| The polycyclic aromatic quinone hypericin, which is capable of photoactivation and is biosynthesized by Hypericum plant species, was examined as a potential agent for chemotherapy and for photodynamic therapy of neoplastic disease. The model systems used to test for activity were a mitochondrial succinoxidase enzyme activity assay, an EMT6 mouse mammary carcinoma clonogenic assay, and an MX-1 human mammary carcinoma antitumor assay. Parallel experiments were conducted in the presence and absence of light. Pure hypericin and acetone extracts of Hypericum perforatum were tested for activity and yielded similar results. In the dark, hypericin inhibited succinoxidase activity in isolated mitochondrial membranes, but had no effect on EMT6 cells or MX-1 tumors. However, photoactivated hypericin exhibited activity in all test systems. In particular, photoactivated hypericin inhibited succinoxidase activity at nmol/mg mitochondrial protein levels, EMT6 cell clonogenicity at ;Experiments designed to elucidate the mechanistic parameters of hypericin's biological activity were also conducted. The results show that photosensitized inhibition of mitochondrial succinoxidase was drug-dose, light-dose, and wavelength dependent with the greatest inhibition occurring by 600 nm light activation. Hypericin photosensitized singlet oxygen generation was also light-dose and wavelength dependent. Unequivocal evidence for hypericin photosensitized singlet oxygen generation was obtained using kinetic isotope ratios of products from the reaction between singlet oxygen and the geminally deuterated singlet oxygen trap, tetramethylethylene. The data are consistent with a type II singlet oxygen mediated mechanisms for hypericin photosensitized succinoxidase inhibition. The photodynamic effect of hypericin on EMT6 cells was examined under hypoxic and aerobic conditions, and photocytotoxicity was found to be oxygen-dependent. The findings suggest that type I oxygen-independent photooxidation reactions do not contribute significantly to hypericin's photodynamic mechanism of action. Fluorescence photomicrographs of hypericin in EMT6 cells show that hypericin is distributed throughout the plasma and cytosolic membranes but is absent from the nucleus, indicating that the events which lead to photocytotoxicity may not involve direct damage to nuclear DNA. |
