Yoon Jung-Hoon, Yoon Hyo-Eun, Kim Ok, Kim Sang Kyum, Ahn Sang-Gun, Kang Keon Wook
Department of Pathology, School of Dentistry, Research Center for Oral Disease Regulation of the Aged, Chosun University, Gwangju 501-759, Korea.
Lasers Surg Med. 2012 Jan;44(1):76-86. doi: 10.1002/lsm.21154. Epub 2012 Jan 3.
5-Aminolaevulinic acid (ALA) and its derivatives act as precursors of the photosensitizer protoporphyrin IX (PpIX). In this study, we compared cytotoxic effects of photodynamic therapy (PDT) with the hexenyl ester of ALA (ALA-hx) between MCF-7 human breast cancer cells and adriamycin-resistant MCF-7 (MCF-7/ADR) cells.
Cell viability and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT), flow cytometry assays. Chick chorioallantoic membrane (CAM) assays were applied to assess in vivo effect of ALA-hx PDT. Molecular analyses using Western blots and minimal reporter constructs containing the antioxidant response element (ARE) region were performed to reveal mechanistic basis for the differential PDT sensitivity of MCF-7 and MCF-7/ADR cells.
In MCF-7/ADR cells, PDT with ALA-hx more efficiently produced reactive oxygen species (ROS) and suppressed cell viability compared to MCF-7 cells. Cell death induced by ALA-hx PDT in MCF-7/ADR cells was mainly due to apoptosis. CAM assays confirmed that the apoptotic activity of PDT in MCF-7/ADR cells was significantly higher than that in control MCF-7 cells. We also found that MCF-7/ADR cells produced lower levels of glutathione (GSH), a major antioxidant, than control MCF-7 cells. Expression of Nrf2-dependent anti-oxidant genes including γ-glutamylcysteine ligase, heme oxygenase-1, and quinone oxidoreductase were down-regulated in MCF-7/ADR cells, and Nrf2 overexpression partially decreased the susceptibility of ALA-hx PDT in MCF-7/ADR cells. Moreover, PpIX synthesis and expression levels of protoporphyrinogen oxidase (PPO) and coproporphyrinogen oxidase (CPO) were much higher in MCF-7/ADR cells than MCF-7 cells.
ALA-hx PDT more potently produced intracellular ROS in MCF-7/ADR cells, which might be due to down-regulation of Nrf2-mediated anti-oxidant gene transcription and up-regulation of PpIX synthesis via the induction of CPO and PPO. These findings suggest that ALA-hx PDT may be usable as a therapeutic alternative for adriamycin-resistant breast cancer.
5-氨基酮戊酸（ALA）及其衍生物作为光敏剂原卟啉IX（PpIX）的前体。在本研究中，我们比较了ALA己酯（ALA-hx）光动力疗法（PDT）对MCF-7人乳腺癌细胞和阿霉素耐药的MCF-7（MCF-7/ADR）细胞的细胞毒性作用。
通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和流式细胞术检测细胞活力和凋亡情况。应用鸡胚绒毛尿囊膜（CAM）试验评估ALA-hx PDT的体内效应。采用蛋白质免疫印迹法和含有抗氧化反应元件（ARE）区域的最小报告基因构建体进行分子分析，以揭示MCF-7和MCF-7/ADR细胞对PDT敏感性差异的机制基础。
与MCF-7细胞相比，在MCF-7/ADR细胞中，ALA-hx PDT能更有效地产生活性氧（ROS）并抑制细胞活力。ALA-hx PDT诱导MCF-7/ADR细胞死亡主要归因于凋亡。CAM试验证实，PDT对MCF-7/ADR细胞的凋亡活性显著高于对照MCF-7细胞。我们还发现，MCF-7/ADR细胞产生的主要抗氧化剂谷胱甘肽（GSH）水平低于对照MCF-7细胞。在MCF-7/ADR细胞中，包括γ-谷氨酰半胱氨酸连接酶、血红素加氧酶-1和醌氧化还原酶在内的Nrf2依赖性抗氧化基因的表达下调，Nrf2过表达部分降低了MCF-7/ADR细胞对ALA-hx PDT的敏感性。此外，MCF-7/ADR细胞中PpIX的合成以及原卟啉原氧化酶（PPO）和粪卟啉原氧化酶（CPO）的表达水平均高于MCF-7细胞。
ALA-hx PDT在MCF-7/ADR细胞中能更有效地产生细胞内ROS，这可能是由于Nrf2介导的抗氧化基因转录下调以及通过诱导CPO和PPO上调PpIX合成所致。这些发现表明，ALA-hx PDT可能作为阿霉素耐药乳腺癌的一种治疗选择。
