In vitro photosensitization of tumour cell enzymes by Photofrin II administered in vivo

Abstract

Dougherty, 1979). After 24-72h, to allow clearance of the photosensitiser from normal tissues, the malignant lesions are exposed to visible light, usually by laser irradiation. Tumour necrosis and regression ensue from photoradiation. It is generally agreed that cytotoxicity is mediated via formation of the highly reactive oxygen species, singlet oxygen, 102, (Weishaupt et al., 1976;Gibson et al., 1984;Parker, 1987). Since the original promising clinical results utilised HpD, a crude preparation composed of at least seven different porphyrin species (Gibson et al., 1984; Kessel, 1986;Moan et al., 1987), subsequent investigations were directed towards determination of the chemical structure of the 'active component' of HpD (Moan et al., 1982;Kessel and Chou, 1983;Dougherty et al., 1984). Methods developed to purify HpD produced a porphyrin mixture enriched in the hydrophobic components, reported to be mainly dihaematoporphyrin ethers or esters (Byrne et al., 1987;Dougherty, 1987;Kessel et al., 1987). This enriched preparation, Photofrin II, is now commercially produced for clinical and laboratory studies.In our earlier studies we utilised HpD as the photosensitiser (Hilf et al., , 1984Gibson et al., 1984). Because of the complex nature of HpD, the intracellular localisation and effects of various photosensitising components relative to time after administration could differ from the pharmacokinetics that would be observed with Photofrin II. We therefore undertook a study of Photofrin II employing the same in vivo-in vitro protocol used previously for HpD (Hilf et al., 1984). In this protocol, the photosensitiser is injected into tumour-bearing animals, tumours are removed and subcellular organelles are prepared. These preparations are then exposed to visible light in vitro, and various biochemical endpoints are analysed, such as site-specific enzyme activities. One advantage of this protocol is that it takes into account any metabolism of the sensitiser by the tumour-bearing host. In this report, using Photofrin II as the photosensitiser, data are presented on the time-course and drug dose response of photosensitisation of selected mitochondrial and cytosolic enzymes in the R3230AC mammary carcinoma. Materials and methods MaterialsPhotofrin II was kindly provided by Photomedica Inc., Raritan, NJ. All other reagents were obtained from Sigma Chemical Co., St Louis, MO, unless otherwise noted. Animals and tumoursThe R3230AC mammary adenocarcinoma was maintained by subcutaneous transplantation in the axillary region of 60-80g female Fischer rats, using the sterile trochar procedure described previously (Hilf et al., 1965).Preparation of subcellular organelles from tumours For in vitro studies, tumour-bearing rats were killed 17-24 days after implantation of the R3230AC mammary adenocarcinoma. From excised tumours, mitochondria were prepared according to methods described earlier . Briefly, R3230AC tumours were removed, weighed, placed in a dish on ice in 0.9% NaCl solution and minced with scissors. Approximately 2g...

Increased efficacy of photodynamic therapy of R3230AC mammary adenocarcinoma by intratumoral injection of Photofrin II

“…Previously, after i.p. administration of either HpD or Photofrin II, a time course of photosensitivity of these organelles in vitro indicated that inhibition of mitochondrial function would be greatest when tumours were exposed to light 24-72 h after drug administration (Gibson et al, 1989). Intratumoral administration of Photofrin II, however, showed a different time-course.…”

“…For study of mitochondrial effects, the in vivo-in vitro protocol was employed, an approach that takes into account any host metabolism and intracellular localisation of the photosensitiser occurring in vivo (Gibson & Hilf, 1983;Gibson et al, 1984bGibson et al, , 1989. From such data, we can derive an estimate of the pharmacokinetics of the administered photosensitiser.…”

“…Power density incident on the tumours was adjusted to 200 mW cm-2 as measured using a power radiometer (RK5200, Laser Precision, Utica, NY, USA). (Gibson et al, 1989). Here we examined whether such a dose relationship existed after Photofrin II was administered intratumorally (i.t.).…”

How Does Photodynamic Therapy Work?

“…Photoperoxidation of membrane cholesterol and other unsaturated phospholipids leads to changes in membrane permeability, loss of fluidity, cross-linking of aminolipids and polypeptides, and inactivation of membrane associated enzyme systems and receptors (Girotti, 1990). While the inhibition of mitochondrial enzymes has frequently been considered a key event in PDT cell lethality (Gibson et al, 1989;Salet and Moreno, 1990), there is evidence that the inactivation of membrane transport systems, depolarization of the plasma membrane and inhibition of DNA repair enzymes may precede inactivation of mitochondrial, cytosolic and lysosoma1 enzymes ; Boegheim et a f . , Specht and Rodgers, 1990).…”

Effects of the inhibitors of energy metabolism, lonidamine and levamisole, on 5-aminolevulinic-acid-induced photochemotherapy

“…Damage to mitochondrial functions leads to irreversible cell inactivation. The photosensitization may inhibit a number of mitochondrial enzymes: cytochrome C oxidase, FoFl ATPase, succinate dehydrogenase, NADH dehydrogenase (Gibson et a/., 1989;Hilf et al, 1984), adenylate kinase and monoamine oxidase (Murant et a/., 1987). Suppression of mitochondrial activity after PCT leads to a significant reduction in the cellular ATP level and, consequently, to a loss of viability (Hilf et al, 1986).…”