Department of General Surgery, The Chinese People's Armed Police Forces Anhui Provincial Corps Hospital, Hefei 230041, China; Research Technology Center, Anhui University of Chinese Medicine, Hefei 230038, China.
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China.
Phytomedicine. 2024 Jul;129:155661. doi: 10.1016/j.phymed.2024.155661. Epub 2024 Apr 21.
Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear.
To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms.
The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice.
Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells.
Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.
胆囊癌（GBC）对人类健康构成重大威胁。其发展受到许多因素的影响，特别是细胞内活性氧物质（ROS）的动态平衡。这种动态平衡对肿瘤细胞的存活至关重要，ROS 的异常调节与许多癌症的发生和发展有关。二氢丹参酮 I（DHT I）是从丹参中分离得到的一种具有生物活性的成分，它通过诱导细胞凋亡对多种肿瘤细胞表现出细胞毒性。然而，DHT I 发挥细胞毒性作用的确切分子机制尚不清楚。
探讨 DHT I 对 GBC 的抗肿瘤作用及其潜在的分子机制。
采用 CCK-8 法、集落形成实验和 EdU 染色等方法检测 GBC 细胞（NOZ 和 SGC-996）的增殖。我们还研究了细胞凋亡、细胞周期进程、ROS 水平和线粒体膜电位的变化，以深入探讨复杂的分子机制。通过定量 PCR（qPCR）、免疫荧光染色和 Western blot 检测在 mRNA 和蛋白水平上的靶基因表达。通过免疫共沉淀检测核因子红细胞 2 相关因子 2（Nrf2）和 Kelch 样 ECH 相关蛋白 1（Keap1）之间的相关性。最后，在裸鼠 GBC 移植瘤模型中研究了 DHT I 的体内作用。
我们的研究结果表明，DHT I 对 GBC 细胞具有细胞毒性作用，包括抑制增殖、破坏线粒体膜电位、诱导氧化应激和凋亡。我们的体内研究证实了 DHT I 对异种移植裸鼠肿瘤生长的抑制作用。在机制上，DHT I 主要通过促进 Keap1 介导的 Nrf2 降解和抑制蛋白激酶 C（PKC）诱导的 Nrf2 磷酸化来靶向 Nrf2。这导致 Nrf2 核易位的抑制和其靶基因表达的减少。此外，Nrf2 的过表达可有效拮抗 DHT I 的抗肿瘤作用，而 Nrf2 的敲低可显著增强 DHT I 对 GBC 的抑制作用。同时，PKC 抑制剂和核导入抑制剂增加了 GBC 细胞对 DHT I 处理的敏感性。相反，Nrf2 激活剂、蛋白酶体抑制剂、抗氧化剂和 PKC 激活剂均拮抗了 DHT I 诱导的 NOZ 和 SGC-996 细胞凋亡和 ROS 生成。
我们的研究结果表明，DHT I 通过调节 Keap1-Nrf2 信号通路和 Nrf2 磷酸化抑制 GBC 细胞的生长。这些研究结果为进一步研究 DHT I 作为 GBC 治疗的潜在治疗剂提供了有力的依据。
