Aminolevulinic acid (ALA) has been approved as an intraoperative molecular imaging probe for protoporphyrin IX (PpIX) fluorescence-guided resection of glioma. Here we explored its potential application for renal cell carcinoma (RCC) that is showing increased incidence in recent years. ALA-mediated PpIX in cell lysates (intracellular) and culture medium was measured in five human RCC cell lines (786-O, 769-P, A-704, Caki-1, Caki-2) and a non-tumor human kidney epithelial cell line HK-2 by spectrofluorometry and flow cytometry. The activity of PpIX bioconversion enzyme ferrochelatase (FECH) and PpIX efflux transporter ABCG2 was determined to correlate with the PpIX level. We found that ALA-PpIX fluorescence was highly variable among RCC cell lines and A-704 was the only RCC cell line exhibiting significantly higher intracellular PpIX than HK-2 cells. Neither the intracellular PpIX level nor the total amount of PpIX (including PpIX in cell lysates and the medium) had significant correlation with the activity of FECH or ABCG2. To enhance the intracellular PpIX, cells were treated with Ko143, a pharmacological inhibitor of ABCG2. Ko143 significantly increased the intracellular PpIX in cell lines with ABCG2 activity, but not in cell lines with little ABCG2 activity. In fact, there was a positive correlation between the ABCG2 activity and Ko143-induced PpIX enhancement across kidney cell lines. To identify clinically relevant ABCG2 inhibitors, small molecule inhibitors targeting various cell signaling pathways, some of which are known to inhibit ABCG2, were evaluated for the enhancement of ALA-PpIX in Caki-2 cells that had the highest ABCG2 activity in the RCC cell panel. Our screening led to the identification of several clinically available inhibitors that significantly increased the intracellular PpIX. Particularly, kinase inhibitor lapatinib exhibited the strongest enhancement effect. These clinical inhibitors can be used for the enhancement of ALA-PpIX fluorescence in tumors with elevated ABCG2 activity.

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.

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