大车前苷介导wnt信号通路对胶质瘤细胞U-251MG增殖、侵袭及裸鼠成瘤的影响

Plantamajoside Inhibits the Proliferation and Invasion of Glioma Cell Line U-251 MG and Tumor Formation of Nude Mice by Regulating Wnt Signaling Pathway

目的探讨大车前苷(Plantamajoside, PMS)介导wnt信号通路对胶质瘤细胞U-251MG增殖,侵袭及裸鼠成瘤的影响。方法将正常培养U-251MG细胞分为对照组和PMS干预组,PMS干预组釆用PMS (50、100和200 μg/ml)处理48 h后。采用克隆形成实验测定细胞增殖情况;流式细胞仪检测各组细胞凋亡情况;Transwell检测各组细胞侵袭能力;Western印迹检测各组增殖相关蛋白Ki67、PCNA、cleavedCaspase-3、cleaved Caspase-9,侵袭相关蛋白 E-cadherin、Vimentin > VEGF, wnt 信号通路蛋白 B-catenin、TCF4、c-Myc表达。皮下注射胶质瘤细胞U-251MG建立裸鼠模型,PMS组采用高中低剂量PMS腹腔注射治疗,检测肿瘤重量,免疫组化检测Ki67、Caspase-3和VEGF的表达,Western印迹检测β-catenin、TCF4和c-Myc的表达。结果与对照组相比,不同剂量PMS干预组U-251MG细胞克隆数明显减少,凋亡率显著增加(P<0.05),侵袭率显著下降(P<0.01),蛋白Ki-67, PCNA、VEGF和Vimentin的表达显著下调(P<0. 01), cleaved Caspase-3 和 cleaved Caspase-9 的表达显著上调(P<0. 05), E-cadherin 表达显著上调(P<0. 01),β-catenin、TCF4和c-Myc蛋白表达明显下调;PMS高、中、低剂量组裸鼠肿瘤质量显著下降(P<0.01),肿瘤组织中 Caspase-3 的表达显著上调(P<0.01), Ki67、VEGF、β-catenin、TCF4 和 c-Myc 的表达显著下调(P<0.01)。结论PMS可能通过抑制wnt信号通路,抑制U-251MG细胞的增殖和侵袭,抑制裸鼠皮下U-251MG移植瘤的生长。

Objective To explore the effects of plantamajoside ( PMS) on proliferation and invasion of glioma cell line U-251 MG and tumor formation of nude mice and the underlying mechanism. Methods U-251 MG cells were cultured and divided into control group and PMS intervention group. Cells in PMS intervention group were treated with PMS (50, 100 and 200 μg/mL) for 48 h. The cell proliferation was determined by colony formation assay. The cell apoptosis was detected in each group by flow cytometry. The invasive ability was detected in each group by Trans well. Western Blot was used to detect the expression levels of proliferation-related proteins Ki67 , PCNA, cleaved Caspase-3 and cleaved Caspase-9 , invasion-related proteins E-cadherin, Vimentin and VEGF and wnt signaling pathway proteins β-catenin, TCF4 and c-Myc in each group. The nude mouse models were established by subcutaneous injection of glioma cell line U-251 MG, and those in PMS group were given intraperitoneal injection of high, medium or low dose of PMS. Tumor mass was measured ,and the expression levels of Ki67 , Caspase-3 and VEGF were detected by immunohistochemistry. The expression levels of P-catenin, TCF4 and c-Myc were detected by Western blotting. Results Compared with control group, the number of clones of U-251 MG cells was significantly decreased, the apoptosis rate was significantly increased (P < 0. 05 ), the invasion rate was significantly decreased ( P < 0. 01 ), the protein expression levels of Ki-67 , PCNA, VEGF and Vimentin were significantly downregulated ( P < 0. 01), the expression of cleaved Caspase-3 and cleaved Caspase-9 was significantly up-regulated ( P < 0. 05 ), the expression of E-cadherin was significantly up-regulated (P <0.01 ), the protein expression levels of P-catenin, TCF4 and c- Myc were significantly down-regulated in different doses of PMS intervention groups. The tumor mass in high, medium and low dose of PMS groups was significantly decreased ( P <0. 01), the expression level of Caspase-3 in tumor tissues was significantly up-regulated (P < 0. 01 ) and the expression levels of Ki67 , VEGF,β-catenin, TCF4 and c-Myc were significantly down-regulated ( P < 0. 01 ). Conclusion PMS may inhibit the proliferation and invasion of U-251 MG cells, and the subcutaneous growth of U-251 MG tumor in nude mice by inhibiting wnt signaling pathway.