甘草次酸通过Wnt/β-catenin通路抑制黑色素瘤恶性生物学行为的机制研究

Mechanism of glycyrrhetinic acid on inhibiting malignant biological behaviors of melanoma by Wnt/β-catenin pathways

目的探讨甘草次酸通过Wnt/β-连环素(β-catenin)通路抑制黑色素瘤恶性生物学行为的可能机制。方法以黑色素瘤细胞B16-F10为研究对象,MTT法进行浓度梯度试验选择0、1、2、4μmol/L为细胞处理浓度,并以顺铂0.01 mmol/L干预为阳性对照。采用Transwell小室法检测细胞侵袭及迁移能力;免疫印迹试验(Western blot)实验检测B16-F10细胞中Wnt/β-catenin通路蛋白和侵袭迁移相关蛋白基质金属蛋白酶(MMP)-2、MMP-9表达水平。构建裸鼠移植瘤模型,实验分为对照组(无药物处理)、甘草次酸组(40 mg/kg),观察移植瘤小鼠的肿瘤组织生长情况及肿瘤组织中Wnt/β-catenin通路蛋白和侵袭迁移相关蛋白表达水平。结果MTT结果显示,甘草次酸以浓度依赖性方式抑制B16-F10细胞增殖,当甘草次酸浓度≥2μmol//L时,其对B16-F10细胞的增殖具有明显的抑制作用(P<0.05);Transwell小室实验结果显示,与对照组相比,甘草次酸2、4μmol/L浓度处理后,B16-F10细胞的侵袭、迁移能力明显下降(P<0.05);WB实验结果显示,与0μmol/L甘草次酸处理相比,甘草次酸2、4μmol//L浓度处理后B16-F10细胞中的MMP-9、MMP-2、Wnt1、β-catenin蛋白表达水平明显下降(P<0.05);荷瘤实验结果显示,与对照组相比,甘草次酸组小鼠肿瘤重量、体积明显下降,瘤体组织中的Wnt1、β-catenin、MMP-9、MMP-2蛋白及表达水平也明显下降(P<0.05)。结论甘草次酸能够显著抑制黑色素瘤的体内外恶性生物学行为,其机制可能是抑制Wnt/β-catenin信号通路激活来发挥作用。

Objective To explore the possible mechanism of glycyrrhetinic acid on inhibiting malignant biological behaviors of melanoma by Wnt/β-catenin pathways.Methods The melanoma cells B16-F10 were selected as the research objects.The concentration gradient tests(0,1,2,4μmol/L)were conducted by MTT.The cells given cisplatin intervention was enrolled as positive controls.The cells invasion and migration were detected by Transwell chamber assay.The expression levels of Wnt/β-catenin pathway proteins,invasion and migration related proteins(MMP-2,MMP-9)in B16-F10 cells were detected by Wester blot.The xenograft models of nude mice were constructed,and they were divided into control group(without drugs treatment)and glycyrrhetinic acid group(40 mg/kg).The growth of tumor tissues,and expression levels of Wnt/β-catenin pathway proteins,invasion and migration related proteins were observed.Results MTT results showed that glycyrrhetic acid could inhibit the proliferation of B16-F10 cells in a concentration-dependent manner.The inhibition effect of glycyrrhetic acid(≥2μmol/L)was significant on the proliferation of B16-F10 cells(P<0.05).The results of Transwell chamber assay showed that compared with control group,invasion and migration abilities of B16-F10 cells were significantly reduced after treatment with glycyrrhetinic acid(2,4μmol/L)(P<0.05).Wester blot results showed that compared with those without glycyrrhetinic acid treatment,expression levels of MMP-9,MMP-2,Wnt1 and β-catenin protein in B16-F10 cells significantly decreased after treatment with glycyrrhetinic acid(2,4μmol/L)(P<0.05).The results of tumor-bearing assay showed that compared with control group,weight and volume of tumors significantly decreased in glycyrrhetinic acid group,and expression levels of Wnt1,β-catenin,MMP-9 and MMP-2 proteins also significantly decreased(P<0.05).Conclusion Glycyrrhetinic acid can significantly inhibit the malignant biological behaviors of melanoma in vitro and vivo.And its mechanism may be related to inhibiting the activation of Wnt/β-catenin signaling pathways.