5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

Abstract

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.

Introduction

High-dose chemotherapy and autologous stem cell transplantation are common treatment modalities for patients with malignant hematopoietic disorders, especially high-risk patients. However, autologous stem cell transplantation is limited by a high relapse rate, which may be attributed to the presence of residual tumor cells in autografts and the absence of graft versus tumor response. Photodynamic therapy (PDT) has been used to eradicate residual tumor cells in autografts (purging) and to reduce contamination of tumor cells [1], [2], [3]. Because of a higher uptake of photosensitizer in malignant cells, subsequent light exposure selectively kills the tumor cells with minimal toxicity to progenitor cells or stem cells [2], [3].

Recently, a different approach using 5-aminolaevulinic acid (ALA) has been introduced to PDT. ALA is a distant precursor of heme. Application of exogenous ALA, followed by many biosynthetic processes, leads to an accumulation of photosensitizer protoporphyrin IX (PpIX) in most tissues in situ, while PpIX accumulates in malignant tissues more than in normal tissues [4], [5]. The tumor selectivity is most likely dependent on the decreased activity of ferrochelatase, a rate-limiting enzyme in the last step of heme biosynthesis pathway [6]. ALA-based PDT (ALA-PDT) has proved successful for the treatment of skin and gastrointestinal malignancies [7], [8], [9], [10], [11].

The selective destruction of leukemia cells by ALA-PDT reported by Grebenova et al. [12] indicate that ALA-PDT may be a possible means for purging residual tumor cells in autologous transplants. However, multidrug resistance (MDR) is a matter of concern associated with purging. MDR is associated with high expression of an mdr-1 gene product, P-glycoprotein (P-gp), which is a cell surface protein that functions as a transmembrane pump [13]. This pump is reported to efflux a variety of such chemotherapeutic drugs as anthracyclines and vinca alkaloids as well as some photosensitizers like porphymer sodium and benzoporphyrin derivative [14]. Chemotherapy, which is generally applied before autologous stem cell transplantation, has been well known to enhance the expression of P-gp in tumor cells. Therefore, a purging technique directed against drug-resistant tumor cells would be beneficial. Although some groups reported that P-gp did not appear to mediate ALA or PpIX efflux [5], [15], [16], ALA-PDT still showed some cross-resistance to chemotherapy in some cell types [16]. It is therefore important to clarify whether ALA-PDT is effective in the treatment of multidrug resistant leukemia cells.

In the present study we investigated the fluorescence kinetics of ALA-induced PpIX product and PDT toxicity in the mdr-1 gene-transducted and doxorubicin-induced multidrug resistant cells in comparison with those of their parent cells.