Morris Rachel L, Varnes Marie E, Kenney Malcolm E, Li Ying-Syi, Azizuddin Kashif, McEnery Maureen W, Oleinick Nancy L
Department of Radiation Oncology, Case Western Reserve University, Cleveland, OH 44106-4942, USA.
Photochem Photobiol. 2002 Jun;75(6):652-61. doi: 10.1562/0031-8655(2002)075<0652:tpbrip>2.0.co;2.
The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.
外周苯二氮䓬受体（PBR）是线粒体外膜上一种18 kDa的蛋白质，它与电压依赖性阴离子通道相互作用，并可能参与通透性转换孔的形成。PBR的生理作用体现在其对内源性配体的高亲和力结合上，这些内源性配体是胆固醇和血红素的代谢产物。血红素的某些卟啉前体可以作为光动力疗法（PDT）的光敏剂，该疗法依赖于卟啉相关大环的可见光激活。由于一系列卟啉类似物对PBR的表观结合亲和力与其使细胞光失活的能力平行，因此有人提出PBR是卟啉衍生的光细胞毒性的分子靶点。酞菁（Pc）光敏剂Pc 4在线粒体中蓄积，且结构与卟啉相似。因此，我们测试了PBR结合对Pc 4 - PDT的相关性。通过与PBR的高亲和力配体3H - PK11195竞争，来测量其与大鼠肾线粒体（RKM）或完整的中国仓鼠卵巢（CHO）细胞结合的亲和力。为了直接评估Pc的结合情况，我们合成了14C标记的Pc 4，发现Pc 4是3H - PK11195与PBR结合的竞争性抑制剂，而PK11195并不抑制14C - Pc 4与RKM的结合。此外，14C - Pc 4与RKM的结合在高达10 microM时未显示出饱和迹象。最后，当加载了Pc 4的CHO细胞暴露于激活红光下时，会诱导细胞凋亡；在过表达抗凋亡蛋白Bcl - 2的同伴细胞系中，Pc 4 - PDT诱导凋亡的效果较差。对于这两种细胞系，PK11195均抑制PDT诱导的凋亡；然而，这种抑制是短暂的，并不扩展至总体细胞死亡，这通过克隆形成试验得以确定。结果表明：（1）PBR上存在Pc 4的低亲和力结合位点；（2）除了影响PK11195结合的位点外，RKM和CHO细胞中还存在Pc 4的多个结合位点；（3）高饱和水平的PK11195能够短暂抑制由Pc 4 - PDT引发的凋亡，对总体细胞杀伤的影响较小。我们得出结论，与其他线粒体事件（如对Bcl - 2的光损伤）相比，Pc 4与PBR的结合与Pc 4 - PDT的光细胞毒性相关性较小，并且观察到的PK11195对Pc 4 - PDT诱导凋亡的抑制可能通过独立于PBR的机制发生。
