X线诱导肺腺癌SPC-A-1细胞发生Parthanatos

X-ray irradiation induces parthanatos in human lung adenocarcinoma SPC-A-1 cells

目的初步探讨X线诱导肺癌SPC-A-1细胞是否发生新的细胞死亡形式Parthanatos。方法以肺腺癌SPC-A-1细胞为研究对象,以1、2、4、6、8、10 Gy梯度剂量辐射细胞,平板克隆法测定X线辐射对人肺癌细胞SPC-A-1的半数致死剂量(LD50),WST-1法进行验证。LD50辐射细胞后4、8、12、24 h,RT-PCR法检测细胞AIF转录水平变化,Western blot检测细胞AIF蛋白表达变化,激光共聚焦观察细胞AIF向细胞核内转移情况,Western blot检测细胞辐射后不同时间AIF在线粒体蛋白组分和核蛋白组分中的分布变化。TUNEL法检测辐射后12 h细胞核DNA的断裂。结果 X线对SPC-A-1细胞的LD50大致为3 Gy。以3 Gy辐射细胞后不同时间AIF在转录、蛋白水平均无显著变化(P>0.05)。辐射后12 h AIF由线粒体向细胞核转位,伴随细胞核的溶解、碎裂。亚细胞组分Western blot检测到12 h后AIF在线粒体蛋白组分中显著下降,由对照组的1.12±0.15下降至12 h组的0.21±0.07(P<0.05),而在核蛋白组分中显著上升,由对照组的0.05±0.02上升至12 h组的0.45±0.07(P<0.05),同时在固缩的细胞核中检测到DNA的断裂。结论 AIF核转位参与了X线诱导的细胞凋亡过程。辐射后12 h AIF由线粒体向细胞核转位显著增多,导致细胞核固缩、碎裂、溶解,细胞发生了Parthanatos。

Objective Parthanatos （thanatos is god of death in Greek mythology） is a new form of cell death, which often occurs under the many kinds of stimulus. We investigate whether it occurs in SPC-A- 1 cells induced by X-ray irradiation. Methods Immunohistoehemical assay and immunofluoreseenee staining were used to confirm the expression of apoptosis inducing factor （AIF） located in normal culture of SPC-A-1 cells. Then the median lethal dose （ LD50 ） of SPC-A-1 cells was determined after the cells were treated byl, 2, 4, 6, 8 and 10 Gy X-rays irradiation by colony-forming assay and then confirmed by WST-1 assay. The expression of AIF at mRNA and protein levels was measured by RT-PCR and Western blot assay respectively after the cells were treated by LD50 X-rays irradiation for 4, 8, 12 or 24 h. At the same time, AIF translocation from the mitochondria to the nucleus was observed by laser scanning eonfocal microscopy. Western blotting was used to detect the changes of AIF redistribution in the mitochondrial and nuclear protein components. The nuclear DNA strand breaks were observed by TUNEL assay after 12 hours＇ treatment. Results The LD50 of SPC-A-1 cells was 3 Gy to X-ray irradiation. No obvious changes were seen in the mRNA or protein levels of AIF at the different time points after irradiation treatment （P 〉0.05 ）. AIF was translocated from the mitochondrium to nucleus, accompanied by nuclear DNA dissolution and fragmentation after 12 hours＇ treatment. The AIF level was decreased from 1.12 ±0.15 to 0.21 ±0.07 in the mitochondrial components after 12 hours＇ treatment （P 〈 0. 05 ）, and that in the nuclear components was increased from 0. 05 ±0.02 to 0.45 ±0.07 （ P 〈 0. 05 ）. The DNA strand breaks were detected in the nuclear at the same time point. Conclusion Nuclear translocation of AIF is involved in the death of SPC-A-1 cells induced by X-ray irradiation. X-ray irradiation of 3 Gy for 12 h results in the translocation of AIF from mitochondrium to nucleus, and then nuclear chromatin condensation, fragmentation and dissolution, and then Parthanatos.