Li Qing, Wang Xiaobing, Wang Pan, Zhang Kun, Wang Haiping, Feng Xiaolan, Liu Quanhong
1 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, China .
Cancer Biother Radiopharm. 2014 Feb;29(1):42-52. doi: 10.1089/cbr.2013.1526. Epub 2013 Nov 9.
The present study aims to investigate the antitumor effect and possible mechanisms of chlorin e6 (Ce6)-mediated sono-photodynamic therapy (Ce6-SPDT) on murine 4T1 mammary cancer cells in vitro.
Cellular uptake and intracellular distribution of Ce6 in 4T1 cells were detected by flow cytometry and confocal microscope. Cells after loading with 1 μg/mL Ce6 were exposed to ultrasound at 1.0 MHz for up to 1 minute with an intensity of 0.36 W/cm2 and laser light with total radiation dose of 1.2 J/cm2. Cell viability and clonogenicity were determined by MTT assay and colony formation assay. Apoptosis was analyzed by DAPI staining, Western blots were used to detect the activity of Caspase-3. DNA damage, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) of 4T1 cells were also evaluated by flow cytometry. FD500 was employed to detect changes of membrane permeability after ultrasound.
Ce6 rapidly entered 4T1 cells within 4 hours after it has been added and displayed a mitochondria-localization pattern. Compared with sonodynamic therapy (SDT) and photodynamic therapy (PDT) alone, the combined SPDT treatment further enhanced cell viability loss, DNA damage, and clonogenicity inhibition. DAPI staining and western blots analysis reflected that cells with apoptotic morphological characteristics and the activity of Caspase-3 were apparently increased in the combined group. Besides, SPDT caused obvious MMP loss and intracellular ROS generation at early 1 hour post treatment. Interestingly, the SPDT induced cell viability loss and cell apoptosis was greatly inhibited by pre-treatment with ROS scavenger N-acetylcysteine and Caspase inhibitor z-VAD-fmk. FD500 detection showed that ultrasound enhanced cell membrane permeability, implying much higher uptake of Ce6 might be involved in PDT therapy by pre-ultrasound treatment.
The findings demonstrated that Ce6-mediated SPDT enhanced the antitumor efficacy on 4T1 cells compared with SDT and PDT alone, a Caspase-dependent apoptosis and loss of MMP, generation of ROS may be involved.
本研究旨在探讨二氢卟吩e6（Ce6）介导的声动力疗法（Ce6-SPDT）对小鼠4T1乳腺癌细胞的体外抗肿瘤作用及可能机制。
采用流式细胞术和共聚焦显微镜检测Ce6在4T1细胞中的摄取及细胞内分布。用1μg/mL Ce6负载细胞后，以0.36W/cm2的强度在1.0MHz频率下超声照射1分钟，并用总辐射剂量为1.2J/cm2的激光照射。通过MTT法和集落形成试验测定细胞活力和克隆形成能力。通过DAPI染色分析细胞凋亡，用蛋白质免疫印迹法检测Caspase-3的活性。还用流式细胞术评估4T1细胞的DNA损伤、线粒体膜电位（MMP）和细胞内活性氧（ROS）。采用FD500检测超声作用后细胞膜通透性的变化。
Ce6加入后4小时内迅速进入4T1细胞，并呈现线粒体定位模式。与单独的声动力疗法（SDT）和光动力疗法（PDT）相比，联合SPDT治疗进一步增强了细胞活力丧失、DNA损伤和克隆形成抑制。DAPI染色和蛋白质免疫印迹分析表明，联合组中具有凋亡形态特征的细胞和Caspase-3的活性明显增加。此外，SPDT在治疗后1小时早期导致明显的MMP丧失和细胞内ROS生成。有趣的是，用ROS清除剂N-乙酰半胱氨酸和Caspase抑制剂z-VAD-fmk预处理可大大抑制SPDT诱导的细胞活力丧失和细胞凋亡。FD500检测表明超声增强了细胞膜通透性，这意味着超声预处理可能使更多的Ce6摄取参与PDT治疗。
研究结果表明，与单独的SDT和PDT相比，Ce6介导的SPDT增强了对4T1细胞的抗肿瘤疗效，可能涉及Caspase依赖性凋亡、MMP丧失和ROS生成。
