背景:胆囊癌(GBC)对人类健康构成重大风险。它的发展受到许多因素的影响,特别是细胞内活性氧(ROS)的稳态。这种稳态对肿瘤细胞的存活至关重要,ROS的异常调控与许多癌症的发生和进展有关。二氢丹参酮I(DHTI),从丹参中分离出的生物有效成分,通过诱导凋亡对各种肿瘤细胞表现出细胞毒性。然而,dhtI发挥细胞毒性作用的确切分子机制尚不清楚.
目的:探讨dhtⅠ对GBC的抗肿瘤作用及其可能的分子机制。
方法:GBC细胞的增殖,NOZ和SGC-996,使用各种测定法进行评估,包括CCK-8测定,集落形成测定和EdU染色。我们还检查了细胞凋亡,细胞周期进程,ROS水平,以及线粒体膜电位的改变,以深入研究复杂的分子机制。定量PCR(qPCR),免疫荧光染色,进行蛋白质印迹以评估mRNA和蛋白质水平的靶基因表达。使用免疫共沉淀法检查了核因子红细胞2相关因子2(Nrf2)和海带样ECH相关蛋白1(Keap1)之间的相关性。最后,使用小鼠胆囊癌异种移植模型研究了dhtI的体内作用。
结果:我们的研究结果表明,dhtI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,诱导氧化应激和细胞凋亡。我们的体内研究证实了dhtI对异种移植裸鼠肿瘤生长的抑制作用。机械上,dhtI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降低。此外,Nrf2过表达有效抵消了dhtI的抗肿瘤作用,而Nrf2敲除显著增强了dhtⅠ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dhtI治疗的敏感性。相反,Nrf2活化剂,蛋白酶体抑制剂,抗氧化剂和PKC激活剂均拮抗dhtI诱导的NOZ和SGC-996细胞凋亡和ROS生成。
结论:我们的发现表明,dhtI通过调节Keap1-Nrf2信号通路和Nrf2磷酸化来抑制GBC细胞的生长。这些见解为进一步研究dhtI作为GBC治疗的潜在治疗剂提供了强有力的理由。
BACKGROUND: 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.
OBJECTIVE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.