D-Aminolaevulinic Acid-Induced Photodynamic Therapy Inhibits
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British Journal of Cancer (1999) 80(7), 998–1004 © 1999 Cancer Research Campaign Article no. bjoc.1998.0454 δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro SL Gibson, JJ Havens, ML Nguyen and R Hilf Department of Biochemistry and Biophysics and the UR Cancer Center, University of Rochester School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Rochester NY 14642, USA Summary Recently, considerable interest has been given to photodynamic therapy of cancer using δ-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after δ-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with δ- aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM δ-aminolaevulinic acid for 27 h accumulated 6.07 × 10–16 mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm–2 irradiation after a 3-h incubation with δ- aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 × 10–16 mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with δ- aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the δ-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation. Keywords: δ-aminolaevulinic acid; protoporphyrin IX; porphobilinogen deaminase; photodynamic therapy Photodynamic therapy (PDT) has been used successfully to (PPIX) (Malik and Lugaci, 1987; Kennedy and Pottier, 1992; Peng control the growth of a variety of human malignancies (Rosenthal et al, 1997). Protoporphyrin IX is formed as the penultimate step and Glatstein, 1994; van Hillegersberg et al, 1994; Fisher et al, of the haem biosynthetic pathway. The promise of this photosensi- 1995; Kriegmeir et al, 1996; Peng et al, 1997). Based on these tizer stems from reports that PPIX accumulates to a greater extent clinical trials, PDT has been approved as a treatment for various in malignant compared to normal tissue after administration of cancers in the US, Canada, France, The Netherlands and Japan. δ-aminolaevulinic acid (δ-ALA) (Dailey and Smith, 1984; van Traditionally, PDT consists of the systemic, topical or intra- Hillegersberg et al, 1992; Malik et al, 1995). Although the basis tumoural administration of a photosensitizer, followed by a time for its concentration in tumour tissue is not known, this observa- interval to allow dye distribution and localization. Malignant tion is being exploited in the use of δ-ALA in PDT. lesions are then exposed to the light of an appropriate photosensi- Earlier, we demonstrated that retreatment of R3230AC rat tizer absorption wavelength. Photoactivation of the dye results in mammary adenocarcinomas that had recurred after an initial course the formation of reactive oxygen species of which singlet oxygen of PDT using Photofrin® as the photosensitizer was as effective in 1 ( O2) is reported to be the major species and the one primarily controlling tumour growth as the original course of therapy. Here, responsible for the ensuing toxicity. Photofrin®, a derivative of as an extension of those earlier studies, we have directed our efforts haematoporphyrin, is currently the photosensitizer that has towards defining the effects that exogenous δ-ALA administration received approval for PDT. and subsequent photosensitization would produce on the enzymes There are numerous ongoing studies aimed at development of in the haem biosynthetic pathway. The administration of δ-ALA ‘new generation’ photosensitizers, and many of these compounds circumvents the initial biosynthetic regulatory step, δ-ALA show promise (Gomer, 1991; Pandey et al, 1995; Fan et al, 1997). synthase (δ-ALA-S), which is feedback inhibited by haem. Recently, there has been considerable interest in the formation Presumably, this subversion of regulation facilitates the intra- of an endogenously produced photosensitizer, protoporphyrin IX cellular accumulation of PPIX after δ-ALA administration (Dailey and Smith, 1984; Batlle, 1993). It is inferred that the second slowest step in the haem biosynthetic pathway, porphobilinogen Received 7 August 1998 deaminase (PBGD) (Healey et al, 1981; Ades, 1990), would be the Revised 28 October 1998 next rate-limiting step after δ-ALA-S is circumvented. In a recent Accepted 27 November 1998 report, we demonstrated a relationship between PBGD activity and Correspondence to: R Hilf the amount of PPIX accumulated, both of which increased in the 998 δ-Aminolaevulinic acid-induced photosensitization 999 δ Table 1 Effects of -ALA-induced photosensitization on cell proliferation Experiment 1 and intracellular CellTrackerTM concentration ALA 27 h . --7 Day Cell number × 105 CellTrackerTM 1 4.97 ± 0.59 0.22 ± 0.05 2 (no light) 10.6 ± 1.60 0.10 ± 0.02 Experiment 2 2 (30 mJ cm–2) 3.76 ± 0.41 0.23 ± 0.06 ALA 3 h * ALA 24 h R3230AC cells were incubated with δ-ALA and CellTrackerTM with or without subsequent light exposure (see Materials and Methods). The data on Day 1 represent baseline values for cell number and CellTrackerTM fluorescence, Experiment 3 expressed as relative fluorescence units per cell, obtained prior to the ALA 3 h ALA 24 h addition of δ-ALA or irradiation of cultures. The data listed for day 2 were obtained 24 h after cells were incubated for 3 h with δ-ALA and either exposed to 30 mJ cm–2 irradiation or maintained in the dark. Each value represents the mean cell number or CellTrackerTM concentration calculated from the results of at least three separate experiments performed in duplicate, ± s.e.m. Experiment 4 . - ..... --·-·-·· ···· -···----- ····--··-·-- ALA 3 h * no ALA +FBS 24 h ALA 24 h presence of exogenous δ-ALA (Gibson et al, 1998). Presently, we extend those findings by examining the effect photosensitization Experiment 5 would have on PBGD activity and PPIX accumulation. The ques- ALA 3 h no ALA +FBS 24 h ALA 24 h tions asked were: (1) are either PBGD activity or PPIX levels ;jJ ------- altered following δ-ALA-induced photosensitization?; (2) are photosensitized cells able to synthesize PPIX?; and (3) can cells be Figure 1 Graphic representation of the experiments employed to study the δ photosensitized with a second round of treatment? effects of -ALA administration and subsequent photoradiation on PPIX levels and PBGD activity in cultured R3230AC cells. The horizontal boxes Our results show that PBGD activity is inhibited by δ-ALA- are not drawn to the exact time scale, but represent the different time periods induced photosensitization with a concomitant decrease in PPIX in each experiment. Experiment 1: cells were incubated in α-MEM–FBS+δ- ALA in the dark for 27 h. Experiment 2: cells were incubated in α- levels. Subsequent treatment of these cells with light resulted in MEM–FBS+δ-ALA for 3 h. The medium containing δ-ALA was then removed, reduced cytotoxicity, indicating that retreatment of lesions with α-MEM–FBS–phenol red–δ-ALA was added for 2.5 min (boxes with *) δ-ALA-based PDT within 24 h after their initial exposure is followed by a 24-h incubation in α-MEM–FBS+δ-ALA. Experiment 4: cells were incubated in α-MEM–FBS+δ-ALA for 3 h, and the medium replaced with compromised due to the inability of the cells to synthesize PPIX. α-MEM–FBS-phenol red–δ-ALA for 2.5 min (box with *). Complete α-MEM was added for 24 h, followed subsequently by a 24-h incubation in α- MEM–FBS+δ-ALA. Experiment 3 was similar to Experiment 2 except that MATERIALS AND METHODS during the 2.5-min interval between δ-ALA incubations, cultures were exposed to 30 mJ cm–2 irradiation (shaded box). Experiment 5 was the same as Experiment 4 except that cultures were exposed to 30 mJ cm–2 (shaded Chemicals and reagents box) after the initial 3-h δ-ALA incubation period. At selected times during each experiment, cells were removed for determination of PPIX levels, All chemicals and reagents were purchased from Sigma Chemical PBGD activity and cell number Co. (St Louis, MO, USA) unless otherwise noted. Cell culture media and antibiotics were obtained from Grand Island Biological (Grand Island, NY, USA). Fetal bovine serum (FBS) was The modifications were made for cells incubated with δ-ALA, α- purchased from Atlanta Biologicals (Atlanta, GA, USA). δ-amino- MEM without FBS containing phenol red (α-MEM–FBS+δ-ALA); laevulinic acid (δ-ALA) was obtained from Porphyrin Products or the irradiation procedure, α-MEM without FBS or phenol red or (Logan, UT, USA) and Cell TrackerTM was purchased from δ-ALA (α-MEM–FBS–phenol red–δ-ALA). Only cells from Molecular Probes (Eugene, OR, USA). passages 1–10 were used for experiments. A stock of cells, from passages 1–4, were stored at –86°C and used to initiate cultures.