Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis

Abstract

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

Figure 1

Accumulation of PpIX after administration of 5-ALA in cancer cells Cancer cells from cell lines MCF-7, HT-29, MKN45 and PC-3 were incubated with 1 mM 5-ALA for 4 h. PpIX fluorescence was detected by fluorescence microscopy. Magnification is 400X.

Figure 2

PpIX accumulation in cells from the JFCR39 cell panel after treatment with 5-ALA or 5-ALA and FTC (A) Intracellular and extracellular PpIX accumulation after incubation with 1 mM 5-ALA. (B) Intracellular and extracellular PpIX accumulation after incubation with 1 mM 5-ALA and 10 μM FTC.

Figure 3

Western blot analysis of ABCG2 and dynamin 2 (A) ABCG2 and dynamin 2 protein expression in the JFCR39 panel cell lines. (B) Correlation between ABCG2 protein expression and the extracellular PpIX level. (C) Correlation between dynamin 2 protein expression and the extracellular PpIX level. (P < 0.005). The Pearson correlation coefficients (r) and the P values (P) are shown.

Figure 4

Effects of dynamin inhibitors on PpIX accumulation Cancer cells were incubated with 1 mM 5-ALA for 4 h in the presence of 1 μM to 16 μM MiTMAB, and then intracellular and extracellular PpIX accumulation was determined. (A) Intracellular PpIX; (B) extracellular PpIX. Data are expressed as the means ± S.D. of multiple experimental replicates (n = 3).

Figure 5

Effects of the double inhibition of ABCG2 and dynamin on PpIX accumulation in the JFCR39 cell lines. (A) Intracellular PpIX accumulation in the JFCR39 cell lines after incubation with 1 mM 5-ALA in the presence of FTC and MiTMAB. (B) Extracellular PpIX accumulation after incubation with 1 mM 5-ALA in the presence of FTC and MiTMAB. Data are expressed as the means ± S.D. of multiple experimental replicates (n = 3). Dunnett test: *P < 0.05; **P < 0.01.

Figure 6

Schematic illustration of PpIX excretion in human cancer cells after the administration of 5-ALA.