. Autophagy was monitored by immunohistochemistry, AUTOdot and immunoblotting (WES).

Results

Treatment with ARN-509 led to cell death of up to 37% with 50 µM and 60% with 100 µM by day 7. The combination of 50 µM ARN-509 with autophagy inhibitors produced a further increase in cell death by day 7. Immunostaining results showed that ARN-509 induced autophagy in LNCaP cells as evidenced by elevated levels of ATG5, Beclin 1 and LC3 punctuation and by an increase in the LC3-II band detected by WES. Autophagic flux was restored by the treatment of cells with Chl, intensifying the LC3-II band. These findings were further supported by an enhanced autophagosome punctuation observed by Autodot staining.

Conclusions

These data demonstrate that treatment with ARN-509 leads to increased autophagy levels in LNCaP cells. Furthermore, in combination with autophagy inhibitors, ARN-509 provided a significantly elevated antitumor effect, thus providing a new therapeutic approach potentially translatable to patients.

中文翻译：

---

阿帕鲁胺与自噬抑制剂组合可改善对前列腺癌细胞的治疗效果。

背景

ARN-509（Apalutamide）是一种独特的雄激素受体（AR）拮抗剂，用于治疗去势抵抗性（CR）前列腺癌（PC）。它抑制AR核易位，DNA结合和AR基因靶标的转录。由于已在PC中检测到自噬失调，因此靶向自噬是克服早期治疗耐药性的一种潜在方法。因此，我们研究了对ARN-509治疗的自噬反应的特征，并评估了结合自噬抑制的潜在作用。

方法

在不含类固醇的培养基中培养人前列腺癌细胞（LNCaP）。分别用ARN-509（50 µM）或与自噬抑制剂3-甲基腺嘌呤（3MA，5 mM）或氯喹（Chl，20 µM）或ATG5 siRNA组合处理细胞。通过流式细胞仪和荧光显微镜法测量细胞活力和凋亡。通过免疫组织化学，AUTOdot和免疫印迹（WES）监测自噬。

结果

到第7天，使用ARN-509进行处理可导致细胞死亡（分别为50 µM和37％的细胞分别达到37％和50％）。50 µM ARN-509与自噬抑制剂的组合在第7天的细胞死亡进一步增加。结果显示ARN-509诱导LNCaP细胞自噬，这可通过ATG5，Beclin 1和LC3标点的升高以及WES检测到的LC3-II条带的增加来证明。通过用Chl处理细胞恢复自噬通量，从而增强了LC3-II谱带。通过Autodot染色观察到的增强的自噬标点进一步支持了这些发现。

结论

这些数据表明，用ARN-509处理会导致LNCaP细胞中的自噬水平升高。此外，与自噬抑制剂联合使用时，ARN-509具有显着提高的抗肿瘤作用，从而提供了可能对患者具有潜在意义的新治疗方法。