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where other ABC transporters like MDR1, MRP2 and BCRP were also expressed. Therefore, in the present study, the kinetics of HY in-corporation into the particular cells overexpressing ABC transporters was measured after 2, 4, 8, 12, 16, 24 and 48 h of incubation of cells with HY. A strong correlation between the level of BCRP and the in-tracellular content of HY was found. The lowest accumulation of HY was observed in cBCRP cells. Moreover, inhibition of BCRP by the specific inhibitor Ko143 significantly increased the accumulation of HY in cBCRP cells. Based on these results, we can conclude that HY is a preferential substrate of BCRP.
HY is not the only PS of which accumulation can be aﬀected by enhanced activity of BCRP. Similar results have been reported with aminolevulinic Pepstatin A (ALA) converted to protoporphyrin IX (PpIX) in urothelial carcinoma cells and triple-negative breast cancer cells [15,16], ALA or its methyl ester (MAL) converted to PpIX in human keratinocytes and esophageal adenocarcinoma cells , and delta-aminolevulinic acid (DALA) converted to PpIX in human proliferating keratinocytes . Jonker et al. showed that pheophorbide a (PhA) is transported eﬀectively by both murine Bcrp1 and human BCRP . Subsequently, Robey et al. confirmed that PhA is a BCRP-specific sub-strate with potential value for measuring BCRP function and expression in clinical samples . Moreover, they demonstrated in their next study that BCRP also transports pyropheophorbide a methyl ester, chlorin e6 and ALA converted to PpIX .
Until now, to the best of our knowledge, there has been no study which has examined a direct link between the modulation of HY ac-cumulation by drug eﬄux transporters and its impact on HY-PDT cy-totoxicity. Therefore, MTT, annexin V/PI and caspase 3/TMRE assays were used to explore the impact of ABC transporter overexpression on the eﬀectiveness of HY-PDT. The cytotoxic eﬀect of HY-PDT was not evident only in cBCRP cells. Thus, we wanted to prove that BCRP is responsible not only for decreased HY accumulation, but also for de-creased activity of HY-PDT, using a specific BCRP inhibitor Ko143. As expected, the inhibition of BCRP by Ko143 reversed the resistance of cBCRP cells to HY-PDT. We have previously shown that the primary cytotoxic action of HY-PDT might be aﬀected by the ability of cells to overcome oxidative stress through the activity of various mechanisms including cellular redox systems [30,35]; however, the results of this study clearly show that the elimination of HY from cancer cells by BCRP is the first key step in aﬀecting the outcome of HY-PDT. These results are in agreement with the findings of other authors who hold BCRP responsible for the decreased intracellular level of various PSs and for lower PDT eﬃciency [10–20].
In summary, the present study clearly shows that HY is a pre-ferential substrate of the ABC transporter BCRP. Moreover, we directly showed that besides lowering HY content in cancer cells, BCRP also negatively aﬀects the cytotoxic action of HY-PDT. On the basis of our findings, we suggest individualized screening for BCRP expression and activity as a useful tool for the improvement of the HY-PDT eﬃcacy and HY-PDD selectivity.
This work was supported by the Slovak Research and Development Agency under contract no. APVV-14-0154, and NEXO II (Network of Excellence in Oncology) under project ITMS 26220120039. The authors are very grateful to Kamila Droppová, Diana Rošková and Viera Balážová for assistance with technical procedures.
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