Evaluation of aminolevulinic acid-mediated protoporphyrin IX fluorescence and enhancement by ABCG2 inhibitors in renal cell carcinoma cells

Abstract

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.

Small molecule kinase inhibitors enhance aminolevulinic acid-mediated protoporphyrin IX fluorescence and PDT response in triple negative breast cancer cell lines

“…Effects of small molecule kinase inhibitors on ALA-PpIX fluorescence and PDT response were evaluated in TNBC cell lines in this study. The results confirmed our previous findings in renal cell carcinoma cell lines 9 and demonstrate in TNBC cell lines that clinically approved kinase inhibitors, such as lapatinib, sunitinib, and gefitinib, increased ALA-PpIX fluorescence. As in the previous study, we found here that lapatinib was the only kinase inhibitor that significantly increased intracellular PpIX and reduced PpIX efflux.…”

“…Our previous evaluation of over 12 kinase inhibitors led to the identification of lapatinib as the most potent drug for enhancing ALA-PpIX fluorescence. 9 This study further demonstrates the effectiveness of lapatinib in enhancing ALA-PDT by inducing apoptosis. Strong enhancement of ALA-PpIX fluorescence and PDT by lapatinib was likely due to its potent inhibition of ABCG2 activity.…”

“… 5 – 7 Tumor cell lines with elevated ABCG2 activity exhibited good responses to Ko143 for the enhancement of PpIX and PDT, but not tumor cell lines with little ABCG2 activity. 8 , 9 These previous studies support the combination of ABCG2 inhibitors and ALA for therapeutic enhancement. However, FTC and Ko143 are not suitable for in vivo application due to neurotoxicity and the instability in serum, respectively.…”

“…Effects of kinase inhibitor lapatinib on FECH activity were determined as described previously. 9 Briefly, cells in 100-mm dishes were treated with lapatinib ( ) for 4 h or untreated for control and lysed with 0.1% Triton X-100 in PBS containing palmitic acid and protease inhibitor cocktail. Lysates were centrifuged to obtain the supernatant, which was incubated with zinc acetate ( ) and various concentrations of PpIX for 10 min at 37°C.…”

“… 13 These ABCG2-interacting kinase inhibitors including imatinib 14 and gefitinib 15 were shown to increase ALA-PpIX fluorescence and PDT effects, suggesting that these drugs may be repurposed for the therapeutic enhancement of ALA. We have evaluated over a dozen small molecule kinase inhibitors and identified six FDA-approved drugs that significantly increased the intracellular PpIX in a renal cell carcinoma cell lines with high ABCG2 activity. 9 Notably, lapatinib was the only one that not only increased intracellular PpIX but also reduced PpIX efflux in the medium. In this study, we further assessed some of these kinase inhibitors for the enhancement of ALA-PpIX fluorescence and PDT effect in triple negative breast cancer (TNBC) cell lines, which are known to be resistant to chemotherapeutics due to the elevation of ABCG2 and other efflux transporters.…”