砷制剂对禽白血病肿瘤细胞Warburg效应及Hedgehog信号通路的影响

Effects of arsenic preparations on the Warburg effect and Hedgehog signaling pathway in avian leukemia tumor cells

【目的】探究三氧化二砷(ATO)对禽白血病(AL)肿瘤细胞Warburg效应及Hedgehog信号通路的影响,明确ATO缓解AL鸡肝脏肿瘤病变的作用机制,为生产上以ATO治疗AL提供科学依据。【方法】开展急性毒性试验,确定ATO对绿壳蛋鸡的安全性;通过p27抗原检测、PCR扩增鉴定、gp85基因测序等对禽白血病病毒(ALV)进行鉴定,并采集病鸡肿瘤组织制作病理组织切片进行病理性诊断;体外培养AL鸡肿瘤细胞,分别加入0.5、1.0和2.0μmol/L ATO进行处理,培养12、24和48 h后收集细胞,分别检测葡萄糖、乳酸、己糖激酶(HK)及葡萄糖转运蛋白1(GLUT1)的含量;以ALV人工感染绿壳蛋鸡构建模型组,再用不同剂量(0.5、1.0和2.0 mg/kg·BW)ATO进行治疗,采用ELISA检测鸡肝脏组织中Hedgehog信号通路Gli1和Shh蛋白水平,并以实时荧光定量PCR检测Shh、Gli1、Gli2、Ptch2和Smo基因的表达情况。【结果】ATO对绿壳蛋鸡的半数致死量(LD_(50))为19.501 mg/kg·BW,LD_(50)-95%可信限为19.501±0.213 mg/kg·BW。从疑似患AL的绿壳蛋鸡中鉴定出1株ALV-J株,命名为GZCS01,其感染形成的肿瘤为肾母细胞瘤。以不同剂量(0.5、1.0和2.0μmol/L)ATO作用肾母细胞瘤细胞,培养48 h后发现细胞液中的葡萄糖、乳酸及HK含量均极显著降低(P<0.01,下同),同时能降低肾母细胞瘤细胞内的GLUT1含量,从而减弱肿瘤细胞Warburg效应;高剂量的ATO(2.0 mg/kg·BW)能极显著或显著抑制Shh和Gli1蛋白表达(P<0.05,下同),极显著或显著抑制Shh、Gli1、Gli2和Smo基因表达,同时极显著上调Ptch2基因表达。【结论】ATO能抑制肿瘤细胞对葡萄糖的摄取和乳酸生成,且降低关键酶生成,从而减弱Warburg效应,抑制肿瘤细胞增殖;ATO还可通过下调Hedgehog信号通路关键因子Shh、Gli1、Gli2和Smo的表达及促进Ptch2表达,从而减轻AL的肝脏病变。可见,使用砷制剂(如阿散酸等)治疗AL具有可行性。

【Objective】To explore the effects of arsenic trioxide(ATO)on Warburg effect and Hedgehog signaling pathway of avian leukemia(AL)tumor cells,to clarify the mechanism of ATO alleviating AL liver tumor lesions in chi-ckens,and to provide scientific basis for ATO treatment of AL in production.【Method】Acute toxicity test was carried out to determine the safety of ATO to green shell laying hens.The pathogen of avian leukemia virus(ALV)was identified by p27 antigen detection,PCR amplification and gp85 gene sequencing,and the pathological diagnosis was made by collec-ting the tumor tissue of infected chickens.AL chicken tumor cells were cultured in vitro and treated with 0.5,1.0 and 2.0μmol/L ATO,respectively.The cells were collected after culture for 12,24 and 48 h,and the contents of glucose,lactic acid,hexokinase(HK)and glucose transporter 1(GLUT1)were detected,respectively.Green shell laying hens were arti‐ficially infected with ALV to construct a model group,and then treated with different doses of ATO(0.5,1.0 and 2.0 mg/kg·BW).ELISA was used to detect the protein levels of Hedgehog signaling pathway Gli1 and Shh in chicken liver tis‐sue.The expression of Shh,Gli1,Gli2,Ptch2 and Smo genes was detected by real-time fluorescence quantitative PCR.【Result】The median lethal dose(LD_(50))of ATO for green shell laying hens was 19.501 mg/kg·BW,and the LD_(50)-95%confidence limit was 19.501±0.213 mg/kg·BW.An ALV-J strain named GZCS01 was identified from green shell laying hens suspected to have AL,and the tumor formed by infection was nephroblastoma.At different doses of ATO(0.5,1.0 and 2.0μmol/L),ATO was applied to wilms tumor cells.After 48 h culture,glucose content,lactate generation and HK contents in cell fluid were extremely significantly decreased(P<0.01,the same below),and GLUT1 content in wilms tu‐mor cells was also decreased.Thus,the Warburg effect of tumor cells was weakened.High dose of ATO(2.0 mg/kg·BW)could extremely significantly or significantly inhibit the expression of Shh and Gli1 protein(P<0.05,the same below),extremely significantly or significantly inhibit the expression of Shh,Gli1,Gli2 and Smo genes,and extremely signifi‐cantly up-regulate the expression of Ptch2 gene.【Conclusion】ATO can inhibit glucose uptake and lactic acid production of tumor cells,and reduce key enzyme production,thus weakening Warburg effect and inhibiting tumor cell proliferation.ATO can also reduce liver lesions in AL by down-regulating the expression of key factors in Hedgehog signaling pathway such as Shh,Gli1,Gli2 and Smo,and promoting the expression of Ptch2.It can be seen that the use of arsenic prepara‐tions(such as arsanic acid)in the treatment of AL is feasible.