光化激活9-羟基脱镁叶绿酸-α诱导人喉鳞状细胞癌HN-3细胞凋亡与迁徙抑制

何培杰; 毛雯静; 张焕康; 周梁

doi:10.13201/j.issn.1001-1781.2015.15.012

光化激活9-羟基脱镁叶绿酸-α诱导人喉鳞状细胞癌HN-3细胞凋亡与迁徙抑制

- 复旦大学附属眼耳鼻喉科医院耳鼻咽喉-头颈外科(上海, 200031)

详细信息

通讯作者: 周梁,E-mail:zhoulent@126.com

中图分类号: R739.62

收稿日期: 2015-03-25

Apoptosis and migration suppression of HN-3human laryngeal squamous cancer cells induced by photo-activation of 9-hydroxypheophorbide-α

- Department of Otolaryngology-Head and Neck Surgery, Affiliated Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, 200031, China

More Information

Corresponding author: ZHOU Liang, E-mail: zhoulent@126.com

Received Date: 25 March 2015

摘要

摘要: 目的:探讨新型光敏剂9-羟基脱镁叶绿酸-α(9-HPbD)介导的光动力疗法(PDT)对HN-3人喉鳞状细胞癌细胞的凋亡诱导、迁徙抑制作用及其机制。方法:以不同浓度的9-HPbD(0.29 μg/ml, 0.59 μg/ml)孵育贴壁的HN-3细胞6 h,664 nm二极管激光(能量密度为2.0 J/cm²)照射光敏化的HN-3细胞15 min。光照后立即行细胞划痕,并于光照后0、12、24、36 h分别在相同划痕位置进行拍照,计算细胞迁徙距离;光照后1 h行H₂DCFDA染色并分别以荧光法及流式细胞术测定活性氧生成(ROS);光照24 h后行MTT实验、Hoechst33342/PI双重染色、蛋白印迹法(Western blotting)评价细胞增殖活力、细胞核形态及eNOS、p-c-Jun、EGFR表达。结果:①9-HPbD-PDT显著抑制HN-3细胞增殖,9-HPbD单独孵育(未进行激光照射)与单纯激光照射均未显示对HN-3细胞的生长抑制。②PDT后HN-3细胞活性氧产生明显增加,细胞核浓缩、碎裂,eNOS、p-c-Jun表达上调。③9-HPbD-PDT显著抑制HN-3细胞迁徙能力,呈光敏剂剂量相关性。PDT后HN-3细胞EGFR表达下调。④以活性氧阻滞剂谷胱甘肽(GSH)、抗坏血酸预孵育HN-3细胞,光动力触发的凋亡诱导、迁徙抑制等被部分阻滞。 结论:①9-HPbD-PDT对HN-3细胞具有光化学毒性,p-c-Jun通路激活及eNOS表达上调、EGFR表达下调可能参与了细胞凋亡与迁徙抑制的诱导。②活性氧生成在9-HPbD-PDT诱导HN-3细胞凋亡与迁徙抑制中发挥重要作用。
- 喉肿瘤 /
- 鳞状细胞癌 /
- 光动力疗法 /
- 凋亡 /
- 迁徙运动
Abstract: Objective: To investigate the effect and potential mechanisms about apoptosis induction and migration suppression of photodynamic therapy with a new photosensitizer, 9-hydroxypheophorbide-α(9-HPbD), and diode laser on HN-3 human laryngeal squamous cancer cells.Method: The attached HN-3 cancer cells were photosesitized with 0.29 μg/ml and 0.59 μg/ml 9-HPbD for 6 h and irradiated by 664 nm diode laser for 15 min at an energy density of 2.0 J/cm² for activating 9-HPbD. Wound healing assay and photographing was respectively performed immediately after laser irradiation. Photographing focusing on the same location was repeated 12 h, 24 h and 36 h after PDT and cells migration distance counted respectively. H₂DCFDA staining was used to assess accumulation of reactive oxygen series (ROS) 1 h after PDT. MTT assay, Hoechst33342/PI double staining, western blotting were respectively performed to assess cellular viability, apoptosis and the expression of Enos, p-c-Jun, EGFR.Result: Phototoxicity and apoptosis on HN-3 cells induced by 9-HPbD-PDT was exhibited in a dose-related manner. Neither 9-HPbD alone nor laser alone was cytotoxic to HN-3 cells. Generation of ROS was initiated immediately after PDT. The apoptotic cells, marked with condensed/ fragmented blue or pink nuclei, and up-regulated expression of eNOS, p-c-Jun were subsequently induced 24 h after PDT. Coupled with a down-regulated expression of EGFR, a photosensitizer dose-ralated cell migration suppression was initiated by PDT. After pretreatment of GSH or ascorbic acid, a kind of antioxidant, the efficacy of PDT-induced apoptosis and migration suppression was partially inhibited.Conclusion: Activation of p-c-Jun, eNOS and down-regulated expression of EGFR may respectively involve in the apoptosis induction and cell migration suppression after 9-HPbD-PDT. Generation of ROS may play an important role in the course of apoptosis induction and migration suppression of HN-3 cells initiated by 9-HPbD-PDT.
- laryngeal neoplasms /
- squamous carcinoma /
- photodynamic therapy /
- apoptosis /
- migration

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