Early release of mitochondrial cytochrome c and expression of mitochondrial epitope 7A6 with a porphyrin-derived photosensitizer: Bcl-2 and Bcl-xL overexpression do not prevent early mitochondrial events but still depress caspase activity.

Certain nonmetallic porphyrins have potent antitumor activity upon visible light irradiation. Treatment of HeLa cells with nanomolar amounts of the photochemo therapeutic agent verteporfin and red light mobilized caspases 2, 3, 6, 7, 8, and 9, caused degradation of specific caspase substrates, and resulted in morphologic changes consistent with apoptosis. Caspase processing was detectable by 1 hour after light irradiation. The mitochondrial 7A6 epitope, recognized by monoclonal antibody APO2.7, became accessible, and cytochrome c was detectable within the cytosolic fraction of cells treated with verteporfin immediately after light irradiation. The general caspase inhibitor benzyloxycarboyl-Val-Ala-Asp-fluoromethylketone did not prevent 7A6 expression produced by photosensitization at peptide concentrations which completely prevented caspase activation and cleavage of caspase-specific substrates. Enforced overexpression of Bcl-2 or Bcl-xL prevented cytochrome c release and 7A6 expression produced by ultraviolet B light treatment, but did not prevent cytochrome c release or 7A6 expression elicited by verteporfin photosensitization. Bcl-2 or Bcl-xL overexpression delayed morphologic changes, depressed caspase activation, and limited substrate degradation, but did not protect against loss of viability after verteporfin photosensitization. This observation indicates that cells overexpressing Bcl-2 or Bcl-xL exhibit resistance to caspase activation even after the appearance of cytochrome c in the cytosol. Porphyrin photosensitizers are effective chemotherapeutic agents that elicit primary proapoptotic mitochondrial events even in the setting of heightened Bcl-2 or Bcl-xL expression.

某些非金属卟啉在可见光照射下具有强大的抗肿瘤活性。用纳摩尔量的光化学治疗剂维替泊芬和红光处理海拉细胞，可激活半胱天冬酶2、3、6、7、8和9，导致特定半胱天冬酶底物降解，并产生与细胞凋亡一致的形态学变化。光照射1小时后即可检测到半胱天冬酶的加工过程。单克隆抗体APO2.7识别的线粒体7A6表位变得可及，光照射后立即在维替泊芬处理的细胞的胞质部分可检测到细胞色素c。通用半胱天冬酶抑制剂苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸 - 氟甲基酮在完全阻止半胱天冬酶激活和半胱天冬酶特异性底物裂解的肽浓度下，不能阻止光致敏产生的7A6表达。强制过表达Bcl - 2或Bcl - xL可阻止细胞色素c释放以及紫外线B光处理产生的7A6表达，但不能阻止维替泊芬光致敏引发的细胞色素c释放或7A6表达。Bcl - 2或Bcl - xL过表达延迟了形态学变化，抑制了半胱天冬酶激活，并限制了底物降解，但不能防止维替泊芬光致敏后的细胞活力丧失。这一观察结果表明，过表达Bcl - 2或Bcl - xL的细胞即使在细胞色素c出现在胞质中后，仍对半胱天冬酶激活具有抗性。卟啉光致敏剂是有效的化学治疗剂，即使在Bcl - 2或Bcl - xL表达升高的情况下，也能引发主要的促凋亡线粒体事件。