Wang H Y, Li D Z, Su J, You X J, Gao J
Laboratory Center of Henan Provincial Hospital of Traditional Chinese Medicine (The Second Affiliated Hospital of Henan University of Chinese Medicine), Zhengzhou 450011, China.
Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Yi Xue Za Zhi. 2025 Jul 8;105(25):2103-2111. doi: 10.3760/cma.j.cn112137-20241018-02359.
To analyze the effect and mechanisms of tigecycline on the proliferation of liver cancer cells in mouse model. Human hepatocellular carcinoma G2 (HepG2), human hepatocellular carcinoma 7 (Huh7), human hepatocellular carcinoma 3B (Hep3B) and Mahler's human hepatocellular carcinoma 97 high metastatic (MHCC97H) were divided into 6 groups respectively: group T0 (without tigecycline), group T1 (1.25 μmol/L tigecycline), group T2 (2.50 μmol/L tigecycline), group T3 (5.00 μmol/L tigecycline), group T4 (10.00 μmol/L tigecycline), and group T5 (20.00 μmol/L tigecycline). The proliferation capabilities of each group were compared after 48 hours of treatment (cell survival rate, the experiment was repeated 3 times). Based on the results of the effect of tigecycline on the proliferation ability of liver tumor cells, the mRNA and protein levels of ferroptosis-related genes [Acyl-CoA synthetase long chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), and solute vector family member 11 (SLC7A11) ] in HepG2 and Hep3B cells in groups T0, T2 and T3 were detected (the experiment was repeated 3 times). Based on the results of the effects of tigecycline on the genes and protein levels of ACSL4, GPX4, and SLC7A11 in HepG2 and Hep3B, HepG2 and Hep3B cells were divided into 4 groups respectively: group T0, group T3, group T3F [5.00 μmol/L tigecycline combined with 10.00 μmol/L Ferrostatin-1 (Fer-1)] and group T3P [5.00 μmol/L tigecycline combined with 10.00 μmol/L ACSL4 specific inhibitor (PRGL493)], to compare the ACSL4 protein levels and cell proliferation abilities among different groups (cell survival rate, the experiment was repeated 3 times). HepG2 or Hep3B cell suspensions were subcutaneously injected into BALB/c nude mice to construct the subcutaneous tumor-bearing model of liver tumors in nude mice. On the 6th day, the mice were randomly divided into 3 groups (5 mice in each group for the HepG2 and Hep3B cell liver tumor nude mouse models): group N (no treatment), group TIG (treated with 100 mg/kg tigecycline), and group TP (treated with 100 mg/kg tigecycline combined with 0.5 mg/kg PRGL493). The effects of tigecycline on the proliferation of liver tumor cells in vivo and the ACSL4 protein in tumor tissues were detected, and the mechanism by which tigecycline inhibits the proliferation of liver tumor cells was analyzed. The survival rates of HepG2, Huh7, Hep3B, and MHCC97H cells in T1, T2, T3, T4, and T5 groups were all lower than that of T0 group (all <0.05); in both HepG2 and Hep3B cells, there was no statistically significant difference in the mRNA levels of ACSL4, GPX4, and SLC7A11, or in the protein levels of GPX4 and SLC7A11 between the T3 and T0 groups (all >0.05). However, the ACSL4 protein levels of HepG2 and Hep3B cells in T3 group were all higher than that of T0 group (all <0.05). The ACSL4 protein levels of HepG2 and Hep3B cells in T3F and T3P groups were lower than that of T3 group (all <0.05), and the cell survival rates of HepG2 and Hep3B cells in T3F and T3P groups were higher than that of T3 group(all <0.05). The tumor volumes of nude mouse subcutaneous tumor models in TIG group on the 21st day were lower than that of N group(all <0.05). The tumor volumes of nude mouse subcutaneous tumor models in TP group were higher than that of TIG group (all <0.05). Tigecycline inhibits the proliferation of liver cancer cells, and its mechanism may be related to promoting ACSL4-mediated ferroptosis.
分析替加环素对小鼠模型中肝癌细胞增殖的影响及其机制。将人肝癌G2（HepG2）、人肝癌7（Huh7）、人肝癌3B（Hep3B）和马勒人肝癌97高转移细胞（MHCC97H）分别分为6组：T0组（不加替加环素）、T1组（1.25 μmol/L替加环素）、T2组（2.50 μmol/L替加环素）、T3组（5.00 μmol/L替加环素）、T4组（10.00 μmol/L替加环素）和T5组（20.00 μmol/L替加环素）。处理48小时后比较各组的增殖能力（细胞存活率，实验重复3次）。根据替加环素对肝肿瘤细胞增殖能力影响的结果，检测T0、T2和T3组中HepG2和Hep3B细胞中铁死亡相关基因[酰基辅酶A合成酶长链家族成员4（ACSL4）、谷胱甘肽过氧化物酶4（GPX4）和溶质载体家族成员11（SLC7A11）]的mRNA和蛋白水平（实验重复3次）。根据替加环素对HepG2和Hep3B细胞中ACSL4、GPX4和SLC7A11基因及蛋白水平影响的结果，将HepG2和Hep3B细胞分别分为4组：T0组、T3组、T3F组[5.00 μmol/L替加环素联合10.00 μmol/L铁死亡抑制剂-1（Fer-1）]和T3P组[5.00 μmol/L替加环素联合10.00 μmol/L ACSL4特异性抑制剂（PRGL493）]，比较不同组间的ACSL4蛋白水平和细胞增殖能力（细胞存活率，实验重复3次）。将HepG2或Hep3B细胞悬液皮下注射到BALB/c裸鼠体内，构建裸鼠肝肿瘤皮下荷瘤模型。第6天，将小鼠随机分为3组（HepG2和Hep3B细胞肝肿瘤裸鼠模型每组5只）：N组（不治疗）、TIG组（用100 mg/kg替加环素治疗）和TP组（用100 mg/kg替加环素联合0.5 mg/kg PRGL493治疗）。检测替加环素对体内肝肿瘤细胞增殖及肿瘤组织中ACSL4蛋白的影响，分析替加环素抑制肝肿瘤细胞增殖的机制。T1、T2、T3、T4和T5组中HepG2、Huh7、Hep3B和MHCC97H细胞的存活率均低于T0组（均<0.05）；在HepG2和Hep3B细胞中，T3组与T0组相比，ACSL4、GPX4和SLC7A1 mRNA水平以及GPX4和SLC7A11蛋白水平均无统计学差异（均>0.05）。然而，T3组中HepG2和Hep3B细胞的ACSL4蛋白水平均高于T0组（均<0.05）。T3F组和T3P组中HepG2和Hep3B细胞的ACSL4蛋白水平低于T3组（均<0.05），T3F组和T3P组中HepG2和Hep3B细胞的细胞存活率高于T3组（均<0.05）。TIG组裸鼠皮下肿瘤模型在第21天的肿瘤体积低于N组（均<0.05）。TP组裸鼠皮下肿瘤模型的肿瘤体积高于TIG组（均<0.05）。替加环素抑制肝癌细胞增殖，其机制可能与促进ACSL4介导的铁死亡有关。
