Zhang Qing-Yu, Li Ran, Zeng Guo-Fang, Liu Bin, Liu Jie, Shu Yang, Liu Zhong-Kao, Qiu Zhi-Dong, Wang Dong-Jun, Miao Hui-Lai, Li Ming-Yi, Zhu Run-Zhi
Qing-Yu Zhang, Ran Li, Guo-Fang Zeng, Bin Liu, Jie Liu, Yang Shu, Zhong-Kao Liu, Zhi-Dong Qiu, Dong-Jun Wang, Hui-Lai Miao, Ming-Yi Li , Run-Zhi Zhu, Key Laboratory of Hepatic Disease, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, Guangdong Province, China.
World J Gastroenterol. 2014 Aug 7;20(29):10082-93. doi: 10.3748/wjg.v20.i29.10082.
To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human hepatic cancer cells.
The hepatoma cell lines SK-Hep-1 and MHCC97L were used in this study. The cells were cultured in RPIM-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. DHM was dissolved in dimethyl sulfoxide and diluted to various concentrations in medium before applying to cells. MTT assays were performed to measure the viability of the cells after DHM treatment. Wound healing and Boyden transwell assays were used to assess cancer cell motility. The invasive capacity of cancer cells was measured using Matrigel-coated transwell chambers. Matrix metalloproteinase (MMP)-2/9 activity was examined by fluorescence analysis. Western blot was carried out to analyze the expression of MMP-2, MMP-9, p-38, JNK, ERK1/2 and PKC-δ proteins. All data were analyzed by Student's t tests in GraphPad prism 5.0 software and are presented as mean ± SD.
DHM was found to strongly inhibit the migration of the hepatoma cell lines SK-Hep-1 (without DHM, 24 h: 120 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 65 ± 10 μmol/L, P < 0.001) and MHCC97L (without DHM, 24 h: 126 ± 7 μmol/L vs 100 μmol/L DHM, 24 h: 74 ± 6 μmol/L, P < 0.001). The invasive capacity of the cells was reduced by DHM treatment (SK-Hep-1 cells without DHM, 24 h: 67 ± 4 μmol/L vs 100 μmol/L DHM, 24 h: 9 ± 3 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 117 ± 8 μmol/L vs 100 μmol/L DHM, 24 h: 45 ± 2 μmol/L, P < 0.001). MMP2/9 activity was also inhibited by DHM exposure (SK-Hep-1 cells without DHM, 24 h: 600 ± 26 μmol/L vs 100 μmol/L DHM, 24 h: 100 ± 6 μmol/L, P < 0.001; MHCC97L cells without DHM, 24 h: 504 ± 32 μmol/L vs 100 μmol/L DHM 24 h: 156 ± 10 μmol/L, P < 0.001). Western blot analysis showed that DHM decreased the expression level of MMP-9 but had little effect on MMP-2. Further investigation indicated that DHM markedly reduced the phosphorylation levels of p38, ERK1/2 and JNK in a concentration-dependent manner but had no impact on the total protein levels. In addition, PKC-δ protein, a key protein in the regulation of MMP family protein expression, was up-regulated with DHM treatment.
These findings demonstrate that DHM inhibits the migration and invasion of hepatoma cells and may serve as a potential candidate agent for the prevention of HCC metastasis.
研究二氢杨梅素（DHM）对人肝癌细胞迁移和侵袭的影响。
本研究采用肝癌细胞系SK-Hep-1和MHCC97L。细胞在补充有10%胎牛血清的RPIM-1640培养基中，于37℃、5%二氧化碳饱和湿度的培养箱中培养。DHM溶解于二甲基亚砜中，在应用于细胞前在培养基中稀释至不同浓度。采用MTT法检测DHM处理后细胞的活力。采用伤口愈合实验和Boyden小室实验评估癌细胞的运动能力。使用基质胶包被的小室检测癌细胞的侵袭能力。通过荧光分析检测基质金属蛋白酶（MMP）-2/9的活性。进行蛋白质免疫印迹法分析MMP-2、MMP-9、p-38、JNK、ERK1/2和PKC-δ蛋白的表达。所有数据采用GraphPad prism 5.0软件中的Student's t检验进行分析，结果以平均值±标准差表示。
发现DHM能强烈抑制肝癌细胞系SK-Hep-1（无DHM，24小时：120±8μmol/L vs 100μmol/L DHM，24小时：65±10μmol/L，P<0.001）和MHCC97L（无DHM，24小时：126±7μmol/L vs 100μmol/L DHM，24小时：74±6μmol/L，P<0.001）的迁移。DHM处理降低了细胞的侵袭能力（SK-Hep-1细胞无DHM，24小时：67±4μmol/L vs 100μmol/L DHM，24小时：9±3μmol/L，P<0.001；MHCC97L细胞无DHM，24小时：117±8μmol/L vs 100μmol/L DHM，24小时：45±2μmol/L，P<0.001）。DHM处理也抑制了MMP2/9的活性（SK-Hep-1细胞无DHM，24小时：600±26μmol/L vs 100μmol/L DHM，24小时：100±6μmol/L，P<0.001；MHCC97L细胞无DHM，24小时：504±32μmol/L vs 100μmol/L DHM 24小时：156±10μmol/L，P<0.001）。蛋白质免疫印迹分析表明，DHM降低了MMP-9的表达水平，但对MMP-2影响不大。进一步研究表明，DHM以浓度依赖性方式显著降低p38、ERK1/2和JNK的磷酸化水平，但对总蛋白水平无影响。此外，PKC-δ蛋白是调节MMP家族蛋白表达的关键蛋白，DHM处理使其上调。
这些发现表明，DHM抑制肝癌细胞的迁移和侵袭，可能作为预防肝癌转移的潜在候选药物。
