阿霉素通过Wnt/β-catenin信号通路抑制口腔鳞癌干细胞迁移和侵袭

Inhibitory Effects of Adriamycin on the Migration and Invasion of Oral Squamous Carcinoma Stem Cells via Wnt/β-catenin Signaling Pathway

目的:探讨阿霉素对口腔鳞癌干细胞迁移、侵袭、凋亡的影响及其可能的机制。方法:体外培养人口腔鳞癌细胞系SCC25,通过流式细胞术分选CD44^(-)和CD44^(+)细胞,RT-PCR检测CD44^(-)和CD44^(+)细胞的Oct4、CD133、CD44和GAPDH的m RNA表达;检测和比较CD44^(-)和CD44^(+)细胞的克隆形成能力。CD44+细胞用阿霉素或β-catenin抑制剂LF3进行处理,分别使用Transwell和细胞划痕检测细胞侵袭和迁移能力,一步法TUNEL检测细胞凋亡水平,WB检测β-catenin和TCF-4的蛋白表达。结果:流式细胞术成功分离CD44^(-)和CD44^(+)细胞,RT-PCR检测CD44+细胞高表达Oct4、CD133和CD44 m RNA,CD44-细胞弱表达Oct4m RNA,不表达CD133和CD44 m RNA;CD44^(+)细胞的克隆形成能力显示显著强于CD44^(-)细胞(P<0.05)。阿霉素显著降低了CD44^(+)细胞的侵袭能力和迁移能力(P<0.05),显著提高了CD44+细胞的凋亡率(P<0.05);阿霉素显著降低了CD44+细胞β-catenin和TCF-4的蛋白表达(P<0.05),LF3对β-catenin和TCF-4蛋白表达的影响与阿霉素比较无显著差异(P>0.05)。结论:阿霉素可能通过抑制Wnt/β-catenin信号通路降低口腔鳞癌干细胞迁移、侵袭能力,促进细胞凋亡。

Objective: To investigate the effects of adriamycin on the migration, invasion and apoptosis of oral squamous cell carcinoma stem cells and its related signal pathways. Methods: Human oral squamous cell carcinoma cell line SCC25 was cultured in vitro.CD44^(-)and CD44^(+)cells were sorted and compared by flow cytometry. RT-PCR was used to detect the m RNA expression levels of Oct4,CD133, CD44, and GAPDH in CD44^(-)and CD44^(+)cells. CD44^(-)and CD44^(+)cells were tested for their ability to form clones. CD44+cells were treated with adriamycin or inhibitor LF3, and Transwell and cell scratches were used to detect cell invasion and migration ability.One-step TUNEL was used to detect apoptosis. WB was used to detect β-catenin and TCF-4 protein expression levels. Results: CD44^(-)and CD44^(+)cells successfully isolated by flow cytometry. RT-PCR detected that CD44^(+)cells strongly expressed Oct4, CD133 and CD44 m RNA. CD44^(-)cells weakly expressed Oct4 m RNA, and did not express CD133 and CD44 m RNA. The results of clonal formation analysis showed that CD44^(+)cells were significantly stronger than CD44^(-)cells(P<0.05). Adriamycin significantly reduced CD44^(+) cell invasion capacity(P<0.05). Adriamycin significantly reduced CD44^(+)cell migration capacity(P<0.05). Adriamycin significantly increased CD44^(+) cell apoptosis levels(P<0.05). Adriamycin significantly reduced β-catenin and TCF-4 protein expression in CD44^(+) cells(P<0.05), and the expression of β-catenin and TCF-4 protein by LF3 culture was not significantly different with adriamycin(P>0.05). Conclusion: Adriamycin reduces the migration and invasion ability of oral squamous cell carcinoma stem cells by inhibiting the Wnt/β-catenin signaling pathway, and promotes apoptosis.