Department of Clinical Medical Sciences, International University of Health and Welfare Graduate School of Medicine, Tokyo, Japan.
Department of Gastroenterology, International University of Health and Welfare School of Medicine, Narita, Japan; Department of Gastroenterology and Hepatology, International University of Health and Welfare Hospital, Nasushiobara, Japan.
Biomed Pharmacother. 2022 Sep;153:113363. doi: 10.1016/j.biopha.2022.113363. Epub 2022 Jul 11.
The improvements of antitumor effects and tolerability on chemotherapy for advanced hepatocellular carcinoma (HCC) are warranted. Here, we aimed to elucidate the mechanism of the combining effect of tyrosine kinase inhibitor sorafenib (SOR) and iron chelator deferasirox (DFX) in human hepatoma cell lines, HepG2 and Huh-7.
The types of programmed cell deaths (PCDs); necrosis/necroptosis and apoptosis, were evaluated by flow cytometry and fluorescent microscopy. Human cleaved caspase-3 was analyzed by ELISA for apoptosis. GSH assay was used for ferroptosis. PCDs inhibition was analyzed by adding apoptosis inhibitor Z-VAD-FMK, ferroptosis inhibitor ferrostatin-1, necroptosis inhibitor necrosulfonamide, respectively. The expression of NF-κB was quantified by Western blotting.
In SOR monotherapy, cleaved caspase-3 expression was increased in all concentrations, confirming the result that SOR induces apoptosis. In SOR monotherapy, GSH/GSSG ratio was decreased on concentration-dependent, showing that SOR also induced ferroptosis. Lipid Peroxidation caused by SOR, corresponding to ferroptosis, was suppressed by DFX. In fluorescence microscopy of SOR monotherapy, apoptosis was observed at a constant rate on all concentrations, while necroptosis and ferroptosis were increased on high concentration. In sorafenib and deferasirox combinations, sub G1 phase increased additively. In SOR and DFX combinations, the cytotoxic effects were not suppressed by ferrostatin-1, but suppressed by Z-VAD-FMK and necrosulfonamide. In each monotherapy, and SOR + DFX combinations, the expression of NF-κB in nucleus was suppressed. Regarding PCD by SOR and DFX combination, ferroptosis was suppressed and both apoptosis and necroptosis became dominant.
Suppression of NF-κB is possibly involved in the effect of DFX. As a result, SOR and DFX combination showed additive antitumor effects for HCC through the mechanism of programed cell deaths and NF-kB signal modification.
提高晚期肝细胞癌（HCC）化疗的抗肿瘤效果和耐受性是必要的。在这里，我们旨在阐明酪氨酸激酶抑制剂索拉非尼（SOR）和铁螯合剂地拉罗司（DFX）联合作用在人肝癌细胞系 HepG2 和 Huh-7 中的机制。
通过流式细胞术和荧光显微镜评估程序性细胞死亡（PCD）的类型；坏死/坏死性凋亡和细胞凋亡。通过 ELISA 分析人天冬氨酸特异性半胱氨酸蛋白酶-3（cleaved caspase-3）用于细胞凋亡。使用 GSH 测定法进行铁死亡。通过分别添加凋亡抑制剂 Z-VAD-FMK、铁死亡抑制剂 Ferrostatin-1、坏死性凋亡抑制剂 Necrostatin-1 来分析 PCD 抑制。通过 Western blot 定量 NF-κB 的表达。
在 SOR 单药治疗中，所有浓度的 cleaved caspase-3 表达均增加，证实了 SOR 诱导细胞凋亡的结果。在 SOR 单药治疗中，GSH/GSSG 比值呈浓度依赖性降低，表明 SOR 也诱导铁死亡。DFX 抑制 SOR 引起的脂质过氧化，对应于铁死亡。在 SOR 单药治疗的荧光显微镜下，在所有浓度下均以恒定速率观察到凋亡，而在高浓度下观察到坏死性凋亡和铁死亡增加。在索拉非尼和地拉罗司联合治疗中，亚 G1 期呈相加性增加。在 SOR 和 DFX 联合治疗中，细胞毒性作用不受 Ferrostatin-1 抑制，但受 Z-VAD-FMK 和 Necrostatin-1 抑制。在每种单药治疗和 SOR + DFX 联合治疗中，核内 NF-κB 的表达均受到抑制。关于 SOR 和 DFX 联合治疗的 PCD，铁死亡受到抑制，凋亡和坏死性凋亡均成为主要作用方式。
NF-κB 的抑制可能参与了 DFX 的作用。结果，SOR 和 DFX 联合通过程序性细胞死亡和 NF-kB 信号修饰机制对 HCC 表现出相加的抗肿瘤作用。
