Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes

Abstract

Summary A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiationdependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium-and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.Keywords: photodynamic therapy; 5-aminolaevulinic acid; differentiation; keratinocyte; protoporphyrin IX Photodynamic therapy (PDT) is a treatment strategy consisting of two components: the photosensitizer (PS) and light (Hasan and Parrish, 1996). Both show negligible toxicity by themselves at the doses used for therapeutic applications, but become cytotoxic once combined at the site of desired activity by a variety of photochemical and cellular mechanisms (Henderson and Dougherty, 1992). Current clinical and experimental protocols involve systemic administration of a PS, usually a tetrapyrrole compound, followed by local irradiation with activating light (Fisher et al, 1995), and considerable experience has been acquired with the use of exogenous porphyrins and porphyrin derivatives (Ortel et al, 1996).A more recent approach exploits the indigenous ability of most cells to synthesize porphyrins from their physiological precursor, 5-aminolaevulinic acid (ALA; Batlle, 1993). The synthesis of ALA, the precur...

Differentiation enhances aminolevulinic acid-dependent photodynamic treatment of LNCaP prostate cancer cells

“…The approach described here arose out of an exploration of the capacity of cells to synthesise PpIX (the photosensitiser) from ALA (the pro-drug), and is based upon the finding that cellular differentiation increases the ability of a variety of cells to synthesise PpIX from exogenous ALA (Ortel et al, 1998). For example, induction of differentiation in skin keratinocytes (by increasing calcium concentrations in the culture medium) leads to an accumulation in PpIX, involving upregulation of the heme-synthetic enzyme coproporphyrinogen oxidase, and results in significantly increased photosensitisation (Ortel et al, 1998).…”

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma

“…Hasan and co-workers have shown that induction of prostate cancer cell differentiation augments intracellular PpIX accumulation (Ortel et al, 1998). Similarly, DMSO induced differentiation of B16 cells enhanced by PpIX accumulation .…”

“…It has been suggested that during differentiation and reduction in heme synthesis, the activity of the third enzyme in the porphyrin synthesis pathway, PBGD, would be altered accordingly (Ortel et al, 1998). Porphobilinogen deaminase during butyrateinduced differentiation of B16 cells was analysed by Western blotting, revealing a time-dependent decrease in total PBGD protein ( Figure 5A).…”

“…From the present results and from earlier studies, two patterns of differentiation can be outlined, one associated with upregulation and the other with downregulation of PBGD expression. Ortel et al (1998) have shown that induction of keratinocyte differentiation augments intracellular PpIX accumulation. Similarly, DMSO was found to induce differentiation of B16 melanoma cells and enhance PpIX accumulation and Mamet et al (1994) demonstrated increased activity of PBGD in malignant cell lines.…”

“…HMBA induces the porphyrin biosynthesis pathway expression of globin genes and differentiation (Marks et al, 1987). Ortel et al (1998) have demonstrated that cellular differentiation increases the ability of a variety of cells to synthesise PpIX from exogenous ALA.…”