Choi Bo-hyun, Ryoo In-geun, Kang Han Chang, Kwak Mi-Kyoung
College of pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, Republic of Korea.
PLoS One. 2014 Sep 16;9(9):e107158. doi: 10.1371/journal.pone.0107158. eCollection 2014.
Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.
光动力疗法（PDT）已成为治疗多种实体瘤的有效方法。转录因子NRF2已知可抵御氧化应激和亲电应激；然而，其在癌症中的组成型活性赋予了癌细胞对抗癌药物的抗性。在本研究中，我们通过使用NRF2基因敲低的乳腺癌MDA-MB-231细胞，研究了NRF2信号通路作为基于脱镁叶绿酸a（Pba）的光动力疗法潜在分子决定因素的作用。稳定敲低NRF2的细胞在光动力疗法后显示出增强的细胞毒性以及凋亡/坏死性细胞死亡，同时单线态氧和活性氧（ROS）水平升高。对荧光Pba的共聚焦显微镜观察表明，NRF2基因敲低的细胞比对照细胞积累更多的Pba。随后对膜药物转运蛋白表达的分析表明，乳腺癌耐药蛋白（BCRP；ABCG2）的基础表达依赖于NRF2。在所检测的药物转运蛋白中，只有BCRP的基础表达因NRF2基因敲低而降低。此外，在用BCRP特异性抑制剂孵育后，两种细胞系中细胞内Pba积累和ROS的差异消失。此外，与对照相比，NRF2基因敲低的细胞过氧化物还原酶3表达水平较低，这意味着线粒体ROS防御系统的减弱可能有助于光动力疗法致敏。NRF2-BCRP通路在Pba光动力疗法反应中的作用在结肠癌细胞HT29中得到进一步证实。具体而言，NRF2基因敲低导致光动力疗法后细胞死亡增加，单线态氧和ROS水平升高，这是由于BCRP表达降低所致。同样，在乳腺癌MCF-7细胞、结肠癌细胞HCT116细胞、肾癌细胞A498细胞和胶质母细胞瘤A172细胞中，用NRF2 shRNA处理后，光动力疗法诱导的ROS生成显著增加。综上所述，这些结果表明，调控NRF2可以增强多种癌细胞对Pba光动力疗法的敏感性。
