金丝桃素介导的光动力学疗法对K562细胞活性与自噬的影响

The effect of Hyp-PDT on K562 activity and autophagy pathway of leukemia cells cultured in vitro

目的:观察金丝桃素介导的光动力学疗法(Hyp-PDT)对慢性髓性白血病细胞(K562细胞株)活性与自噬的影响。方法:体外培养K562细胞株经过含金丝桃素(0.4μg/mL)的培养液避光孵育后,采用0.3 m W/cm^2光强度及照射4 min实施Hyp-PDT,CCK-8法测定细胞活性。收集照射前,照射后4、8、16 h的细胞悬液,分别在光镜和电镜下观察细胞形态学及自噬体数量,Western blot检测自噬相关蛋白LC3、Beclin-1、p-Akt、Akt的表达水平。结果:金丝桃素组光照后细胞活性随时间延长逐渐降低,与光照前比较,差异均有统计学意义(P<0.01),且与正常细胞组、DMSO细胞组比,所有时间点的细胞活性均显著降低(均P<0.01)。Hyp-PDT干预后,金丝桃素组光照后部分细胞出现细胞肿胀变性甚至溶解破裂。透射电镜显示K562细胞照射前即存在自噬体,照射后8 h DMSO组中的自噬体数量开始增多,但金丝桃素组的自噬体数量明显减少。Western blot结果显示:照射后8 h和16 h时金丝桃素组LC3蛋白表达量减少,而DMSO组LC3蛋白含量呈上升趋势;照射后16 h,与DMSO组比,金丝桃素组的Beclin-1蛋白含量明显下降,p-Akt蛋白表达量明显减少(均P<0.01)。结论:Hyp-PDT可能通过抑制p-Akt磷酸化,影响PI3K-Akt通路,减少K562自噬,促进细胞死亡,起到杀伤白血病肿瘤细胞的作用。

Objective:To explore the effect of Hyp-PDT(Hypericin-mediated photodynamic therapy)on K562 activity and autophagy pathway of leukemia cells cultured in vitro.Methods:Human leukemia cell line K562 was cultured in vitro.Hyp-PDT was performed with 0.3 mW/cm2 light intensities and 4 min irradiation times.The CCK8 analysis was applied and the results were analyzed.Cell protein was collected instantly before irradiation,4 h,8 h and 16 h after irradiation respectively for Western blot determination of autophagy related protein.Morphological changes of K562 were observed under phase contrast microscope and electron microscopy.Results:Hyp-PDT with the light intensity of 0.3 mW/cm2 significantly decreased cell viabilities in K562 post irradiation at different time points(all P<0.01).After Hyp-PDT intervention,some cells in hypericin group showed swelling,degeneration and even dissolution after light irradiation.There were several cases of autophagy in K562 cells before irradiation,8 h after irradiation,and autophagy increased in cells treated with DMSO but decreased obviously or even disappeared in Hyp group.The expression of LC3 protein decreased in Hyp group but increased slightly in DMSO group 8 h and 16 h after irradiation.The expression of Beclin-1 decreased 16 h after Hyp-PDT and a synchronous decrease of p-Akt protein was observed. Conclusion: Hyp-PDT of K562 invitro can promote cell fatality by inhibiting autophagy in K562 cells. It may be related to the inhibition of Aktpathway.