Zhang Yu, Qin Shukui, Chao Jiaojiao, Luo Yan, Sun Yandi, Duan Jianxin
Department of Graduate School, Nanjing University of Chinese Medicine, Nanjing, China.
Department of Medical Oncology Center, BaYi Affiliated Hospital, Nanjing, China.
Front Oncol. 2022 Jul 22;12:885139. doi: 10.3389/fonc.2022.885139. eCollection 2022.
Primary liver cancer (PLC) is a common and highly lethal malignancy in the world. Approximately 85% of PLC is hepatocellular carcinoma (HCC), and this study mainly focuses on HCC. The onset of liver cancer is insidious and often complicated with basic liver disease. Meanwhile, its clinical symptoms are atypical, and the degree of malignancy is high. What is worse is that its treatment is difficult, and the prognosis is poor. All these factors make its mortality close to its incidence. AST-3424 is a prodrug of a potent nitrogen mustard, which targets the tumor by its specific and selective mode of activation and results in the concentration of the drug in the tumor and plays a higher intensity of antitumor effect with reduced side effects. The purpose of this study was to explore the antitumor activity and mechanism of AST-3424 monotherapy and combination therapy with oxaliplatin (OXA) or 5-fluorouracil (5-Fu). Moreover, it can provide an experimental basis for further studies.
Tumor growth of HCC cells was examined by using the Cell Counting Kit-8 (CCK-8), flow cytometry, and clone formation assays. Tumor migration of HCC cells was examined by using the Transwell assay. The antitumor activity of AST-3424 monotherapy and combination therapy with OXA and 5-Fu was quantified by growth and metastasis inhibition rate. The underlying molecular mechanism was investigated by using Western blotting.
The inhibiting effects of AST-3424 were significant in both HepG2 cells and PLC/PRF/5 cells. Moreover, HepG2 cells showed higher sensitivity to AST-3424. With increasing AST-3424 concentration, AKR1C3 protein expression level was downregulated significantly. The inhibition of AST-3424 was significantly higher than OXA, 5-Fu, Sor (sorafenib), and Apa (apatinib) in both HCC cells. AST-3424 monotherapy and combination therapy with OXA or 5-Fu all strongly inhibited the proliferation of HCC cells, blocked HCC cells in the S phase, promoted apoptosis induction, and suppressed the migration of HCC cells. Among them, the antitumor effect of AST-3424 in combination with OXA was obviously enhanced. Western blotting analysis demonstrated the regulation of P21, Bax, Caspase3, PARP, MMP-2, MMP-9, and p-Smad proteins in the presence of AST-3424 monotherapy and combination therapy with OXA or 5-Fu, indicating that its antitumor mechanisms may be associated with the regulation of the TGF-β signaling cascade.
The studies revealed that AST-3424 in combination with both OXA and 5-Fu showed an increased antitumor effect, and the combination with OXA resulted in a synergistic effect. Together with the results, additional and studies are warranted to further certify its antitumor effects and explore more potential antitumor mechanisms.
原发性肝癌（PLC）是全球常见且致死率极高的恶性肿瘤。约85%的PLC为肝细胞癌（HCC），本研究主要聚焦于HCC。肝癌起病隐匿，常伴有基础肝脏疾病。同时，其临床症状不典型，恶性程度高。更糟糕的是，其治疗困难，预后较差。所有这些因素使得其死亡率接近发病率。AST - 3424是一种强效氮芥前体药物，它通过其特异且选择性的激活方式靶向肿瘤，使药物在肿瘤中富集，并在副作用降低的情况下发挥更高强度的抗肿瘤作用。本研究的目的是探究AST - 3424单药治疗以及与奥沙利铂（OXA）或5 - 氟尿嘧啶（5 - Fu）联合治疗的抗肿瘤活性及机制。此外，可为进一步研究提供实验依据。
采用细胞计数试剂盒 - 8（CCK - 8）、流式细胞术和克隆形成实验检测HCC细胞的肿瘤生长情况。采用Transwell实验检测HCC细胞的肿瘤迁移情况。通过生长和转移抑制率量化AST - 3424单药治疗以及与OXA和5 - Fu联合治疗的抗肿瘤活性。采用蛋白质免疫印迹法研究潜在的分子机制。
AST - 3424对HepG2细胞和PLC/PRF/5细胞均有显著抑制作用。此外，HepG2细胞对AST - 3424表现出更高的敏感性。随着AST - 3424浓度增加，AKR1C3蛋白表达水平显著下调。在两种HCC细胞中，AST - 3424的抑制作用均显著高于OXA、5 - Fu、索拉非尼（Sor）和阿帕替尼（Apa）。AST - 3424单药治疗以及与OXA或5 - Fu联合治疗均强烈抑制HCC细胞增殖，将HCC细胞阻滞于S期，促进细胞凋亡诱导，并抑制HCC细胞迁移。其中，AST - 3424与OXA联合的抗肿瘤作用明显增强。蛋白质免疫印迹分析表明，在AST - 3424单药治疗以及与OXA或5 - Fu联合治疗时，P21、Bax、Caspase3、PARP、MMP - 2、MMP - 9和p - Smad蛋白受到调控，表明其抗肿瘤机制可能与TGF -β信号级联的调控有关。
研究表明，AST - 3424与OXA和5 - Fu联合均显示出增强的抗肿瘤作用，与OXA联合产生协同效应。结合这些结果，需要更多研究进一步证实其抗肿瘤作用并探索更多潜在的抗肿瘤机制。
