Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, China.
Cancer Biother Radiopharm. 2013 Apr;28(3):207-17. doi: 10.1089/cbr.2012.1190. Epub 2013 Mar 18.
The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT).
HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm(2) in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4'-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP).
Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment.
The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly induced apoptosis of U937 cells, suggesting that HMME may be a good sonosensitizer, and HMME-SDT might be a potential therapeutic strategy for cancer treatment.
本研究旨在探讨血卟啉单甲醚（HMME）介导的声动力学疗法（SDT）诱导 U937 细胞凋亡的作用。
HMME 浓度保持在 10μg/mL。肿瘤细胞悬浮在无血清 RPM1640 中，在 1.1MHz 超声下作用 60 秒，强度为 1W/cm²，有无 HMME 存在。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化盐（MTT）试验测定细胞活力。采用 Annexin V-PE/7-ADD 染色流式细胞术以及 4'-6-二脒基-2-苯基吲哚（DAPI）染色荧光显微镜分析细胞凋亡。用流式细胞术分析 U937 细胞的 DNA 损伤、细胞内活性氧（ROS）和线粒体膜电位（MMP）。Western blot 和逆转录-聚合酶链反应用于分析 caspase-3 和多聚（ADP-核糖）聚合酶（PARP）的蛋白和 mRNA 表达水平。
荧光成像显示 HMME 主要定位于线粒体。MTT 试验显示 SDT 后 4 小时细胞存活率为 55.6%。流式细胞术分析显示，HMME 介导的 SDT 使 U937 细胞早期和晚期凋亡细胞群显著增加（35.6%）。与对照组、超声组和 HMME 组相比，联合 SDT 组细胞内 ROS 和 MMP 损失显著增加。DAPI 染色也发现明显的核浓缩，SDT 治疗后 2 小时 DNA 片段增加到 33.9%。免疫荧光染色显示 SDT 后 Bax 明显易位。Western blot 显示 caspase-3 和 PARP 明显裂解。此外，SDT 后 U937 细胞 caspase-3 和 PARP mRNA 表达显著增加。
研究结果表明，HMME 介导的声动力学作用（HMME-SDT）显著诱导 U937 细胞凋亡，提示 HMME 可能是一种良好的声敏剂，HMME-SDT 可能是癌症治疗的一种潜在治疗策略。
