Phototoxicity of Protoporphyrin IX, Diarginine Diprotoporphyrinate and N,N-Diphenylalanyl Protoporphyrin Toward Human Fibroblasts and Keratinocytes In Vitro: Effect of 5-Methoxypsoralen ¶

Abstract

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N-diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (h = 365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)z > PP(Phe)> A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg), and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1-0.5 pA4 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)z and 5-MOP concentrations lower than 0.75 pM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)2 is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg), phototoxic effect in the presence of !?-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, Abbreviations: DMEM, Dulbecco's modified minimum essential medium;EMEM, Eagle's minimum essential medium with Earle's salts; FCS, fetal calf serum; HBSS, Hank's buffered saline solution: ID,,, dose inhibiting 50% of membrane integrity; 5-MOP, 5-methoxypsoralen; NR, neutral red; PBS, phosphate-buffered saline solution; PDT, photodynamic therapy; PP(Arg),, diarginine diprotoporphyrinate; PPIX, protoporphyrin IX, PP(Phe),, N,N-diphenylalanyl protoporphyrin; PUVA, psoralens + ultraviolet-A, UV-A, ultraviolet-A. 0 2004 American Society for Photobiology 003 1 -8655/04 $5.00+0.00 has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(ArgI2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)t phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg), in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.

Breakthroughs in Melanoma Research

“…However, further studies will be necessary to evaluate the impact of these drugs on RANK/RANKL signaling physiological functions, more specifically during growth, and to deal with these drugs potential wrong impact on immune system. singlet oxygen), that are potentially cytotoxic causing lethal damage to the cell and/or inhibits angiogenesis (Bugaj et al, 2004). Various photosensitizing drugs have been developed, such as 5-aminolevulinic acid (ALA) that unlike other PDT drugs is not a photosensitizer.…”

“…PDT emerges like a hopeful treatment for cancer and other diseases (Paras, 2003), is a low invasive treatment based on photosensitizer drugs that are administrated to the patients and can be retained on a selective mode for the ill tissue more than the normal tissue (Mang, 2004). This therapeutic procedure has been applied in the treatment of many hyper proliferative diseases of keratinocytes (Bugaj et al, 2004). The action of photodynamic therapy is based on the use of visible light or near infrared, a photosensitizer and the presence of molecular oxygen (O 2 ) on transformed cells (like tumor cells); light is absorbed for the photosensitizer and this action is used to activate the oxygen (Bonnett, 2000) present into cancerous cells and to generate reactive oxygen species (mainly 2.3 Photodynamic treatment 30,000 cells/well were seeded in 96 well plates and these were exposed to 0, 25,50,75,100,125, 150 µg of ALA/mL of culture medium serum free for 4h.…”

Isolation of Cellular Clones of Murine Melanoma Resistants to the Photodynamic Therapy and Characterization of Some Mechanisms Involved in the Radioresistance

“…PDT emerges like a hopeful treatment for cancer and other diseases (Paras, 2003), is a low invasive treatment based on photosensitizer drugs that are administrated to the patients and can be retained on a selective mode for the ill tissue more than the normal tissue (Mang, 2004). This therapeutic procedure has been applied in the treatment of many hyper proliferative diseases of keratinocytes (Bugaj et al, 2004). The action of photodynamic therapy is based on the use of visible light or near infrared, a photosensitizer and the presence of molecular oxygen (O 2 ) on transformed cells (like tumor cells); light is absorbed for the photosensitizer and this action is used to activate the oxygen (Bonnett, 2000) present into cancerous cells and to generate reactive oxygen species (mainly singlet oxygen), that are potentially cytotoxic causing lethal damage to the cell and/or inhibits angiogenesis (Bugaj et al, 2004).…”

“…This therapeutic procedure has been applied in the treatment of many hyper proliferative diseases of keratinocytes (Bugaj et al, 2004). The action of photodynamic therapy is based on the use of visible light or near infrared, a photosensitizer and the presence of molecular oxygen (O 2 ) on transformed cells (like tumor cells); light is absorbed for the photosensitizer and this action is used to activate the oxygen (Bonnett, 2000) present into cancerous cells and to generate reactive oxygen species (mainly singlet oxygen), that are potentially cytotoxic causing lethal damage to the cell and/or inhibits angiogenesis (Bugaj et al, 2004). Various photosensitizing drugs have been developed, such as 5-aminolevulinic acid (ALA) that unlike other PDT drugs is not a photosensitizer.…”