Low-Dose Methotrexate Enhances Aminolevulinate-Based Photodynamic Therapy in Skin Carcinoma Cells In vitro and In vivo

Abstract

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.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…S8). The lack of NHEK susceptibility to PpIX-based photodynamic cell killing under different conditions has previously been reported [40,41].…”

Noninvasive fluorescence monitoring of protoporphyrin IX production and clinical outcomes in actinic keratoses following short-contact application of 5-aminolevulinate

“…Pattern b might be explained by the "field cancerization," a clinically well-accepted phenomenon in which preneoplastic changes within the skin extend well beyond the clinically detectable lesion. 13 Last, 23% of regions had lower readings within the lesion than in the surrounding skin ͑Fig. 4, pattern c͒, although these readings were still higher than readings from normal skin or photobleached lesions ͑Fig.…”

Optimizing photodynamic therapy by liposomal formulation of the photosensitizer pyropheophorbide-a methyl ester: In vitro and ex vivo comparative biophysical investigations in a colon carcinoma cell line

“…This de-excitation can occurs by a Type I mechanism that corresponds to a transfer of charge from PPME* leading to the creation of O 2 ∑ -. PPME* can also pass its excess energy to molecular oxygen, in a reaction known as a Type II mechanism, leading this time to the creation of 1 Interaction between OH ∑ , EtOH and POBN ends in the formation of the POBN-ethoxy adduct that we observed by ESR. For each quencher used to block one-by-one all the stages of the HW mechanism (SOD, DES and CAT respectively for I a , I b and I c ), a decrease of the signal intensity of nearly 30% was measured.…”