No abstract

Photodynamic Therapy (PDT) uses a photosensitizing drug in combination with visible light to kill cancer cells. PDT has an advantage over surgery or ionizing radiation because PDT can eliminate tumors without causing fibrosis or scarring. Disadvantages include the dual need for drug and light, and a generally lower efficacy for PDT versus surgery. This minireview describes basic principles of PDT, photosensitizers available, and aspects of tumor biology that may provide further opportunities for treatment optimization. An emerging biomodulatory approach, using methotrexate or Vitamin D in combination with aminolevulinate-based PDT, is described. Finally, current clinical uses of PDT for solid malignancies are reviewed.

CCAAT-enhancer binding proteins (C/EBP) are basic region/leucine zipper (bZIP) transcription factors selectively expressed during the differentiation of liver, adipose tissue, blood cells, and the endocrine pancreas. Here we show that C/EBP isoforms are differentially expressed in the skin. BALB/MK keratinocytes incubated in 0.12 mM calcium medium undergo a differentiation program featuring growth-arrest at 24-48 h, keratin K10 gene expression beginning at 24 h, and apoptosis commencing at 48 h. Within this framework, western immunoblot analysis and immunohistochemistry reveal that C/EBP alpha increases 5-fold at 1-2 d and remains elevated, C/EBP beta rises 2-fold at 2-4 d and gradually falls, and CHOP rises 9-fold in the first 24 h then returns rapidly to baseline. Several products of alternative translation are observed in BALB/MK cells, i.e., 42 kDa and 30 kDa forms of C/EBP alpha, and 32 kDa and 20 kDa forms of C/EBP beta. By immunohistologic examination of human, rat, and mouse skin, all three transcription factors are highly expressed within epithelial compartments in a spatially restricted distribution. C/EBP alpha is concentrated in the upper epidermis in a predominantly cytoplasmic location within cells, whereas the highest levels of C/EBP beta and CHOP are seen in the mid-epidermis, mainly within nuclei. High levels of C/EBP beta and CHOP (but not C/EBP alpha) are also observed in hair follicles and sebaceous glands. The identity of these factors in the epidermis is confirmed by western immunoblot analyses. In summary, C/EBP are expressed in a differentiation-associated manner in the skin, and may play an important role in regulating one or more aspects of the epidermal differentiation program.

Photodynamic therapy, mediated by exogenously administered aminolevulinic acid (ALA-PDT) followed by exposure to a laser or broadband light source, is a promising modality for treatment of many types of cancers, but it remains inadequate to treat large, deep solid tumors. Here we report that calcitriol, the active form of Vitamin D3, can be administered prior to ALA as a non-toxic preconditioning regimen to markedly increase the efficacy of ALA-PDT. Using mouse models of squamous skin cancer for preclinical proof of concept, we showed that calcitriol delivered topically or intraperitoneally increased tumoral accumulation of the PDT-activated ALA product protoporphyrin-IX (PpIX) up to 10-fold, mainly by altering expression of the porphyrin synthesis enzymes coproporphyrinogen oxidase (increased) and ferrochelatase (decreased). Calcitriol-pretreated tumors underwent enhanced apoptotic cell death following ALA-based PDT. Mechanistic studies documented activation of the extrinsic apoptotic pathway, with specific cleavage of caspase-8 and increased production of TNFα in tumors preconditioned by calcitriol treatment before ALA-PDT. Very low doses of calcitriol (0.1–1 µg/kg body weight) were sufficient to elicit tumor-selective enhancement to ALA-PDT efficacy, rendering toxicity concerns negligible. Our findings define a simple, non-toxic and highly effective preconditioning regimen to enhance the response of epithelial tumors to ALA-PDT, possibly broadening its clinical applications by selectively enhancing accumulation of photosensitizer PpIX along with TNFα in tumors.

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l À1 , 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers.

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...

Purpose: To improve treatment efficacy and tumor cell selectivity of δ-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) via pretreatment of cells and tumors with methotrexate to enhance intracellular photosensitizer levels. Experimental Design: Skin carcinoma cells, in vitro and in vivo, served as the model system. Cultured human SCC13 and HEK1 cells, normal keratinocytes, and in vivo skin tumor models were preconditioned with methotrexate for 72 h and then incubated with ALA for 4 h. Changes in protoporphyrin IX (PpIX) levels and cell survival after light exposure were assessed. Results: Methotrexate preconditioning of monolayer cultures preferentially increased intracellular PpIX levels 2-to 4-fold in carcinoma cells versus normal keratinocytes. Photodynamic killing was synergistically enhanced by the combined therapy compared with PDT alone. Methotrexate enhancement of PpIX levels was achieved over a broad methotrexate concentration range (0.0003-1.0 mg/L; 0.6 nmol/L-2 mmol/L). PpIX enhancement correlated with changes in protein expression of key porphyrin pathway enzymes, ∼4-fold increase in coproporphyrinogen oxidase and stable or slightly decreased expression of ferrochelatase. Differentiation markers (E-cadherin, involucrin, and filaggrin) were also selectively induced by methotrexate in carcinoma cells. In vivo relevance was established by showing that methotrexate preconditioning enhances PpIX accumulation in three models: (a) organotypic cultures of immortalized keratinocytes, (b) chemically induced skin tumors in mice; and (c) human A431 squamous cell tumors implanted subcutaneously in mice. Conclusion: Combination therapy using short-term exposure to low-dose methotrexate followed by ALA-PDT should be further investigated as a new combination modality to enhance efficacy and selectivity of PDT for epithelial carcinomas.

A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.