Hwangbo Hyun, Kim So Young, Lee Hyesook, Park Shin-Hyung, Hong Su Hyun, Park Cheol, Kim Gi-Young, Leem Sun-Hee, Hyun Jin Won, Cheong Jaehun, Choi Yung Hyun
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 46241, Republic of Korea.
Anti-Aging Research Center, Dong-eui University, Busan 47340, Republic of Korea.
Biomol Ther (Seoul). 2020 Sep 1;28(5):443-455. doi: 10.4062/biomolther.2020.122.
The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.
硫氧还蛋白（Trx）系统在调节细胞内氧化还原水平以及保护生物体免受氧化应激方面发挥着关键作用。最近的研究表明，硫氧还蛋白还原酶（TrxR）在各种类型的人类癌细胞中过表达，这表明Trx-TrxR系统可能是抗癌药物开发的一个潜在靶点。本研究使用Hep3B细胞研究了金诺芬（一种TrxR特异性抑制剂）对萝卜硫素介导的凋亡性细胞死亡的协同作用。结果表明，与单独治疗相比，萝卜硫素通过抑制TrxR活性和细胞增殖显著增强了金诺芬诱导的细胞凋亡。金诺芬和萝卜硫素对细胞凋亡的协同作用通过膜联蛋白V阳性细胞和亚G1期细胞的增加得到证实。联合治疗诱导的细胞凋亡导致线粒体膜电位（ΔΨm）丧失和Bax上调。此外，联合治疗中半胱天冬酶（-3、-8和-9）的蛋白水解活性以及活化的半胱天冬酶-3的底物蛋白聚（ADP-核糖）聚合酶的降解也更高。此外，联合治疗诱导了活性氧（ROS）的过量产生。然而，用ROS清除剂N-乙酰-L-半胱氨酸处理可减少联合治疗诱导的ROS产生和细胞凋亡。因此，这些结果推断ROS在金诺芬和萝卜硫素诱导的细胞凋亡中起关键作用。此外，通过抑制磷酸肌醇3-激酶（PI3K）/Akt途径，金诺芬和萝卜硫素诱导的细胞凋亡显著增加。综上所述，本研究表明TrxR活性的下调通过ROS产生和PI3K/Akt信号通路的抑制促成了金诺芬和萝卜硫素对细胞凋亡的协同作用。
