【作者】 胡倩；

【导师】 赵秋；

【作者基本信息】 武汉大学 ， 内科学及消化内科， 2022， 博士

【摘要】 目的:铁死亡是一种新型非凋亡的程序性细胞死亡。其特征表现是铁积累和致死量的脂质过氧化物堆积。由于癌症中常常存在铁超载和高上皮间质转化的情况,铁死亡诱导有望作为癌症的潜在治疗方式。尽管很多研究都证明了经典的铁死亡激动剂Erastin和RSL3在多种肿瘤中的治疗价值,包括肺癌、卵巢癌、胰腺癌等,但是铁死亡在结直肠癌中的作用和应用前景尚不清楚。目前的研究显示,很多结直肠癌细胞系都表现出铁死亡抵抗,然而关于结直肠癌中发生铁死亡抵抗的原因和应对措施却知之甚少。一些研究表明谷胱甘肽还原酶4(glutathione peroxidase 4,GPX4)的高表达可能是结直肠癌铁死亡抵抗的部分原因,然而这很难解释部分GPX4低表达的结直肠癌细胞仍然对铁死亡抵抗。最近有研究报道GTP环化水解酶1/四氢生物蝶呤(GTP cyclohydrolase-1/tetrahydrobiopterin,GCH1/BH4)代谢通路可以独立于GPX4在细胞内发挥强大的抗铁死亡的作用。本研究试图明确GCH1/BH4是否也可以在结直肠癌中发挥抗铁死亡的作用和阐明其背后的机制,从而为将来更好得应用铁死亡激动剂治疗结直肠癌提供更有效的靶点。方法:首先,我们在四株结直肠癌细胞系中分别给予铁死亡激动剂,检测细胞活力。然后应用高效液相色谱法(High performance liquid chromatography,HPLC)检测不同细胞系中BH4的含量。利用细胞实验和生物信息学技术初步评估GCH1/BH4代谢和铁死亡的关系。其次,通过GCH1敲低和BH4补充实验,评估GCH1/BH4代谢对结直肠癌铁死亡的影响。本研究在体外实验中通过检测以下指标评价铁死亡的状态:细胞活力、脂质过氧化水平、胞质内和线粒体内游离铁染色等。为进一步探究GCH1/BH4代谢参与铁死亡调控的机制,我们应用蛋白免疫印记(Western blot)技术进一步检测了铁自噬相关蛋白的变化,然后用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3MA)进行干预。最后,在体外和裸鼠皮下成瘤的体内实验中,探究GCH1药物抑制剂DAHP与铁死亡激动剂Erastin联用的效果。结果:四株结直肠癌细胞的铁死亡敏感性存在差异,其中HCT116和HT29细胞的敏感性最差,造成其对铁死亡激动剂反应性差的原因可能是BH4而非GPX4的高表达。敲低GCH1基因,可以显著降低细胞内的BH4含量,促进由Erastin诱导的细胞死亡、脂质过氧化和游离铁增加。与此同时,补充BH4可以逆转上述变化。敲低GCH1时,铁蛋白下调,核蛋白受体共激活因子4(Nuclear receptor coactivator 4,NCOA4)上调,LC3B-Ⅰ更多的剪切为LC3B-Ⅱ,因而铁自噬被激活。与此相符,3MA抑制剂可以逆转GCH1敲除促进的铁死亡。最后,联合应用DAHP和Erastin可以在体内、体外诱导铁死亡,并且显著抑制皮下瘤的生长。结论:GCH1/BH4代谢与结直肠癌铁死亡抵抗有关,靶向BH4代谢的关键限速酶GCH1可以活化铁蛋白自噬,从而增加结直肠癌的铁死亡敏感性。联合应用GCH1抑制剂和铁死亡激动剂有望成为新的结直肠癌治疗策略。更多还原

【Abstract】 Objective Ferroptosis is a novel non-apoptotic form of regulated cell death,characterized by iron accumulation and lethal lipid peroxidation production.Since iron overload and high levels of epithelial mesenchymal transformation(EMT)exists in various cancer cells,it makes ferroptosis induction a promising strategy in cancer therapy.Although many studies have demonstrated that erastin and RSL3,the classical ferroptosis inducers,widely inhibit tumor growth such as lung cancer,ovarian cancer,and pancreatic cancer.The role and potent application of ferroptosis in colorectal cancer(CRC)are elusive.Several CRC cell lines have been reported to be more resistant to ferroptosis than other cancer cells.However,few study figures out the reason of CRC ferroptotic resistance and copes strategy to ameliorate the resistance.Currently,some studies confirmed that the CRC ferroptotic resistance is partly due to high expression of glutathione reductase 4(GPX4).However,those studies fail to explain why some CRC cells with low GPX4 levels are resistant to ferroptosis induction.Recent studies have reported that GTP cyclohydrolase-1/tetrahydrobiopterin(GCH1/BH4)prevents cells from lipid peroxidation damage during ferroptosis induction;it is parallel to the GPX4 redox system.Nonetheless,the role of GCH1/ BH4 metabolism in CRC ferroptosis is unknown.Thus,our study sought to investigate the function and underlying regulatory mechanism of GCH1/BH4 metabolism in CRC cells that are resistant to ferroptosis,which provide novel efficient target for triggering ferroptosis in individualized tumor therapy.Methods Firstly,we administrated ferroptosis agonists to four CRC cell lines and evaluated the cell viability.Then we used high performance liquid chromatography(HPLC)to detected the content of BH4 in CRC cells.Combining cell experiments and bioinformatics technology,we roughly judged the correlation between GCH1/BH4 metabolism and ferroptosis.Secondly,we confirmed the role of GCH1/BH4 metabolism in CRC ferroptosis by GCH1 knockdown and BH4 supplementation treatment.Several ferroptotic parameters including cell viability,lipid peroxidation level,intracellular and mitochondrial ferrous iron were used to determine the activation of ferroptosis in vitro.Mechanistically,we further detected the changes of ferritinophagy related proteins by Western blot,and we used 3MA,an autophagy inhibitor,to reverse ferroptosis.Finally,we combined use of DAHP,a GCH1 inhibitor and ferroptosis inducer in vitro and in vivo,in order to determine the antitumor effects of combination treatment.Results The sensitivity of ferroptosis were diverse in four CRC cells,among which HCT116 and HT29 cells possessed poor reactivity.The poor response to ferroptosis agonist in HCT116 and HT29 cells seemed relative to high expression of BH4 rather than GPX4.Knockdown of GCH1 dramatically diminished the bio-production of BH4 in CRC and promoted cell death,lipid peroxidation and ferrous iron release induced by erastin.Meanwhile,supplementation of BH4 reversed the above changes.Besides,silencing GCH1 decreased ferritin but upregulated nuclear receptor coactivator 4(NCOA4)and promoted the transform of LC3B-Ⅰ to LC3B-Ⅱ.Correspondently,applying the autophagy inhibitor of 3-methyladenine(3MA)reversed the ferroptosis promoted by GCH1 knockdown.Thus,ferritinophagy was dramatically activated by GCH1 knockdown.Finally,the combination of DAHP and erastin synergistically induced ferroptosis in vivo and in vitro,and significantly inhibited the growth of subcutaneous tumors.Conclusion GCH1/BH4 metabolism negative relative to ferroptosis in CRC.Inhibition of GCH1/BH4 metabolism promoted erastin-induced ferroptosis by activating ferritinophagy,which suggests that combining GCH1 inhibitor with erastin in the treatment of CRC is a novel therapeutic strategy.更多还原