Abstract
Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.
Highlights
Photodynamic diagnosis (PDD) and photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissue [1,2]. 5-Aminolevulinic acid (ALA) is the naturally occurring metabolic precursor of an endogenously synthesized photosensitizer, protoporphyrin IX (PpIX) [2,3,4]
We found that ALA-mediated PpIX accumulation correlated negatively with the expression level of the PpIX transporter ATP-binding cassette transporter G2 (ABCG2), but not with that of PEPT1, PEPT2 or FECH (Fig. 1)
These results suggested that ABCG2 distributed in the mitochondrial fraction plays an important role in the regulatory mechanism of ALA-mediated PpIX accumulation
Summary
Photodynamic diagnosis (PDD) and photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissue [1,2]. 5-Aminolevulinic acid (ALA) is the naturally occurring metabolic precursor of an endogenously synthesized photosensitizer, protoporphyrin IX (PpIX) [2,3,4]. Despite the importance of PpIX in ALA-mediated PDD and PDT, the mechanism of accumulation of this metabolite in cancer cells remains unclear. With respect to protoporphyrin traffic, we reported previously the serum-dependent export of PpIX by ABCG2 in T24 cells [18]. In this context, it was reported that ABC transporters in mitochondria have important roles in the intracellular traffic of heme and various protoporphyrin intermediates of heme synthesis [19]. The transport processes that mediate the uptake and efflux of heme and porphyrin have been studied, it still remains unknown whether ABCG2 distributed in the mitochondrial fraction is functional for the efflux of PpIX from mitochondria. We described that functionally active ABCG2 is distributed in mitochondria and that Ko143, a specific inhibitor of ABCG2, increased the accumulation of ALAmediated PpIX in mitochondria
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