ABCG2抑制剂对ALA-PDT诱导皮肤癌细胞凋亡的影响

Effect of ABCG2 inhibitor on apoptosis of different skin cancer cells induced by ALA-PDT

目的探讨三磷酸腺苷结合转运蛋白G超家族成员2(ATP-binding cassette transporter group 2,ABCG2)抑制剂Ko-143对5-氨基乙酰丙酸-光动力疗法(5-aminolaevulinic acid-photodynamic therapy,ALA-PDT)诱导人皮肤基底癌细胞TE354.T和皮肤鳞癌细胞SCL-1凋亡的影响。方法体外培养TE354.T和SCL-1细胞,分为对照组、ALA-PDT组(2 mmol·L^(-1)5-ALA+PDT)、Ko-143组(10μmol·L^(-1)Ko-143)和ALA-PDT+Ko-143组(2 mmol·L^(-1)5-ALA+10μmol·L^(-1)Ko-143+PDT),应用MTT法检测细胞增殖,应用流式细胞术检测细胞凋亡,应用ELISA法检测细胞中谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性和丙二醛(malonydialdehyed,MDA)含量,应用蛋白印迹(Western blot)法检测细胞中原卟啉IX(protoporphyrin IX,PPIX)、ABCG2、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)和活化型半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)蛋白的表达。结果与对照组相比,ALA-PDT组和Ko-143组TE354.T、SCL-1细胞增殖抑制率、凋亡率、细胞中MDA的含量及PPIX和Cleaved caspase-3蛋白的表达水平均显著升高(P<0.05),细胞中GSH-Px的活性及Bcl-2/Bax蛋白的表达水平均显著降低(P<0.05),ALA-PDT组TE354.T和SCL-1细胞中ABCG2表达水平的差异均无统计学意义(P>0.05),Ko-143组TE354.T和SCL-1细胞中ABCG2蛋白的表达水平均显著降低(P<0.05);与ALA-PDT组相比,ALA-PDT+Ko-143组TE354.T、SCL-1细胞增殖抑制率、凋亡率及细胞中MDA含量、PPIX和Cleaved caspase-3蛋白表达水平显著升高(P<0.05),细胞中GSH-Px活性及细胞中ABCG2和Bcl-2/Bax蛋白的表达水平显著降低(P<0.05)。结论Ko-143可抑制ABCG2的表达,导致PPIX蛋白在细胞中的蓄积水平升高,进而提高ALA-PDT诱导TE354.T、SCL-1细胞氧化应激损伤和凋亡水平。

Objective To investigate the effect of ATP-binding cassette transporter group 2(ABCG2)inhibitor Ko-143 on apoptosis of human skin basal carcinoma cell line TE354.T and skin squamous cell carcinoma cell line SCL-1 induced by 5-aminolaevulinic acid-photodynamic therapy(ALA-PDT).Methods TE354.T and SCL-1 cells were cultured in vitro and divided into control group,ALA-PDT group(2 mmol·L^(-1)5-ALA+PDT),Ko-143 group(10μmol·L^(-1)Ko-143)and ALA-PDT+Ko-143 group(2 mmol·L^(-1)5-ALA+10μmol·L^(-1)Ko-143+PDT).MTT assay and flow cytometry were used to detect the cell proliferation and apoptosis.The activity of glutathione peroxidase(GSH-Px)and the content of malondialdehyde(MDA)were detected by ELISA.The protein expression of protoporphyrin IX(PPIX),ABCG2,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cleaved caspase-3 were detected by Western blot.Results Compared with those in the control group,the proliferation inhibition rate and apoptosis rate of TE354.T and SCL-1 cells,the content of MDA,the protein levels of PPIX and cleaved caspase-3 in ALA-PDT group and Ko-143 group were significantly higher(P<0.05),the activity of GSH-Px and protein level of Bcl-2/Bax were significantly lower(P<0.05),there was no significant difference in ABCG2 in TE354.T and SCL-1 cells in ALA-PDT group(P>0.05),the level of ABCG2 protein in TE354.T and SCL-1 cells in Ko-143 group were significantly lower(P<0.05).Compared with those in ALA-PDT group,the proliferation inhibition rate and apoptosis rate of TE354.T and SCL-1 cells,the content of MDA,the protein levels of PPIX and cleaved caspase-3 in ALA-PDT+Ko-143 group were significantly higher(P<0.05),and the activity of GSH-Px and protein levels of ABCG2 and Bcl-2/Bax were significantly lower(P<0.05).Conclusion Ko-143 can inhibit the expression of ABCG2,increase the accumulation level of PPIX protein in cells,and then increase the oxidative stress injury and apoptosis level of TE354.T and SCL-1 cells induced by ALA-PDT.