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

Abstract

Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may be applied to the treatment of neoplasms in a variety of organs. In order to enhance existing regimens of photodynamic therapy, we investigated the effects of adding differentiation therapy to photodynamic therapy in human prostate cancer cells in vitro. The objective of differentiation therapy per se is to reverse the lack of differentiation in cancer cells using pharmacological agents. The motivation for this study was to exploit the differentiation-dependent expression of some heme enzymes to enhance tumour cell toxicity of ALA-photodynamic therapy. A short course of differentiation therapy was applied to increase PpIX formation during subsequent ALA exposure. Using the synthetic androgen R1881, isomers of retinoic acid, and analogues of vitamin D for 3 to 4 days, exogenous ALA-dependent PpIX formation in LNCaP cells was increased, along with markers for growth arrest and for differentiation. As a consequence of higher PpIX levels, cytotoxic effects of visible light exposure were also enhanced. Short-term differentiation therapy increased not only the overall PpIX production but also reduced that fraction of cells that contained low PpIX levels as demonstrated by flow cytometry and fluorescence microscopy. This study suggests that it will be feasible to develop protocols combining short-term differentiation therapy with photodynamic therapy for enhanced photosensitisation.

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

“…Enhancement occurred consistently in eight out of eight experiments, with some variability in magnitude between experiments; typically, a threefold increase in PpIX was observed in MTX-conditioned vs nonconditioned cells ( Figure 3A). The PpIX-elevating effect of MTX was only slightly less robust than that of a positive control, the androgen R1881 shown previously to induce PpIX levels in this cell line (Ortel et al, 2002).…”

“…We had previously described a strong increase in PpIX accumulation in epithelial keratinocytes that were differentiating in response to high calcium (Ortel et al, 1998), or in LNCaP cells in response to androgens (Ortel et al, 2002). To establish whether such a correlation might exist if MTX were used as the prodifferentiating agent, LNCaP cells were pretreated for 72 h with a cytostatic dose of MTX (1 mg l À1 ; see details below), then exposed to ALA for the last 4 h, and analysed for the amount of PpIX synthesised within the cells.…”

“…The ability to promote differentiation, in particular, suggested to us the idea of using MTX in combination with ALA-PDT. Two other agents (androgens and vitamin D) are known to promote cellular differentiation and at the same time to enhance PpIX accumulation in LNCaP cells (Ortel et al, 2002). We decided to test MTX for a similar capability.…”

“…Methotrexate-treated dishes were plated at a higher initial density of B250 000 cells dish À1 , to give a final cell density that was roughly equal to the controls after 72 h. Proliferation was estimated as the ratio (percentage) of the final trypsinised cell number divided by the number of cells plated. Growth arrest was assessed by upregulation of p27/kip-1, and differentiation by upregulation of E-cadherin in MTX-treated LNCaP cells, as described (Ortel et al, 2002).…”

“…Because MTX can induce cellular differentiation in some types of tumour cells, and because we have recently shown that agents capable of inducing cellular differentiation will also cause an elevation in the level of PpIX photosensitiser in epithelial cells of the skin (Ortel et al, 1998) and prostate (Ortel et al, 2002), we became interested in the notion of combination therapy involving MTX and ALA-PDT. Specifically, we reasoned that if MTX could promote differentiation and PpIX accumulation in carcinoma cells, then MTX might serve as a means to augment the efficacy of ALA-PDT for tumour cell killing.…”