【作者】 王兴杰；

【导师】 李国荣；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2022， 硕士

【摘要】 癌症是危害人类健康的重要因素,临床常用的抗癌药物对人体产生了不可忽视的毒副作用,因此,探寻毒性小的抗肿瘤化合物用于癌症治疗是一种潜在的治疗手段。双氢青蒿素是青蒿素类衍生物,在体外和体内研究中表现出有效的抑癌作用。癌细胞倾向于通过糖酵解来提供增殖和迁移所需的能量和酸性微环境,这使得靶向糖酵解代谢有可能成为癌症治疗的有效手段。作为双氢青蒿素的一个重要靶点,翻译控制的肿瘤蛋白（translationally controlled tumor protein,TCTP）促进癌细胞增殖、迁移与侵袭,而双氢青蒿素靶向TCTP的下游抑癌机制却鲜有报道。AMP激活蛋白激酶（AMP-activated protein kinase,AMPK）作为细胞能量状态的传感器负向调控糖酵解,钙/钙调蛋白依赖性蛋白激酶激酶2（calcium/calmodulin-dependent protein kinase kinase 2,Ca MKK2）是激活AMPK的上游激酶。表皮生长因子受体（epidermal growth factor receptor,EGFR）在多种癌细胞中高表达,在生长因子等刺激下发生自磷酸化后激活细胞内信号通路,促进癌细胞增殖与迁移。TCTP是否调控Ca MKK2/AMPK和EGFR通路尚不明确。乳腺癌和肺癌分别是发病率和死亡率最高的癌症,本文利用乳腺癌细胞MDA-MB-231与肺癌细胞A549和H1299探究双氢青蒿素靶向TCTP抑制癌细胞增殖与迁移的机制。我们通过CCK-8实验检测了不同浓度的双氢青蒿素（0、20、40、80、160和200μM）处理MDA-MB-231、A549和H1299细胞24 h对细胞活力的影响,结果显示,双氢青蒿素以剂量依赖的方式抑制了细胞活力,并在80μM达到半抑制浓度（IC50）,因此,我们后续采用80μM处理癌细胞24 h探究其作用机制。用低浓度双氢青蒿素（0、4和8μM）处理MDA-MB-231细胞10天进行克隆形成实验,结果显示,双氢青蒿素以剂量依赖的方式抑制了癌细胞克隆形成。我们进一步通过鸡胚尿囊膜异种移植瘤模型检测了双氢青蒿素对体内肿瘤的抑制作用,发现800μM双氢青蒿素处理肿瘤7天显著抑制了移植瘤的生长。接着,我们通过划痕实验检测了双氢青蒿素对癌细胞迁移能力的影响,结果显示,双氢青蒿素显著抑制了MDA-MB-231、A549和H1299细胞的划痕闭合率。为探究双氢青蒿素是否通过抑制糖酵解来抑制癌细胞增殖和迁移,我们用双氢青蒿素处理MDA-MB-231、A549和H1299细胞后,通过生化试剂盒以及western blot实验检测双氢青蒿素对癌细胞糖酵解的影响,结果显示,双氢青蒿素降低了癌细胞中丙酮酸和乳酸的含量,并且下调了控制葡萄糖摄取的葡萄糖转运蛋白4（glucose transporter 4,GLUT4）和以及糖酵解反应的第一个限速酶—己糖激酶2（hexokinase 2,HK2）的表达。使用糖酵解抑制剂2-DG和双氢青蒿素单独处理H1299细胞后,细胞增殖标志物PCNA的蛋白表达和细胞迁移率均显著降低,说明双氢青蒿素通过抑制糖酵解抑制癌细胞的增殖和迁移。为探究双氢青蒿素是否通过TCTP调控癌细胞的糖酵解,我们用双氢青蒿素处理MDA-MB-231、A549和H1299细胞后发现TCTP的表达显著下调,在H1299和MDA-MB-231细胞中用si RNA敲低TCTP显著降低了HK2的蛋白表达和癌细胞的迁移率,提示双氢青蒿素通过下调TCTP抑制糖酵解进而抑制癌细胞迁移。Ca MKK2激活AMPK,该过程受到Ca²⁺浓度调控。缺氧诱导因子（hypoxia inducible factor-1α,HIF-1α）通过激活糖酵解基因的转录促进糖酵解,AMPK能够负向调控HIF-1α介导的糖酵解。为探究双氢青蒿素是否通过Ca MKK2/AMPK通路调控癌细胞糖酵解,我们用钙离子探针Fluo-4AM检测MDA-MB-231与H1299细胞中的Ca²⁺浓度,发现双氢青蒿素升高了癌细胞内的Ca²⁺浓度。双氢青蒿素处理MDA-MB-231、A549和H1299细胞后,增加了p-Ca MKK2（Ser511）和p-AMPK（Thr172）的表达,即激活了Ca MKK2/AMPK通路。随后我们检测双氢青蒿素处理后HIF-1α的表达,结果显示,双氢青蒿素显著降低了HIF-1α蛋白水平。当用AMPK的抑制剂Compound C处理MDA-MB-231细胞后,HIF-1α的表达增加,而Compound C与双氢青蒿素联合处理反转了双氢青蒿素对HIF-1α表达的降低作用,说明双氢青蒿素通过激活Ca MKK2/AMPK通路下调HIF-1α的表达。为探究TCTP是否调控Ca MKK2/AMPK通路,我们在敲低TCTP后检测相关蛋白表达,结果显示,在MDA-MB-231和H1299细胞中敲低TCTP,p-Ca MKK2和p-AMPK的表达升高,HIF-1α的表达降低。以上结果表明双氢青蒿素通过下调TCTP激活Ca MKK2/AMPK通路进而抑制了HIF-1α介导的糖酵解。EGFR激活黏着斑激酶（focal adhesion kinase,FAK）后能够增加细胞内基质金属蛋白酶（matrix metalloproteinases,MMPs）的表达和分泌来促进癌细胞迁移。我们用双氢青蒿素处理MDA-MB-231、A549和H1299细胞后,发现EGFR、p-EGFR、FAK、p-FAK、MMP2和MMP9的蛋白表达显著降低。为探究双氢青蒿素是否通过TCTP抑制EGFR信号通路,我们在MDA-MB-231和H1299细胞中敲低TCTP检测EGFR的表达及其活性变化,western blot结果显示,EGFR和p-EGFR的表达均降低。当用EGFR抑制剂Erlotinib处理MDA-MB-231和H1299细胞后,FAK、p-FAK以及MMP2/9的表达降低,提示EGFR通过FAK/MMP2/9促进癌细胞迁移。另外,我们也检测了EGFR对癌细胞增殖的影响,发现Erlotinib处理后,癌细胞中PCNA的表达显著降低。这些结果表明双氢青蒿素通过下调TCTP抑制EGFR信号通路而抑制了癌细胞增殖与迁移。在本文中,我们首次证明了双氢青蒿素通过下调TCTP激活Ca MKK2/AMPK信号通路和抑制EGFR信号通路,从而抑制癌细胞的增殖与迁移,为双氢青蒿素的抑癌作用提供新的分子靶标及实验依据。更多还原

【Abstract】 Cancer is an important factor endangering human health.The commonly used anticancer drugs have side effects on human body can′t be neglected,therefore it is a potential treatment method to explore the anti-tumor natural compounds with low toxicity for human body.Dihydroartemisinin,an artemisinin derivative,shows effective antitumor effect in vitro and in vivo research.Cancer cells rely on glycolysis to provide energy and acidic microenvironment for proliferation and migration,which makes targeting glycolysis potentially an effective method for cancer treatment.As a target of dihydroartemisinin,translationally controlled tumor protein（TCTP）promotes the proliferation,migration and invasion of cancer cells.However,the anti-cancer mechanism of dihydroartemisinin through TCTP is rarely reported.AMP-activated protein kinase（AMPK）,a main sensor of cell energy state,negatively regulates glycolysis,and calcium/calmodulin-dependent protein kinase kinase 2（Ca MKK2）is an upstream kinase to activate AMPK.Epidermal growth factor receptor（EGFR）autophosphorylation stimulated by growth factors activates intracellular signaling pathways to participate in proliferation,migration and invasion of cancer cells.Whether TCTP regulates Ca MKK2/AMPK and EGFR pathways to promote cancer is unclear.Breast and lung cancer are the cancers with the highest incidence and mortality respectively.In this study,breast cancer cell lines MDA-MB-231 and lung cancer cell lines A549 and H1299 were used to investigate the anti-cancer mechanism of dihydroartemisinin through TCTP.MDA-MB-231,A549 and H1299 cancer cells were exposed to different concentrations of dihydroartemisinin（0,20,40,80,160 and 200μM）,and CCK-8 assay was performed to test cell viability.The results showed that dihydroartemisinin inhibited cancer cell proliferation in a dose-dependent manner and reached the half inhibitory concentration（IC50）at 80μM.Therefore,we subsequently treated cancer cells with 80μM dihydroartemisinin for 24 h to explore the anti-cancer mechanism.MDA-MB-231 cells were treated with dihydroartemisinin（0,4 and 8μM）for 10 days to test the ability of colony formation,and the results showed that dihydroartemisinin inhibited the colony formation of cancer cells in a dose-dependent manner.We further tested the anti-tumor effect of dihydroartemisinin in vivo through chicken embryo allantoic membrane xenograft tumor model,and the results showed that the volume of transplanted tumor was significantly reduced with dihydroartemisinin（800μM）treatment for 7 days.Then,we tested the effect of dihydroartemisinin on the migration ability of cancer cells by scratch test.The results showed that dihydroartemisinin significantly inhibited the wound closures of cancer cells in MDA-MB-231,A549 and H1299 cancer cells.Due to the high glycolysis rates,glycolysis related proteins in cancer cells are usually highly expressed,such as glucose transporter 4（GLUT4）,which controls glucose uptake,and hexokinase 2（HK2）,the first rate-limiting enzyme of glycolysis reaction.The energy and acidic microenvironment generated by glycolysis promote the proliferation and migration of cancer cells.We further explored whether dihydroartemisinin inhibits cancer cell proliferation and migration by inhibiting glycolysis.Exposed the cancer cells to dihydroartemisinin,and the effect of dihydroartemisinin on glycolysis was detected by related kits and western blot.The results showed that dihydroartemisinin decreased the levels of pyruvate and lactate,and down-regulated the expression of GLUT4 and HK2 in cancer cells.After H1299 cancer cells were treated with the glycolysis inhibitor（2-DG）or dihydroartemisinin,both the protein level of PCNA and migration capacity were significantly reduced.These results suggested that dihydroartemisinin inhibited cancer cell proliferation and migration by inhibiting glycolysis.Next,to investigate whether dihydroartemisinin affects glycolysis through TCTP,we detected protein level of TCTP in cancer cells after dihydroartemisinin treatment.The results showed that the expression of TCTP was significantly down-regulated after dihydroartemisinin treatment.TCTP knockdown decreased protein expression of HK2 and migration capacity in H1299 and MDA-MB-231 cancer cells transfected with TCTP si RNA,indicating that dihydroartemisinin inhibits glycolysis by down-regulating TCTP.Ca MKK2,one of the upstream of AMPK,is regulated by Ca²⁺concentration.Transcription factor hypoxia inducible factor-1α（HIF-1α）promotes glycolysis by activating glycolytic gene transcription,and AMPK negatively regulates HIF-1αmediated glycolysis.To explore whether dihydroartemisinin regulates glycolysis through Ca MKK2/AMPK pathway,we detected the Ca²⁺concentration by Fluo-4 AM in MDA-MB-231 and H1299 cells and found that dihydroartemisinin increased the intracellular Ca²⁺concentration.Dihydroartemisinin treatment increased the expression of p-Ca MKK2（Ser511）and p-AMPK（Thr172）in MDA-MB-231,A549 and H1299 cells,which activated the Ca MKK2/AMPK pathway.Subsequently,we detected the expression of HIF-1αin cancer cells,and found that protein levels of HIF-1αwere significantly reduced after dihydroartemisinin treatment.When MDA-MB-231 cells were treated with Compound C,an AMPK inhibitor,HIF-1αexpression was increased,while Compound C combined with dihydroartemisinin reversed the reducing effect of dihydroartemisinin on HIF-1αexpression.These results suggested that dihydroartemisinin inhibits HIF-1αexpression by activating the Ca MKK2/AMPK pathway.To investigate whether TCTP regulates Ca MKK2/AMPK signaling pathway,TCTP knockdown in MDA-MB-231 and H1299 cells to detect the expression of related proteins.The results showed that,TCTP knockdown increased the expression of p-Ca MKK2 and p-AMPK,and decreased the expression of HIF-1α.Above results suggested that dihydroartemisinin activated the Ca MKK2/AMPK pathway by down-regulating TCTP and then inhibits HIF-1αmediated glycolysis.EGFR activates focal adhesion kinase（FAK）,which increases the expression and secretion of matrix metalloproteinases（MMPs）to promote cancer cell migration by degrading extracellular matrix.After MDA-MB-231,A549 and H1299 cells were treated with dihydroartemisinin,the protein levels of EGFR,p-EGFR,FAK,p-FAK and MMP2/9 were significantly reduced.To investigate whether dihydroartemisinin inhibits EGFR signaling pathway through TCTP,we knocked down TCTP in MDA-MB-231 and H1299 cells and found that the protein levels of EGFR and p-EGFR were decreased.When MDA-MB-231 and H1299cells were treated with EGFR inhibitor Erlotinib,the protein levels of FAK,p-FAK and MMP2/9were decreased,suggesting that EGFR promotes cancer cell migration through FAK/MMP2/9pathway.In addition,we detected the effect of EGFR on cancer cell proliferation,and the expression of PCNA was reduced after EGFR inhibitor Erlotinib treatment in cancer cells.These results suggested that dihydroartemisinin inhibited the EGFR signaling pathway by down-regulating TCTP,and then suppressed the proliferation and migration of cancer cells.In this study,we proved for the first time that dihydroartemisinin activates the Ca MKK2/AMPK signaling pathway and inhibits the EGFR signaling pathway by down-regulating TCTP,and then inhibits cancer cell proliferation and migration,which provides a new molecular target and experimental basis for the anticancer effect of dihydroartemisinin.更多还原