铁皮石斛酚 A 是一种天然的硫氧还蛋白还原酶 1（TrxR1）抑制剂，通过 ROS 介导的内质网应激和 DNA 损伤协同增强索拉非尼在肝癌细胞中的抗肿瘤疗效。

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

机构信息

Department of Pharmacology, School of Medicine, Taizhou University, Taizhou, Jiaojiang 318000, Zhejiang, China.

Municipal Hospital Affiliated to Taizhou University, Taizhou, Jiaojiang 318000, Zhejiang, China.

出版信息

Phytomedicine. 2024 Jun;128:155317. doi: 10.1016/j.phymed.2023.155317. Epub 2023 Dec 25.

DOI:10.1016/j.phymed.2023.155317

PMID:38537439

Abstract

BACKGROUND

Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC.

METHODS

Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts.

RESULTS

Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues.

CONCLUSION

Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

摘要

背景

索拉非尼（Sora）是一种多靶点酪氨酸激酶抑制剂，被广泛认为是晚期肝细胞癌（HCC）的标准化疗药物。然而，耐药机制阻碍了其抗癌疗效。从睡茄中提取的 Withaferin A（WA）作为一种天然生物活性化合物，具有显著的抗肿瘤特性。本研究旨在探讨 Sora 和 WA 联合治疗对 HCC 的影响机制。

方法

通过集落形成和 MTT 测定评估细胞增殖。流式细胞术用于测定细胞凋亡和活性氧（ROS）水平。采用免疫组织化学染色和蛋白质印迹法评估凋亡相关蛋白水平、DNA 损伤和内质网应激。此外，使用 caspase 抑制剂 Z-VAD-FMK、ATF4 siRNA、ROS 清除剂 N-乙酰半胱氨酸（NAC）和 TrxR1 shRNA 来阐明潜在的信号通路。为了验证 Sora/WA 联合治疗的抗肿瘤作用，最终使用 Huh7 异种移植进行了体内实验。

结果

Sora/WA 联合治疗在体内和体外均显示出显著的协同抗肿瘤作用。机制上，WA 通过抑制 TrxR1 活性从而增加 Sora 的抗肿瘤作用，导致 ROS 积累。此外，ROS 的产生激活了 DNA 损伤和内质网（ER）应激途径，最终引发细胞凋亡。用抗氧化剂 NAC 预处理可显著抑制 Sora/WA 联合治疗引起的 ROS 生成、ER 应激、DNA 损伤和凋亡。此外，通过小干扰 RNA（siRNA）抑制 ATF4 可减弱 Sora/WA 联合治疗诱导的凋亡。体内，Sora/WA 联合治疗显著抑制 HCC 异种移植模型中的肿瘤生长并降低肿瘤组织中的 TrxR1 活性。