【作者】 张哲；

【导师】 王艳红；

【作者基本信息】 复旦大学 ， 内科学， 2009， 硕士

【摘要】 目的:研究不同转移潜能的人肝癌细胞的pERK表达与细胞转移潜能及索拉菲尼体外杀伤效应之间的关系。方法:选用4种具有不同转移潜能的人肝癌细胞系:SMMC-7721（非转移）、MHCC97-L（低转移性）、MHCC97-H（高转移性）、HCCLM6（高转移性）为靶细胞。以索拉菲尼为干预药物,以5-氟尿嘧啶（5-FU）为干预对照药物;用U0126作为pERK蛋白抑制剂。（1）应用免疫细胞化学定量分析和Western Blot方法检测各细胞系分别于索拉菲尼、5-FU及U0126作用前后的pERK蛋白表达,并计算pERK蛋白平均光密度值（MOD）定量分析;（2）应用细胞增殖与毒性实验,检测药物的半抑制浓度值(IC₅₀),并与pERK的MOD值进行相关性分析,评价细胞对索拉非尼药物体外杀伤作用的敏感性与pERK蛋白表达的关系。（3）先予U0126抑制肝癌细胞基础pERK蛋白表达后,再应用索拉菲尼干预,进一步评价细胞对索拉非尼药物体外杀伤作用敏感性的变化。结果:（1）免疫细胞化学和图像分析以及Western Blot结果显示,SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞的pERK蛋白平均光密度（MOD）分别为0.042±0.006、0.081±0.007、0.329±0.037和0.463±0.084,存在显著的统计学差异（P＜0.0001,n=6）。提示,细胞基础pERK蛋白表达含量随着肝癌细胞转移潜能的增加而依次递增。（2）分别给予5、10、20μmol/L的索拉非尼作用细胞24h后,SMMC-7721细胞的pERK蛋白表达率分别下降至（81.88±7.65）%、（71.63±10.80）%和（17.47±1.34）%;HCCLM6细胞分别下降至（78.06±4.66）%、（28.12±1.36）%和（3.99±0.19）%。与各自对照组比较,均存在统计学差异（P=0.0043和P＜0.0001,One-Way ANOVA,n=6）。提示,5～20μmol/L浓度范围内的索拉非尼均能抑制4种细胞系细胞ERK磷酸化。与pERK蛋白相对高表达的3种细胞系相比,索拉非尼对低表达pERK蛋白的SMMC-7721细胞的ERK磷酸化抑制程度明显较弱（P＜0.0001,Two-Way ANOVA,n=6）,提示:索拉非尼对4种细胞ERK磷酸化抑制程度存在差异,并且与其基础pERK表达水平有关。（3）以MHCC97-H细胞为代表,分别给予不同浓度的5-FU（10,20,50mg/L）作用48h,与对照组相比,pERK蛋白表达率分别为:（102.3±7.88）%,（110.8±6.60）%,（101.1±5.12）%,均无统计学差异（One-Way ANOVA,P＞0.05,n=6）。提示,5-FU对细胞的ERK磷酸化无抑制作用。（4）索拉非尼对SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞系的IC₅₀分别为:20.85±2.81μmol/L、10.38±1.52μmol/L、10.70±2.35μmol/L和9.11±2.44μmol/L。SMMC-7721细胞组与其他三组细胞比较有显著差异,（Two-Way ANOVA,SPSS 13.0,P=0.0018,n=4）。提示,索拉非尼对pERK低表达细胞的体外杀伤作用明显低于pERK高表达细胞。相关性分析显示:索拉非尼对各细胞系的IC₅₀与各细胞系pERK蛋白的平均光密度（MOD）呈负相关,具有明显的统计学意义,（Spearman r=-0.8671,P=0.0003,n=12）。提示,索拉非尼的药物敏感性与pERK蛋白表达相关。（5） 5-FU对SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞系的IC₅₀分别为:4.24±0.87mg/L,79.71±24.49mg/L,41.21±21.55mg/L和187.45±78.05mg/L,存在明显差异（Two-Way ANOVA,P＜0.0001,n=4）。提示,pERK蛋白低表达细胞（SMMC-7721）对5-FU反应敏感,而pERK高表达细胞（MHCC97-L、MHCC97-H、HCCLM6）则表现出明显的耐药性。相关性分析显示:5-FU对各细胞系的IC₅₀与各细胞系pERK蛋白的平均光密度（MOD）呈正相天,具有显著的统计学意义,（Spearman r=0.7846,P=0.0009,n=12）。提示,5-FU的耐药性与pERK蛋白表达相关。（6）浓度范围为0～100μmol/L的U0126体外作用细胞6h对细胞的直接杀伤作用轻微;而20μmol/L的U0126作用细胞6h后,细胞pERK蛋白表达率下降约60%。细胞经U0126预处理后,对索拉非尼的IC₅₀为17.31±1.62μmol/L,与对照组（10.81±1.24μmol/L）比较,有显著性差异（P=0.0281,n=6）。提示RAF/MEK/ERK信号通路的活性状态在索拉非尼的细胞杀伤作用中具有重要意义;索拉非尼药物敏感性与该信号通路的激活状态及pERK蛋白的表达直接相关。结论:本实验研究进一步证实,在肝细胞癌中,pERK可以作为一个潜在的生物标记物,预测索拉非尼的药物敏感性。RAF/MEK/ERK级联通路的激活,可能参与肝癌细胞侵袭、转移潜能和传统化疗药物耐药性的增强。更多还原

【Abstract】 Objective:To investigate the relationship between effects of sorafenib on cell proliferation and basal phosphorylated extracellular signal-regulated kinase（pERK） levels in different HCC cell lines.Methods:The effects of sorafenib and 5-Fluorouracil on cell proliferation were evaluated by cell viability assay in four HCC cell lines（SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6）,with different metastatic potential and basal pERK expression. Expression levels of pERK were determined by immunocytochemical quantification along with Western Blot analysis and pERK density values were also calculated. Correlation analyses were then carried out between the IC₅₀ values of drugs and pERK density values.After basal ERK phosphorylation was down-regulated with U0126 in MHCC97-H cells,cellular responsiveness to sorafenib was then assessed by cell viability assay.Results:（1） The results of immunocytochemistry and image quantification,confirmed by Western Blot analysis,revealed that baseline pERK was differentially expressed in these HCC cell lines（P＜0.0001,n=6） and seemed to be correlated with their metastatic potential.The pERK density in SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6 cells was 0.042±0.006,0.081±0.007,0.329±0.037 and 0.463±0.084, respectively.In metastatic MHCC97-H and HCCLM6 cells,pERK levels were significantly higher than that in non-metastatic SMMC-7721 cells（P＜0.0001,n=6）. Even among the three metastatic cell lines,pERK levels were differentially expressed and increased stepwise with their metastatic potential（P＜0.0001,n=6）,which indicated that the RAF/MEK/ERK pathway may be involved in tumor invasion and metastasis in HCC. （2） In this study,sorafenib could inhibit ERK phosphorylation in all four HCC cell lines dose-dependently at a concentration between 5 and 20μM.Take SMMC-7721 and HCCLM6 cells for instance,after exposure to 5,10 or 20μM sorafenib for 24 hours,the expression rate of pERK in SMMC-7721 fell gradually to 81.88±7.65%,71.63±10.80%and 17.47±1.34%,respectively,and in HCCLM6 to 78.06±4.66%,28.12±1.36%and 3.99±0.19%,respectively.The expression rates in both cell lines were significantly reduced when compared to each DMSO control group（P=0.0043,n=6 and P＜0.0001,n=6,respectively）.However,further statistical analyses revealed the significant difference in the degree of the sorafenib effects in these HCC cell lines.Interestingly,the sorafenib pERK inhibition effect in SMMC-7721 cells with lower level of pERK was significantly weaker when compared to the other three HCC cell lines with relatively higher basal pERK levels （P＜0.0001,n=6）.These results suggested that the effects of sorafenib on ERK phosphorylation inhibition were significantly associated with basal pERK levels in HCC cell lines.（3） On the contrary,no significant change was observed after 5-FU treatment in MHCC97-H cells,pERK expression rate was 102.3±7.88%,110.8±6.60%, 101.1±5.12%,respectively,after exposure to 10,20 or 50mg/l 5-FU for 48 hours,with no statistical difference to the control group（P＞0.05,n=6）.（4） Effects of sorafenib on cell proliferation were measured by the CCK-8 cell viability assay.According to our results,sorafenib inhibited all four HCC cell lines proliferation in a dose-dependent characteristic,with an IC₅₀ of 20.85±2.81μM, 10.38±1.52μM,10.70±2.35μM and 9.11±2.44μM in SMMC-7721,MHCC97-L, MHCC97-H and HCCLM6 cells,respectively.As expected,SMMC-7721 cells containing lower level of pERK were significantly less sensitive to sorafenib-mediated growth inhibition than the other three HCC cell lines with higher basal pERK levels（P=0.0018,n=4）.Meanwhile,a significant negative correlation （Spearman r=-0.8671,P=0.0003,n=12） was observed between the IC₅₀ values of sorafenib in these HCC cell lines and their pERK density values,indicating that the effects of sorafenib on cell proliferation were significantly correlated with basal pERK levels in these HCC cell lines.（5） While opposite results were observed in treatment with traditional chemotherapy drug 5-FU.5-FU inhibited HCC cell proliferation with an IC₅₀ of 4.24±0.87mg/l,79.71±24.49mg/l,41.21±21.55mg/l and 187.45±78.05mg/l in SMMC-7721,MHCC97-L,MHCC97-H and HCCLM6 cells,respectively,with significant statistical differences（P＜0.0001,n=4）.The SMMC-7721 cells with lower pERK expression demonstrated a higher-sensitivity to 5-FU.However,MHCC97-L, MHCC97-H and HCCLM6 cells with higher pERK expression exhibited more resistance to this drug.The ultimate inhibition rate before reaching platform in these three cell lines was about 35%,40%and 45%,respectively,each compared with the control group.Furthermore,a significant correlation（Spearman r=0.7846,P=0.0009, n=12） was observed between the IC₅₀ values of 5-FU and their pERK density values, indicating that the resistance to 5-FU was significantly associated with basal pERK expression in these HCC cell lines.（6） To more directly determine the relationship between pERK expression and sensitivity to sorafenib,we inhibited the MEK/ERK pathway and reduced basal pERK expression in MHCC97-H cells via U0126,a selective inhibitor of MEK1 and MEK2, and then compared cellular responsiveness to sorafenib with that of the untreated cells. Quantification of cellular pERK level by immunocytochemical analysis indicated that constitutive ERK phosphorylation was strongly reduced in MHCC97-H cells after treatment with 20μM U0126 for 6 hours relative to the level observed in the untreated cells（60%inhibition）,which induced almost no detectable systemic toxicity on cell proliferation.In the following experiments,we compared sorafenib responsiveness of MHCC97-H cells pretreated with 20μM U0126 for 6 hours to an untreated control. Cell viability assay revealed that the pretreated cells were significantly less sensitive to sorafenib-mediated growth inhibition,with an IC₅₀ of 17.31±1.62μM versus 10.81±1.24μM（P=0.0281,n=6）.These results confirmed that the RAF/MEK/ERK signaling pathway was essential for sorafenib-mediated growth inhibition,and that the sensitivity to sorafenib was directly related to the activation of this pathway and basal pERK expression in MHCC97-H cells.Conclusion:In this vitro study,pERK was confirmed to be a useful biomarker predictive of sensitivity in treating HCC with sorafenib.The RAF/MEK/ERK pathway may be involved in invasion,metastasis and drug resistance to traditional chemotherapy in HCC.更多还原