背景:索拉非尼(Sora),多靶点酪氨酸激酶抑制剂,被广泛认为是晚期肝细胞癌(HCC)的标准化学疗法。然而,耐药机制阻碍了其抗癌功效。来自Withaniasomnifera,WithaferinA(WA)作为天然生物活性化合物具有显着的抗肿瘤特性。这项研究旨在研究Sora和WA共同治疗对HCC影响的机制。
方法:通过集落形成和MTT测定评价细胞增殖。流式细胞术用于确定细胞凋亡和活性氧(ROS)水平。细胞凋亡相关蛋白水平的评价,DNA损伤,利用IHC染色和蛋白质印迹法进行内质网应激。此外,caspase抑制剂Z-VAD-FMK,ATF4siRNA,ROS清除剂N-乙酰半胱氨酸(NAC),和TrxR1shRNA用于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异种移植物进行体内实验。
结果:Sora/WA共治疗在体内和体外均显示出显著的协同抗肿瘤作用。机械上,通过抑制TrxR1活性来增强WA对Sora的抗肿瘤作用,导致ROS积累。此外,ROS产生诱导DNA损伤和内质网(ER)应激途径的激活,最终引发细胞凋亡。抗氧化剂NAC预处理显著抑制ROS的生成,ER压力,DNA损伤,和Sora/WA共同处理诱导的细胞凋亡。此外,小干扰RNA(siRNA)对ATF4的抑制减弱了Sora/WA共同治疗诱导的细胞凋亡。在体内,Sora/WA共同治疗可显着抑制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。
结论:我们的研究表明,WA协同增强了Sora的抗肿瘤作用,为不断发展的HCC治疗方法提供了有希望的意义。
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.
CONCLUSIONS: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.