摘要
背景:结直肠癌是全球癌症死亡的主要原因之一,因此迫切需要开发用于治疗结直肠癌的新药。地高辛用于治疗心力衰竭和房性心律不齐已有多年历史。零星的报道提示,地高辛可能对结直肠癌具有抗肿瘤功效。本研究旨在研究地高辛对人结直肠肿瘤细胞的抑瘤作用及其潜在机制。方法:采用MTT法和平板克隆形成实验检测地高辛对HCT8和SW620细胞增殖的影响;采用流式细胞术分析地高辛对HCT8和SW620细胞周期分布和凋亡的影响;采用划痕实验和transwell实验评估地高辛对肿瘤细胞转移的抑制作用;采用MTT法、划痕实验和小管形成实验评估地高辛对人脐静脉内皮细胞(HUVEC)血管生成的抑制作用。采用蛋白免疫印迹法、酶联免疫吸附实验和明胶酶谱实验进行机制研究。结果:地高辛能有效抑制HCT8和SW620结直肠肿瘤细胞的增殖,并将这两种细胞株的细胞周期分别阻滞于G1期和G2/M期。地高辛处理后,HCT8和SW620细胞中未见明显凋亡。地高辛可抑制HCT8细胞的迁移和侵袭,并降低HCT8细胞中基质金属蛋白酶2(MMP2)、MMP9、磷酸化的整合素β1的表达。地高辛能有效抑制HUVEC的增殖、迁移和小管形成,降低HCT8细胞中缺氧诱导因子1α(HIF1α)的表达和血管内皮生长因子A(VEGF-A)的分泌,提示其具有抑制血管生成的作用。此外,地高辛作用于SW620/Ad300细胞还能显著逆转ABCB1介导的多药耐药。结论:研究结果提示,地高辛可抑制人结直肠肿瘤细胞的增殖和转移,并逆转ABCB1介导的多药耐药,有望成为一种抗肿瘤药物。
Background Colorectal cancer(CRC)is one of the leading causes of cancer death worldwide.Novel drugs for CRC therapy are urgently needed.Digoxin has been in clinical use for treatment of heart failure and atrial arrhythmias for many years.Fragmentary reports suggested that digoxin might have antitumor efficacy on CRC.Here,we aimed to investigate the antitumor effect of digoxin on human CRC cells and the underlying mechanism.Methods Cell viability was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay and plate colony formation assay.The effects of digoxin on cell-cycle distribution and apoptosis were analysed by flow cytometry.The anti-metastatic effect on tumor cells was determined by wound-healing assay and transwell assay.Anti-angiogenic effect was examined by determining the inhibition against proliferation,migration,and tube formation of human umbilical vein endothelial cells(HUVECs).Mechanism study was performed by Western blot,enzyme-linked immunosorbent assay(ELISA),and gelatin-zymography assay.Results Digoxin potently inhibited cell proliferation,induced G1-phase and G2/M-phase arrest in colorectal-cancer HCT8 and SW620 cells,respectively.No obvious apoptosis was observed in the treated cells.Anti-metastatic activities were shown on HCT8 cells by inhibiting the migration and invasion.Meanwhile,the expression of MMP2,MMP9,and phosphorylated Integrinb1 were decreased.Digoxin inhibited the proliferation,migration,and tube formation of HUVECs and reduced HIF1a expression and vascular endothelial growth factor A(VEGF-A)secretion in HCT8 cells,suggesting anti-angiogenic activity.Furthermore,digoxin significantly reversed ABCB1-mediated multidrug resistance on SW620/Ad300 cells.Conclusion Our findings suggest that digoxin has the potential to be applied as an antitumor drug via inhibiting proliferation and metastasis as well as reversing the ABCB1-mediated multidrug resistance of colorectal cancer.
基金
supported by the Science&Technology Development Fund of Tianjin Education Commission for Higher Education[2017KJ230].
