2. 盐城市第一人民医院肿瘤科，江苏 盐城 224005
2. Dept of Oncology, the First People′s Hospital of Yancheng City, Yancheng Jiangsu 224005, China
胃癌是影响我国人民生命健康最严重的恶性肿瘤之一，是癌症死亡的主要原因。根治性手术是目前治疗胃癌的最有效方法，但手术不能完全避免局部复发和远处转移的可能，防止肿瘤复发和转移的辅助治疗越来越受到国内外学者的重视。熊果酸（ursolic acid,UA）是广泛存在于白花蛇舌草、女贞子、乌梅和夏枯草等天然植物中的一种五环三萜类化合物，具有抑制肿瘤细胞增殖、诱导细胞凋亡、抗血管生成、抗促癌、抗突变和调节氧化应激等作用，近年来已成为肿瘤化学预防研究的热点[1-3]。临床前研究显示，UA在转基因肿瘤动物模型和人癌裸鼠移植模型中的抑瘤作用明显[3-4]，具有良好的临床应用前景。我们的前期研究发现，UA促进胃癌细胞内活性氧（reactive oxygen species,ROS）生成，诱导单磷酸腺苷激活的蛋白激酶（AMPactivated protein kinase,AMPK）磷酸化，抑制信号转导与转录活化因子3（signal transducer and activator of transcription 3,STAT3）磷酸化和环氧化酶-2（cycloxygenase-2,COX-2)表达[5-6]。UA通过下调COX-2表达而抑制胃癌细胞增殖、诱导凋亡[6-7]。然而，UA抑制胃癌细胞COX-2表达的信号转导通路尚不清楚。
本研究选择2株不同分化程度但COX-2高表达的SGC-7901和MKN-45胃癌细胞株作为研究对象，观察ROS/AMPK/STAT3信号转导通路在UA影响SGC-7901和MKN-45胃癌细胞株COX-2表达中的作用，进一步探讨UA抑制胃癌细胞COX-2表达的具体机制。
1 材料与方法 1.1 药物和试剂UA、AMPK激活剂5氨基咪唑4甲酰胺核苷酸（AICAR）、二甲基亚砜（DMSO）、碘化丙啶（PI）、苯甲磺酰氟（phenylmethanesulfonyl fluoride，PMSF） 和抑肽酶为美国SigmaAldrich公司产品；AMPK抑制剂compound C和STAT3抑制剂WP1066为德国Merck公司产品；RPMI 1640培养液和胎牛血清为美国Gibco BRL公司产品；蛋白质定量BCA试剂盒为美国Pierce公司产品；抗氧化剂N乙酰L半胱氨酸(NAC)为江苏碧云天生物技术公司产品；兔抗人AMPK和磷酸化AMPK(p-AMPK)、乙酰辅酶A羧化酶（ACC）和磷酸化ACC(p-ACC)、STAT3和磷酸化STAT3（p-STAT3）、COX-2和β-ac-tin单克隆抗体为美国Cell Signaling Technology公司产品；辣根过氧化物酶标记的山羊抗兔IgG抗体为美国Bioworld Technology公司产品。PVDF膜为美国Millipore公司产品；ECL发光试剂盒为英国Amersham公司产品；其它试剂为国产分析纯级。
1.2 细胞和培养人胃腺癌中分化细胞株SGC-7901购自中国科学院上海生科院细胞资源中心，人胃腺癌低分化细胞株MKN-45购自南京凯基生物科技发展有限公司。SGC-7901和MKN-45细胞常规传代培养于含10%胎牛血清、100 kU·L-1青霉素和100 mg·L-1链霉素的RPMI 1640培养液中，37℃、5% CO2及饱和湿度的二氧化碳培养箱中培养生长。隔天换液，3 d传代1次。
1.3 药物配制UA、AICAR、compound C和WP1066先以DMSO溶解，NAC用超纯水溶解，而后均以RPMI 1640培养液稀释至所需浓度，DMSO在培养液中的浓度不超过0.1%，0.22 μm的微孔滤膜过滤除菌后4℃保存备用。
1.4 实验分组将传代后处于对数生长期的细胞分为对照（control）组、UA（30 μmol·L-1）组、抗氧化剂（NAC,5 mmol·L-1）组、NAC+UA组、AMPK激活剂（AICAR,0.5 mmol·L-1）组、AICAR+UA组、AMPK抑制剂（compound C,2.5 μmol·L-1）组、compound C+UA组、STAT3抑制剂（WP1066,5 μmol·L-1）组、WP1066+UA组。NAC+UA组NAC预处理30 min后UA再干预培养24 h；AICAR+UA组AICAR预处理2 h后UA再干预培养24 h；compound C+UA组compound C预处理1 h后UA再干预培养24 h；WP1066+UA组WP1066预处理1 h后UA再干预培养24 h。
1.5 细胞蛋白提取和Western blot检测各组干预培养后的细胞用预冷的PBS洗涤3次，以100 μL细胞裂解液（PBS内含：Nonidet P-40 1%，脱氧胆酸钠5 g·L-1，SDS 1 g·L-1，PMSF 0.1 g·L-1和抑肽酶10 mg·L-1）4℃处理60 min。细胞裂解物经11 000×g 4℃离心10 min后取上清，BCA试剂盒测定其蛋白浓度。常规进行SDSPAGE电泳后转印至PVDF膜，室温封闭2 h，分别加入一抗（APMK抗体、p-AMPK抗体、ACC抗体、p-ACC抗体、STAT3抗体、p-STAT3抗体、COX-2抗体和β-ac-tin抗体），4℃孵育过夜，辣根过氧化物酶标记的山羊抗兔IgG抗体为第二抗体，4℃孵育2 h，ECL发光，使用ImageJ（National Institute of Health,Bethesda,MD）图像分析软件对蛋白电泳带的灰度进行半定量分析。
1.6 统计学处理实验结果以x±s表示，所有数据均应用SPSS 17.0统计软件包进行分析，多组间均数的比较采用单因素方差分析（ANOVA），组间两两比较用LSD法检验。
2 结果 2.1 UA和抗氧化剂NAC对胃癌细胞AMPK、STAT3磷酸化和COX-2表达的影响Western blot检查结果显示，抗氧化剂NAC有效抑制UA诱导的AMPK磷酸化，ACC是AMPK的下游直接效应靶蛋白，p-ACC被认为是观察AMPK被激活的最佳指标[8]（Fig 1），UA抑制STAT3磷酸化和COX-2表达的作用被NAC所逆转（Fig 2）。
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| Fig 1 Effects of NAC on ursolic acid(UA)-induced AMPK and ACC phosphorylation in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoAMPK(p-AMPK) or phosphoACC(p-ACC) compared with total AMPK or total ACC respectively. All data represent the x±s of six independent experiments.*P<0.05 vs control.#P<0.05,##P<0.01 vs UA treatment without NAC pre-incubation. |
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| Fig 2 Effects of NAC on UAinhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoSTAT3(p-STAT3) or COX-2 compared with total STAT3 or β-ac-tin respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05,##P<0.01 vs UA treatment without NAC pre-incubation. |
Western blot检查结果显示，AMPK激活剂AICAR单用或联合UA均可诱导SGC-7901和MKN-45细胞AMPK磷酸化（Fig 3），抑制STAT3磷酸化和COX-2表达，联合作用大于单药作用（Fig 4）。
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| Fig 3 Effects of AMPK activator AICAR(5-amino-4-imidazolecarboxamide ribosid-1-b-Dribofuranoside) on UA -induced AMPK and ACC phosphorylation in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoAMPK(p-AMPK) or phosphoACC(p-ACC) compared with total AMPK or total ACC respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05,##P<0.01 vs UA treatment without AICAR pre-incubation. |
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| Fig 4 Effects of AMPK activator AICAR on UAinhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoSTAT3(p-STAT3) or COX-2 compared with total STAT3 or β-ac-tin respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05,##P<0.01 vs UA treatment without AICAR pre-incubation |
AMPK抑制剂compound C有效抑制UA诱导的AMPK磷酸化（Fig 5），UA抑制STAT3磷酸化和COX-2表达的作用被compound C所逆转（Fig 6）。
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| Fig 5 Effects of AMPK inhibitor compound C on UA-induced AMPK and ACC phosphorylation in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoAMPK(p-AMPK) or phosphoACC(p-ACC) compared with total AMPK or total ACC respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05,##P<0.01 vs UA treatment without compound C pre-incubation. |
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| Fig 6 Effects of AMPK inhibitor Compound C on UAinhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells he top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoSTAT3(p-STAT3) or COX-2 compared with total STAT3 or β-ac-tin respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05,##P<0.01 vs UA treatment without Compound C pre-incubation. |
STAT3抑制剂WP1066对UA诱导的AMPK磷酸化无明显影响（Fig 7），WP1066单用或联合UA均可抑制SGC-7901和MKN-45细胞STAT3磷酸化和COX-2表达，且联合作用大于单药作用（Fig 8）。
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| Fig 7 Effects of STAT3 inhibitor WP1066 on UA-induced AMPK and ACC phosphorylation in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoAMPK(p-AMPK) or phosphoACC(p-ACC) compared with total AMPK or total ACC respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control. |
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| Fig 8 Effects of STAT3 inhibitor WP1066 on UAinhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells The top panels show a representative immunoblot of six separate experiments undertaken. The histograms at the bottom represent the relative expression of phosphoSTAT3(p-STAT3) or COX-2 compared with total STAT3 or β-ac-tin respectively. All data represent the x±s of six independent experiments.*P<0.05,**P<0.01 vs control.#P<0.05 vs UA treatment without WP1066 pre-incubation. |
ROS是氧在机体代谢过程中产生的中间产物及其衍生物，参与细胞增殖、分化、转化和凋亡以及细胞内重要信号途径的转导[9]，近年来研究表明，抗肿瘤药物诱导细胞凋亡与其引起肿瘤细胞内ROS水平增加密切相关[10-12]。AMPK是细胞能量代谢的主要调节器[13],激活AMPK可诱导胃癌和胰腺癌等肿瘤细胞发生凋亡[14-15]，UA和白藜芦醇（resveratrol）的抗肿瘤作用与ROS依赖的AMPK活化有关[5, 16-17]。
AMPK除参与细胞内的能量代谢调节外还调控一些基因的转录和表达[18]，本研究结果显示，UA诱导AMPK磷酸化，抑制STAT3磷酸化和COX-2表达；抗氧化剂NAC能逆转UA对STAT3磷酸化和COX-2表达的抑制作用，提示ROS是UA抑制STAT3活性和COX-2表达的重要介质[5]。AMPK激活剂AICAR是AMP类似物，明显抑制STAT3磷酸化和COX-2表达，UA与AICAR联合使用对STAT3磷酸化和COX-2表达的抑制作用和单用UA组相比差异有显著性，提示UA联合AICAR后AMPK得到进一步的激活。而AMPK抑制剂compound C则逆转UA对STAT3磷酸化和COX-2表达的抑制作用，进一步证明UA通过AMPK信号通路抑制胃癌细胞STAT3活化和COX-2表达。
我们前期研究发现，JAK2/STAT3和PI3K/Akt信号途径能诱导胃癌细胞COX-2的表达[19]，而UA能阻断STAT3通路抑制多发性骨髓瘤细胞增殖[20]。STAT3在各种类型的人胃癌细胞株和胃癌组织中都有较高的活性，JAK/STAT信号转导途径可能在胃癌的发生、发展中起重要的作用[21]。因此，我们推测UA通过ROS/AMPK/STAT3通路下调COX-2表达。当然，由于细胞内信号转导机制十分复杂，UA抑制胃癌细胞COX-2表达尚不排除有其它通路的存在，也不排除不同通路之间存在关联（如crosstalk等），要全面了解其机制需大量而深入的研究。
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