Abstract
We have shown that addition of exogenous delta-aminolaevulinic acid (ALA) to rat pancreatoma AR4-2J cells in culture leads to the increased production of porphobilinogen (PBG) and the accumulation of photoactive protoporphyrin IX (PPix) in these cells. Exposure to light (lambda > 400 nm) at an intensity of 0.2 mW cm-2 for 8 min resulted in an ALA dose-dependent cytolysis of the cells, with an EC50 of 6.6 +/- 0.7 microM. This cytolytic effect was light intensity dependent, with greater cell destruction after exposure to light at an intensity of 0.47 mW cm-2 than at 0.2 mW cm-2; it was also dependent on the duration of illumination, cell survival decreasing with increasing illumination times. The photodestruction of the AR4-2J cells following exposure to ALA can be attributed to the production of endogenous PPix, a photoactive porphyrin that we have shown to generate singlet oxygen upon illumination, whereas ALA itself does not. Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. In contrast, both the isoquinoline carboxamide PK11195 and the benzodiazepine Ro 5-4864 ligands, displaying a high affinity for the MBR, did affect ALA-mediated phototoxicity, each markedly increasing the EC50 for cell photodestruction and thus exerting a photoprotective effect. It is concluded that the MBR may play an important role in the expression of ALA-mediated PPix phototoxicity and that MBR ligands, by diminishing the actions of endogenous PPix, have the potential to rescue cells from porphyrin-induced photolysis.
Highlights
Photodynamic porphyrins are found to occur naturally in many cell types
The biosynthesis of protoporphyrin IX (PPix) can be enhanced by the exogenous administration of the amino acid 6-aminolaevulinic acid (ALA), especially in tumour cells, a pathway that offers promise for exploitation in photodynamic therapy (Pottier et al, 1986; Malik and Lugaci, 1987)
The purpose of the present investigation was to determine whether endogenous PPix produced from exogenous ALA in rat pancreatoma AR4-2J cells can achieve a sufficient concentration to serve as a cytolytic agent following photoactivation
Summary
Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. Keywords photodynamic action; 6-aminolaevulinic acid (ALA); rat pancreatoma cells; mitochondrial benzodiazepine receptor; endogenous protoporphyrin IX; singlet oxygen. It has been proposed that the physiological dicarboxylic porphyrins, notably PPix, are endogenous ligands for the mitochondrial benzodiazepine receptor (MBR), an 18 kDa protein located on the outer mitochondrial membrane and mediating a wide range of physiological effects (Verma et al, 1987), including modulation of mitochondrial respiratory control (Hirsch et al, 1988), inhibition of cellular proliferation (Stepien et al, 1991) and induction of cellular differentiation (Wang et al, 1984a).
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