Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid‐mediated Protoporphyrin <scp>IX</scp> by Iron Chelator Deferoxamine

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.

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“…FECH activity was determined as described previously (17). Cells were implanted in 100mm dishes and allowed to grow to 70% confluency.…”

Section: Fech Activity Assaymentioning

confidence: 99%

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

Howley

Mansi

Shinde

et al. 2020

Journal of Photochemistry and Photobiology B: Biology

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“…Our results show that DFO enhances PpIX fluorescence in cell monolayers, but it does not when the same cells of adherent monolayer nature are forced into suspension. Different mechanisms, such as uptake rates, enzymatic activities or cell-to-cell interaction, have been identified as playing a role in the 5-ALA-induced PpIX photoactivation following exogenous DFO administration [24,35,36]. Yet, our attempt at enhancing the metabolic activities and maintaining membrane integrity did not increase the PPIX fluorescence of suspended cells any further.…”

Section: Discussionmentioning

confidence: 78%

Probing Hexaminolevulinate Mediated PpIX Fluorescence in Cancer Cell Suspensions in the Presence of Chemical Adjuvants

Chan

Gleadle

Vasilev

et al. 2020

IJMS

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Exogenous administration of hexaminolevulinate (HAL) induces fluorescent protoporphyrin IX (PpIX) accumulation preferentially in cancer cells. However, the PpIX fluorescence intensities between noncancer and cancer cells are highly variable. The contrast between cancer and noncancer cells may be insufficient to reliably discriminate, especially at the single cell level in cancer diagnostics. This study examines the use of the chemical adjuvants dimethylsulphoxide (DMSO) or deferoxamine (DFO) to enhance the HAL induced PpIX accumulation in cancer cells. Our results showed that in some of the incubation conditions tested, the addition of DFO with HAL significantly increased PpIX 21 fluorescence of adherent monolayer cancer cells, but this was never the case for cells in suspension. Permeabilisation with DMSO did not increase PpIX fluorescence. Cell-to-cell interaction may well play an important role in the PpIX accumulation when suspended cells are treated in HAL and adjuvant chemicals.presence of intracellular PpIX. 5-ALA based PDD is used in the detection of a wide range of cancers, such as brain [5], ovarian [6], colorectal [7], breast [8] and bladder [9]. In urology, blue light cystoscopy PPD is now recommended by several guidelines around the world. Yet the underlying mechanisms responsible for the specificity of PpIX accumulation in tumour cells remains poorly understood [10]. To date, it has been attributed to the distinctive nature of cancer cells, primarily, to reduced FECH activity [11] and to the limited availability of iron in cancer cells [12,13] or to the reduced availability of nicotinamide adenine dinucleotide phosphate (NADPH) provision. However, the difference in PpIX fluorescence intensity between normal and cancer cells is highly variable from one cell type to another [14]. In fact, the contrast is, in some cases, insufficient to reliably discriminate between the two, especially when applying PDD principles ex vivo, at the single cell level [15,16]. To enable the development of noninvasive diagnostic methods using PDD on body fluid sediments, such as urine [17][18][19][20], practical ways to enhance the PpIX fluorescence contrast ex vivo are required [21]. Strategies to do so may well be different from those that are successful in vivo, simply because the cell microenvironments are drastically different in those two scenarios.

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“…Other potential therapies include ferrochelatase inhibitors (e.g. iron chelators [ 38 ] or manganese chloride [ 39 ]) and ABCG2 inhibitors [ 40 ]. Differentiating agents such as vitamin D may be the easiest first step as they are already clinically available and are not expected to enhance uptake in normal pancreas as this should already be well differentiated.…”

Section: Discussionmentioning

confidence: 99%

5-Aminolevulinic acid for fluorescence-guided surgery in pancreatic cancer: Cellular transport and fluorescence quantification studies

Labib

Yaghini

Davidson

et al. 2021

Translational Oncology

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5-Aminolevulinic acid (ALA) is a potential contrast agent for fluorescence-guided surgery in pancreatic ductal adenocarcinoma (PDAC). However, factors influencing ALA uptake in PDAC have not been adequately assessed. We investigated ALA-induced porphyrin fluorescence in PDAC cell lines CFPAC-1 and PANC-1 and pancreatic ductal cell line H6c7 following incubation with 0.25–1.0 mM ALA for 4–48 h. Fluorescence was assessed qualitatively by microscopy and quantitatively by plate reader and flow cytometry. Haem biosynthesis enzymes and transporters were measured by quantitative polymerase chain reaction (qPCR). CFPAC-1 cells exhibited intense fluorescence under microscopy at low concentrations whereas PANC-1 cells and pancreatic ductal cell line H6c7 showed much lower fluorescence. Quantitative fluorescence studies demonstrated fluorescence saturation in the two PDAC cell lines at 0.5 mM ALA, whereas H6c7 cells showed increasing fluorescence with increasing ALA. Based on the PDAC:H6c7 fluorescence ratio studies, lower ALA concentrations provide better contrast between PDAC and benign pancreatic cells. Studies with qPCR showed upregulation of ALA influx transporter PEPT1 in CFPAC-1, whereas PANC-1 upregulated the efflux transporter ABCG2. We conclude that PEPT1 and ABCG2 expression may be key contributory factors for variability in ALA-induced fluorescence in PDAC.

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Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid‐mediated Protoporphyrin IX by Iron Chelator Deferoxamine

Cited by 20 publications

References 39 publications

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

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

Probing Hexaminolevulinate Mediated PpIX Fluorescence in Cancer Cell Suspensions in the Presence of Chemical Adjuvants

5-Aminolevulinic acid for fluorescence-guided surgery in pancreatic cancer: Cellular transport and fluorescence quantification studies

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