异甘草素通过阻断 p38 MAPK 调控的自噬抑制胰腺癌进展。

Isoliquiritigenin inhibits pancreatic cancer progression through blockade of p38 MAPK-regulated autophagy.

机构信息

Teaching and Research Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China; Department of Biology, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong SAR, China; Golden Meditech Centre for NeuroRegeneration Sciences, Hong Kong Baptist University, Hong Kong SAR, China.

出版信息

Phytomedicine. 2022 Nov;106:154406. doi: 10.1016/j.phymed.2022.154406. Epub 2022 Aug 20.

DOI:10.1016/j.phymed.2022.154406

PMID:36029643

Abstract

BACKGROUND

Pancreatic cancer has been characterized by poor prognosis, early metastasis and dissatisfactory treatment outcome. The high basal level of autophagy in tumor cells leads to chemoresistance and tumor progression. Thus, it is imminent to explore novel effective chemotherapeutic adjuvants to increase patients' survival rate. Isoliquiritigenin (ISL) is a bioactive flavonoid obtained from the Traditional Chinese herbal medicine Glycyrrhiza glabra, and it possesses a broad range of pharmacological effects. In this study, the anti-cancer effect of ISL in pancreatic cancer treatment and the underlying mechanism are investigated.

METHODS

MTT assay, colony formation and EdU analysis were performed to explore the growth inhibition of ISL on pancreatic cancer cells. Apoptosis were analyzed using TUNEL and flow cytometry. The formations of autophagosomes were analyzed by immunofluorescence microscopy and transmission electron microscopy. RFP-GFP-LC3B probe was applied to detect the autophagy flux. To assess the structural interaction of ISL with p38 protein, molecular docking assays were performed. The molecular mechanism was elucidated by using western immunoblotting. Subsequently, the inhibition of ISL on tumor growth was determined in vivo using pancreatic tumor mice model.

RESULTS

ISL inhibited pancreatic cancer cell growth and induced apoptosis, both in vitro and in vivo. ISL caused accumulation of autophagosome through blockade of late stage autophagic flux. Moreover, autophagy inducer rapamycin enhanced ISL-evoked cell growth inhibition and promoted apoptosis, while inhibition of autophagosome formation by siAtg5 attenuated ISL-induced apoptosis. It is remarkable that ISL synergistically sensitized the cytotoxic effect of gemcitabine and 5-fluorouracil on pancreatic cancer cells as both drugs induced autophagy. Molecular docking analysis has indicated that ISL acted by direct targeting of p38 MAPK, which was confirmed by ISL-induced phosphorylation of p38. The autophagy flux induced by p38 inhibitor SB203580 was blocked by ISL, with further increasing toxicity of ISL in pancreatic cancer cells.

CONCLUSION

The results have revealed that ISL inhibited pancreatic cancer progression by blockade of autophagy through p38 MAPK signaling.

摘要

背景

胰腺癌的预后较差，早期转移，治疗效果不理想。肿瘤细胞中自噬的基础水平较高导致化疗耐药和肿瘤进展。因此，探索新的有效的化疗辅助药物以提高患者的生存率迫在眉睫。甘草素（ISL）是一种从传统中药甘草中提取的生物活性类黄酮，具有广泛的药理作用。本研究探讨了 ISL 对胰腺癌治疗的抗癌作用及其潜在机制。

方法

采用 MTT 法、集落形成实验和 EdU 分析研究 ISL 对胰腺癌细胞生长的抑制作用。用 TUNEL 和流式细胞术分析细胞凋亡。用免疫荧光显微镜和透射电子显微镜分析自噬体的形成。用 RFP-GFP-LC3B 探针检测自噬流。通过分子对接实验研究 ISL 与 p38 蛋白的结构相互作用。用 Western 免疫印迹法阐明其分子机制。随后，用胰腺肿瘤小鼠模型评估 ISL 对肿瘤生长的抑制作用。

结果

ISL 抑制胰腺癌细胞的生长并诱导细胞凋亡，无论是在体外还是体内。ISL 通过阻断晚期自噬流导致自噬体的积累。此外，自噬诱导剂雷帕霉素增强了 ISL 引起的细胞生长抑制和促进凋亡，而 siAtg5 抑制自噬体形成则减弱了 ISL 诱导的凋亡。值得注意的是，ISL 与吉西他滨和 5-氟尿嘧啶协同增强了对胰腺癌细胞的细胞毒性作用，因为这两种药物均诱导了自噬。分子对接分析表明，ISL 通过直接靶向 p38 MAPK 发挥作用，这一作用被 ISL 诱导的 p38 磷酸化所证实。p38 抑制剂 SB203580 诱导的自噬流被 ISL 阻断，进一步增加了 ISL 在胰腺癌细胞中的毒性。