Mppa介导的光动力疗法诱导人卵巢癌细胞凋亡

Methyl pyropheophorbide-a-mediated photodynamic therapy induces apoptosis in human ovarian cancer cells

目的探讨焦脱镁叶绿酸甲酯(methyl pyropheophorbide-a,Mppa)介导的光动力疗法(Mppa-PDT)抑制人卵巢癌SKOV3细胞活性、触发其凋亡的机制。方法将处于对数生长期的人卵巢癌SKOV3细胞随机分为Mppa-PDT处理组(加光敏剂Mppa和LED光照处理)和对照组(空白对照、仅加光敏剂Mppa单药对照、仅接受LED单光对照)。Mppa-PDT作用于人卵巢癌SKOV3细胞后,CCK-8法检测细胞活性;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率;DAPI染色观察细胞凋亡的核的形态学改变;DCFH-DA染色观察细胞内活性氧(ROS)的产生;单细胞凝胶电泳观察DNA损伤情况;蛋白质印迹法检测p53、Caspase-3、Bax、Bcl-2蛋白的表达变化。结果 (1)Mppa-PDT能显著抑制人卵巢癌SKOV3细胞的活性,其抑制作用具有一定的剂量依赖性;(2)Mppa-PDT诱导的人卵巢癌SKOV3细胞凋亡率高于空白对照、单药对照和单光对照组(P<0.05),且空白、单药、单光3组对照组间凋亡率差异无统计学意义(P>0.05);(3)Mppa-PDT作用于人卵巢癌SKOV3细胞后,DAPI细胞核荧光染色可见细胞核深染的凋亡细胞;DCFH-DA荧光染色发现Mppa-PDT组细胞内ROS水平高于3个对照组;单细胞凝胶电泳显示Mppa-PDT组的DNA损伤情况高于3个对照组;蛋白质印迹法检测发现p53、Caspase-3、Bax蛋白表达升高,Bcl-2蛋白表达降低(P<0.05)。结论 Mppa介导的光动力疗法能够抑制人卵巢癌SKOV3细胞活性并诱发其凋亡,且伴随有DNA损伤及线粒体凋亡途径的激活。

Objective To investigate the mechanism by which methyl pyropheophorbide-a-mediated photodynamic therapy （Mppa-PDT） inhibit cell viability and induce apoptosis in human ovarian cancer cell line SKOV3. Methods Human ovarian cancer cells SKOV3 at the logarithmic growth phase were divided into Mppa-PDT treated group （both Mppa and PDT treated group） and control groups （the blank group, the only Mppa treated group and only PDT treated group）. After Mppa-PDT treatment, the cell viability was examined with CCK-8 assay; cell apoptosis was detected by flow cytometry with Annexin V- FITC/PI; and nuclear morphological changes during cell apoptosis was detected by DAPI staning. Moreover, the celluar reactive oxygen species （ROS） were detected by DCFH-DA staining; DNA damage was observed by single cell gel electrophoresis; and the protein expression of p53, Caspase-3, Bax, and Bcl-2 were assessed by Western blotting analysis. Results （1） Mppa-PDT could greatly suppress the cell viability of human ovarian cancer cells SKOV3 in a dose-dependent manner. （2） The cell apoptosis rate of Mppa-PDT treated group was significanlty higher than those of three control groups （blank group, Mppa group and PDT group） （P^0. 05）, and there was no difference among the three control groups （P〉0. 05）. （3） After treating with Mppa-PDT, DAPI staining showed strongly stained nuclei of the apoptotic cells; DCFH-DA staining displayed higher level of ROS than those of the three control groups; single cell gel eleetrophoresis showed greater DNA damage than those of the three control groups; and Western blotting analysis showed that the expression of p53, Caspase-3 and Bax protein was increased and Bcl-2 protein was decreased （P〈0. 05）. Conclusion Mppa-PDT can significantly suppress cell viability and induce apoptosis in human ovarian cancer cell SKOV3, accompanied by DNA damage and the activation of mitochondrial apoptosis pathway.