Analysis of Renal Cell Carcinoma Cell Response to the Enhancement of 5-aminolevulinic Acid-mediated Protoporphyrin IX Fluorescence by Iron Chelator Deferoxamine†

Abstract

As a tumor photodiagnostic agent, 5-aminolevulinic acid (ALA) is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) with fluorescence. ALA-PpIX fluorescence was evaluated in human renal cell carcinoma (RCC) cell lines and non-tumor HK-2 cell lines. We found that extracellular PpIX level was correlated with ABCG2 activity, illustrating its importance as a PpIX efflux transporter. Extracellular PpIX was also related to the K_m of ferrochelatase (FECH) that chelates PpIX with ferrous iron to form heme. The V_max of FECH was higher in all RCC cell lines tested than in the HK-2 cell line. TCGA dataset analysis indicates a positive correlation between FECH expression and RCC patient survival. These findings suggest FECH as an important biomarker in RCC. Effects of iron chelator deferoxamine (DFO) on the enhancement of PpIX fluorescence were assessed. DFO increased intracellular PpIX in both tumor and non-tumor cells, resulting in no gain in tumor/non-tumor fluorescence ratios. DFO appeared to increase ALA-PpIX more at 1-h than at 4-h treatment. There was an inverse correlation between ALA-PpIX fluorescence and the enhancement effect of DFO. These results suggest that enhancement of ALA-PpIX by DFO may be limited by the availability of ferrous iron in mitochondria following ALA administration.

Figure 1.

Analysis of intracellular and extracellular ALA-PpIX and FECH activity in RCC and non-tumor HK-2 cell lines. (A) Cells were incubated with 1 mM ALA for 1 h or 4 h. Intracellular (in cell lysates) and extracellular (in cell culture medium) PpIX were measured with spectrofluorometry (n = 3 or 4). All bars indicate the standard deviation (SD). (B) Extracellular PpIX level was positively correlated with ABCG2 activity (n = 3 or 4, Bars, SD). (C) FECH enzyme kinetics (n =3-5, Bars, SD). (D) Correlation between extracellular PpIX and the K_m of FECH (n = 3 or 4, Bars, SD). (E) Correlation between FECH enzymatic parameters and ABCG2 activity. r: Pearson correlation coefficient.

Figure 2.

Effects of DFO on ALA-PpIX fluorescence by spectrofluorometry. (A, B) RCC and non-tumor HK-2 cells were incubated with either 1 mM ALA alone or in combination with 1 mM DFO for 1 h (A) or 4 h (B). Intracellular (in cell lysates) and extracellular (in cell culture medium) PpIX were measured with spectrofluorometry (n = 3 or 4, Bars, SD). (C) The percent change of intracellular PpIX fluorescence induced by ALA + DFO treatments compared to ALA treatment alone (n = 3 or 4, Bars, SD). ***P < 0.001, compared with the corresponding data at 1 h. (D, E) The ratio of intracellular PpIX between tumor and non-tumor (HK-2) cells at 1 h (D) and 4 h (E), n = 3 or 4, Bars, SD. **P < 0.01, compared with the corresponding ALA alone.

Figure 3.

Effects of DFO on ALA-PpIX fluorescence by flow cytometry. (A) RCC and non-tumor HK-2 cells were incubated with either 1 mM ALA alone or in combination with 1 mM DFO for 4 h. Cell fluorescence was measured with a flow cytometer in the FL3 channel (n = 3, Bars, SD). ** P < 0.01, ***P < 0.001, compared with the corresponding ALA alone. (B) Fluorescence measured with flow cytometry was positively correlated with the intracellular PpIX measured with spectrofluorometry. (C) The percent change of fluorescence induced by ALA + DFO treatments compared to ALA treatment alone (n = 3, Bars, SD). (D) The percent change of ALA-PpIX fluorescence induced by DFO treatment was negatively correlated with ALA-PpIX fluorescence measured with either flow cytometry or spectrofluorometry. (E) Tumor to non-tumor (HK-2) fluorescence ratios (n = 3, Bars, SD). *P < 0.05, compared with the corresponding ALA alone. r: Pearson correlation coefficient.