Almasoud Hadeel, Alzahrani Fares A, Alarifi Saud, Aldahmash Badr, Almutairi Bader, AlKahtane Abdullah A, Yaseen Khadijah N, Aljuhani Bashayer, Alkahtani Saad
Department of Zoology, College of Science, King Saud University, P. O. Box 2455, 11451, Riyadh, Saudi Arabia.
Saudi Pharm J. 2025 Apr 29;33(1-2):6. doi: 10.1007/s44446-025-00001-x.
Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with limited therapeutic options and poor prognosis for advanced stages. This study investigates the synergistic anticancer effects of Ramucirumab (RAM), a VEGFR-2 inhibitor, and 5-Azacytidine (5-Aza), a hypomethylating agent, on HCC cells, focusing on mechanisms of cytotoxicity, DNA damage, apoptosis, and cell cycle modulation. HuH-7 cells were treated with RAM and 5-Aza, alone and in combination, across varying concentrations. Cell viability was assessed using the Neutral Red Uptake assay, while DNA damage and apoptosis were evaluated through the TUNEL assay and protein array analysis. The expression of cell cycle and inflammatory genes was analyzed using quantitative real-time PCR (qRT-PCR). Result shows combination treatment significantly enhanced cytotoxicity compared to individual dose-dependent therapies. DNA damage was markedly increased in RAM-treated cells, with upregulation of apoptotic proteins CAS3, BID, BAD, p53, and FAS observed. In contrast, apoptotic proteins were markedly decreased in combination-treated cells. Cell cycle arrest was evident through the downregulation of key regulatory genes, including MCM2, MCM3, cyclin B1, and CDK2. Inflammatory cytokines IL-1β and IL-6 were repressed, while COX2 expression was elevated, suggesting oxidative stress as a possible mechanism. In conclusion, the synergistic effects of RAM and 5-Aza in HCC cells are mediated through increased damage to DNA, apoptosis, and arrest of cell cycle, offering potential treatment strategy for advanced HCC. Further experiments conducted in vivo are warranted to validate these findings and optimize treatment regimens.
肝细胞癌（HCC）仍然是癌症相关死亡的主要原因，晚期的治疗选择有限且预后较差。本研究调查了血管内皮生长因子受体2（VEGFR-2）抑制剂雷莫西尤单抗（RAM）和去甲基化药物5-氮杂胞苷（5-Aza）对肝癌细胞的协同抗癌作用，重点关注细胞毒性、DNA损伤、凋亡和细胞周期调控机制。用不同浓度的RAM和5-Aza单独及联合处理HuH-7细胞。使用中性红摄取试验评估细胞活力，同时通过TUNEL试验和蛋白质阵列分析评估DNA损伤和凋亡。使用定量实时PCR（qRT-PCR）分析细胞周期和炎症基因的表达。结果显示，与单独的剂量依赖性疗法相比，联合治疗显著增强了细胞毒性。在接受RAM治疗的细胞中，DNA损伤明显增加，观察到凋亡蛋白CAS3、BID、BAD、p53和FAS上调。相反，联合治疗的细胞中凋亡蛋白明显减少。通过关键调控基因（包括微小染色体维持蛋白2（MCM2）、微小染色体维持蛋白3（MCM3）、细胞周期蛋白B1和细胞周期蛋白依赖性激酶2（CDK2））的下调，细胞周期停滞明显。炎症细胞因子白细胞介素-1β（IL-1β）和白细胞介素-6受到抑制，而环氧合酶2（COX2）表达升高，提示氧化应激可能是一种机制。总之，RAM和5-Aza在肝癌细胞中的协同作用是通过增加DNA损伤、凋亡和细胞周期停滞来介导的，为晚期肝癌提供了潜在的治疗策略。有必要进行进一步的体内实验来验证这些发现并优化治疗方案。
