2018, Vol. 38
肝细胞癌(HCC)是世界第六大常见恶性肿瘤[1]。流行病学数据显示, 全球每年新发HCC病例超过78万, 其中我国约占50%[2]。HCC已成为肿瘤相关死亡的第二大原因[3-4]。近年来, 尽管HCC的诊治已取得巨大进步, 但其远期生存率仍欠佳[5-6]。原因在于大多数患者在确诊时已属晚期。索拉菲尼是晚期HCC的标准治疗, 能够延长患者的生存期, 但总体效果仍欠佳[7-9]。HCC的分子靶向治疗是目前研究的热点[9-13]。极光激酶(AURK)在调控细胞周期方面起重要作用[14-15]。极光激酶A和B (AURKA、AURKB)在肝癌组织中存在过度表达, 与肝癌的侵袭性、术后复发、化疗耐药及预后欠佳等相关[16-20]。因此, AURKA及AURKB可作为潜在的抗肝癌靶点[21-23]。Danusertib (Danu)是近年来新合成的小分子化合物, 对AURK具有良好的抑制作用[24-26]。但Danu对HCC的作用和机制尚不明确。本研究旨在探讨Danu对HCC细胞株HepG2的增殖、细胞周期、凋亡和自噬的影响及其机制, 为体内实验奠定基础。
1 材料和方法 1.1 主要材料和试剂极光激酶抑制剂Danu (Selleck), DMEM培养基(Corning Cellgro)。胎牛血清、碘化丙啶(PI)、核糖核酸酶A、MTT、DMSO (Sigma), 自噬检测试剂盒Cyto-ID® (ENZ-51031-K200)(Enzo Life Sciences)。Annexin V/ PE细胞凋亡检测试剂盒(BD Biosciences)。RIPA蛋白裂解液、磷酸酶抑制剂、蛋白酶抑制剂、BCA蛋白定量试剂盒、SDS-PAGE配制试剂、显影液(Thermo Scientific)。聚偏氟乙烯膜(PVDF)(Bio-Rad), 电泳仪、转膜仪及成像系统(ChemiDocTM XRS)(Bio-Rad)。流式细胞仪为Becton Dickinson Immunocytometry系统。细胞周期相关蛋白抗体如Cyclin B1、CDK1/CDC2、p21、p53, 细胞凋亡相关蛋白抗体如细胞色素C (cytochrome C)、Bax、Bcl-xl、Bcl-2、cleaved caspase-3、cleaved caspase-9、cleaved PARP、PUMA, 以及自噬相关蛋白抗体如Beclin 1、LC3-I和LC3-II、PI3K、p-PI3K (Tyr458)、Akt、p-Akt (Ser473)、mTOR、p-mTOR (Ser2448)、PTEN、AMPK、p-AMPK (Thr172)等(CST)。β-actin及二抗(Santa Cruz)。
1.2 细胞、细胞培养及药物浓度人肝癌细胞株HepG2(ATCC), 用含10%胎牛血清、1×105 U/L青霉素和100 mg/L的DMEM培养基, 置于37℃、5% CO2培养箱中常规培养。取对数生长期细胞用于实验。当细胞生长密度在75%左右时加药处理。Danu母液终浓度为50 mmol, 用新鲜DMEM培养基配制不同浓度的溶液。浓度效应实验药物浓度设为0.01、0.1、0.5 μmol, 作用时间24 h。时间效应实验设4、8、12、24、48、72 h组, Danu浓度0.5 μmol。
1.3 MTT法检测Danu对HepG2细胞的增殖抑制作用实验方法主要参照已发表文献[27]。实验组药物浓度为0.01、0.1、0.5、1、5、25和50 μmol。每组设6个复孔, 药物作用时间为24 h或48 h。
1.4 流式细胞仪检测Danu对HepG2细胞周期、凋亡和自噬的影响实验方法主要参照已发表文献[27]。药物作用足够时间后收集细胞, 随后用流式细胞仪进行检测。细胞周期实验用PBS-PI液染色。凋亡实验用PE和7AAD进行染色(按Annexin V/PE凋亡试剂盒说明书进行)。自噬实验用Cyto-ID®进行孵育(按Cyto-ID®自噬检测试剂盒说明书进行)。
1.5 Western blot检测Danu对HepG2细胞周期、凋亡及自噬相关蛋白表达的影响实验方法主要参照已发表文献[27]。在ChemiDocTM XRS成像系统上加入化学发光底物, 曝光显影。用Image Lab 3.0和Image J软件对条带进行分析。
1.6 MTT法检测抑制自噬后HepG2细胞的活力细胞接种同前。实验组设单纯Danu (0.5 µmol)组、单纯氯喹组(CQ组, 20 µmol)及联合组(0.5 µmol Danu+20 µmol CQ), 对照组不加药物。药物作用时间24 h。联合组药物同时孵育。余步骤同前。
1.7 统计学方法采用GraphPad Prism 7软件进行绘图和统计处理。实验结果以均数±标准差表示。多组间数据的比较采用完全随机设计方差分析(ANOVA, Tukey's), P < 0.05认为差异具有统计学意义。
2 结果 2.1 Danu对细胞增殖的影响Danu作用24 h和48 h后, HepG2细胞的增殖受到显著抑制。24 h和48 h的IC50分别为39.4 μmol和14.4 μmol (图 1)。
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图 1 MTT法检测不同浓度Danu对HepG2细胞增殖活力的影响 Fig.1 Effect of danusertib at different concentrations on the viability of HepG2 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs control (0 μmol) group. |
Danu能够导致HepG2细胞出现G2/M周期阻滞和异倍体, 呈浓度和时间依赖。Danu作用细胞24 h后, 0.1 μmol组及0.5 μmol组G2/M期细胞比例分别为58.2%和66.5%, 异倍体比例分别为51.9%和44.5%, 显著高于对照组(图 2)。0.5 μmol Danu作用时间超过4 h后, G2/M期细胞及异倍体比例逐渐升高, 和对照组相比, 均具有统计学意义(图 3)。
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图 2 流式细胞仪检测不同浓度Danu作用24 h后HepG2细胞周期的变化 Fig.2 Effect of treatment with danusertib at different concentrations for 24 h on cell cycle distribution of HepG2 cells. ***P < 0.001 vs 0 μmol group. Dip: Diploid; An1: Aneuploid. |
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图 3 流式细胞仪检测0.5 μmol的Danu作用不同时间后HepG2细胞周期的变化 Fig.3 Effect of 0.5 μmol danusertib treatment for 0-72 h on cell cycle distribution of HepG2 cells. ***P < 0.001 vs 0 μmol group. |
0.5 μmol Danu能使肝癌细胞Cyclin B1及CDC2的表达下调41.3%和30.4%。0.1 μmol及0.5 μmol Danu使p53的表达上调96.4%和74.3%, p21的表达则被上调1.2倍和1.95倍(图 4)。
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图 4 Western blot检测不同浓度Danu作用24 h后Cyclin B1、CDC2、p53及p21表达的变化 Fig.4 Effect of danusertib at different concentrations on expressions of cyclin B1, CDC2, p53 and p21 in HepG2 cells. **P < 0.01, ***P < 0.001 vs 0 μmol group. |
Danu能够诱导HepG2细胞凋亡, 呈浓度和时间依赖。Danu作用细胞24 h后, 0.1 μmol组及0.5 μmol组的总凋亡率分别为10.8%和12.0%, 显著高于对照组(4.9%)。0.5 μmol Danu作用24、48、72 h后, 实验组的凋亡率分别为3.9%、4.1%和11.4%, 和对照组(1.1%)相比, 具有统计学意义(图 5)。
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图 5 流式细胞仪检测不同浓度的Danu及不同作用时间对HepG2细胞凋亡的影响 Fig.5 Effect of danusertib on apoptosis of HepG2 cells. **P < 0.01 vs 0 μmol group. |
0.5 μmol Danu作用24 h能使cleaved caspase-3、cleaved caspase-9及cleaved PARP的表达分别上调72.4%、65.1%和42.3%, 促凋亡蛋白Bax和Puma的表达分别上调0.6和0.9倍, 凋亡抑制蛋白Bcl-xl和Bcl-2的表达则分别下调31.0%和45.5%。0.1 μmol及0.5 μmol组的胞浆细胞色素C表达升高0.9倍和1.5倍(图 6)。
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图 6 Western blot检测不同浓度Danu作用24 h后凋亡相关蛋白表达的变化 Fig.6 Effect of danusertib at different concentrations on expressions of apoptosis-related proteins. *P < 0.05, **P < 0.01, ***P < 0.001 vs 0 μmol group. |
Danu能够导致HepG2细胞发生自噬, 呈浓度和时间依赖。Danu作用细胞24 h后, 0.1 μmol组及0.5 μmol组的自噬细胞比例分别为21.5%和30.0%, 显著高于8.9%。0.5 μmol Danu作用12、24、48及72 h后, 实验组的凋亡率分别为3.2%、15.2%、23.8%和30.7%, 和对照组(8.6%)相比, 具有统计学意义(图 7)。
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图 7 流式细胞仪检测不同浓度Danu及不同作用时间对HepG2细胞自噬的影响 Fig.7 Effect of danusertib at different concentrations and for different durations on autophagy of HepG2. *P < 0.05, **P < 0.01, ***P < 0.001 vs 0 μmol group. |
0.5 μM Danu使自噬标志蛋白Beclin 1的表达上调38.2%, 使LC3-II/LC3-I的比值上调40.7%。p-PI3K/ PI3K、p-AKT/AKT及p-mTOR/mTOR的比值分别被下调54.0%、28.2%和41.6%。PI3K/Akt/mTOR通路的负调节蛋白PTEN则被上调51.5%。p-AMPK/AMPK的比值亦被上调71.0%(图 8)。
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图 8 Western blot检测不同浓度Danu作用24 h后自噬相关蛋白表达的变化 Fig.8 Effect of danusertib treatment at different concentrations for 24 h on expressions of autophagy-related proteins. P < 0.05, ***P < 0.001 vs 0 μmol group. |
采用氯喹(20 μmol)抑制自噬后, 联合组细胞活力为72.0%, 显著低于Danu单药组(88.9%)以及氯喹单药组(82.4%)(图 9)。可见, 抑制自噬可增强Danu杀灭细胞的作用, 说明Danu所引起的自噬起到了保护细胞的作用。
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图 9 MTT法检测采用氯喹抑制自噬后HepG2细胞的活力 Fig.9 Effect of 0.5 μmol danusertib on the viability of HegG2 cells after chloroquine-mediated autophagy inhibition. *P < 0.05, ***P < 0.001 vs 0 μmol group. |
中晚期HCC的生存期短仍旧是不争的事实[4, 28]。索拉菲尼在延长进展期HCC患者生存期方面具有一定优势, 但总体效果仍欠佳[5, 6, 29]。研发新药、寻求新的治疗方案是临床上亟待解决的难题[30]。研究表明, AURK在肝癌组织中的过度表达具有重要的临床意义[16-18], AURKA及AURKB可作为抗肝癌靶点。临床前研究和部分临床研究显示Danu对多种肿瘤具有较好的抗肿瘤效果[31-33], 本项Danu体外抗HCC实验表明, Danu能够有效抑制细胞增殖, 通过调控p53及p21的表达引起细胞出现G2/M细胞周期阻滞和异倍体, 通过活化线粒体依赖通路诱导细胞出现凋亡, 并通过抑制PI3K/Akt/mTOR及活化AMPK通路诱导细胞发生自噬。
AURK属于丝氨酸-苏氨酸激酶, 在调控细胞周期方面起重要作用[34]。本实验结果表明, Danu是通过抑制AURK和p53-p21信号轴来影响HepG2细胞周期分布的。AURK活性受到抑制后, 中心体成熟受限, 纺锤体非正确组装并延迟分裂退出, 最终导致HepG2细胞阻滞于G2/M期。此外, 胞质分裂被阻止, 导致异倍体的出现。Danu上调了p53和p21的表达, 进而抑制Cyclin B1-CDC2复合体的活性, 最终导致HepG2细胞阻滞于G2/M期。
凋亡和自噬是癌症细胞程序性死亡的两种形式, 凋亡主要受外源性死亡受体通路和线粒体介导通路的调控[35]。本研究显示, Danu是通过活化线粒体介导通路诱导HepG2细胞凋亡的。促凋亡蛋白Bax和凋亡抑制蛋白Bcl-xl及Bcl-2在Danu的作用下表达失衡, 进而引起细胞色素C从线粒体释放至胞浆中, 激发caspase依赖性凋亡信号, 最终导致HepG2细胞出现凋亡。在多数情况下, 自噬是维持细胞稳态的(即维持细胞生存), 而抑制自噬则可以阻断细胞利用消耗自身能量物质维持生存的过程[36]。本研究发现Danu能够引起HepG2细胞发生自噬, 且自噬起到了细胞保护作用, 提示联合应用自噬抑制剂可以促进Danu的抗肿瘤效果。PI3K/Akt/mTOR通路抑制和AMPK通路活化能够引起细胞自噬[37]。本研究发现Danu能够下调p-PI3K/PI3K、pAKT/AKT及p-mTOR/mTOR的比值, 以及上调pAMPK/AMPK的比值, 可见Danu抑制了PI3K/Akt/ mTOR通路的磷酸化, 同时促进AMPK的磷酸化, 最终引起HepG2细胞发生自噬。
本研究结果表明, Danu具有较好的体外抗HCC效果, 为下一步进行体内实验奠定了基础。Danu引起的自噬起到了保护细胞的作用, 提示联合使用自噬抑制剂能够增强AURK抑制剂的抗HCC效果。此外, Danu联合索拉菲尼等现有的靶向药物从而增强抗HCC效果可能是日后的研究方向。
总之, Danu能够抑制HepG2细胞增殖, 引起G2/M细胞周期阻滞, 导致细胞凋亡和自噬, 具有良好的体外抗肿瘤效果。Danu能够引起细胞保护性自噬。为增强Danu的抗HCC效果, 联用用药可能是一种选择。
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