br any of the inhibitors used Similarly intracellular fluore
any of the inhibitors used. Similarly, intracellular fluorescence of HY was not aﬀected by PSC833 in cMDR1 cells, or by INDO in cMRP1 cells. However, as expected, Ko143 significantly increased the accumulation of HY in cBCRP Biotintyramide (Fig. 4).
3.6. Inhibition of BCRP by Ko143 reverses the resistance to HY-PDT in cBCRP cells
To prove that BCRP-mediated modulation of HY content may con-tribute to resistance of cancer cells to HY-PDT, annexin V/PI double staining was performed. The concentration of HY for PDT was chosen to cause low phototoxicity in the absence of inhibitors. Therefore, cells were incubated with 50 nM HY for 16 h before HY activation by light at
a total dose of 3.15 J cm−2. Phosphatidylserine externalization and cell viability were analyzed 24 h after HY-PDT. Appropriate inhibitors were added to the equivalent cells 30 min before HY treatment. PSC833, INDO and Ko143 alone did not aﬀect phosphatidylserine externaliza-tion and cell viability in HL-60 cells (Fig. 5D). The same eﬀect was observed for the combination of PSC833 and INDO with HY-PDT in cMDR1 and cMRP1 subclones, respectively (Fig. 5B and C). However, Ko143 significantly enhanced the sensitivity of cBCRP cells to HY-PDT. Inhibition of BCRP by Ko143 before HY-PDT increased the percentage of annexin V+/PI+ cells to the same level as in the case of the HY-PDT eﬀect alone in HL-60 cells (Fig. 5A and D).
To date, the influence of drug eﬄux transporters on HY accumula-tion and subsequent toxicity of HY-PDT has been evaluated only by us by nonspecific inhibition of the transport proteins examined. We pre-viously demonstrated that the application of proadifen (SKF-525A), a well-known cytochrome P450 monooxygenase (CYP450) inhibitor, af-fects the function of MRP1 and BCRP transport proteins, increases in-tracellular HY content and potentiates the eﬀect of HY-PDT . In-creased HY accumulation and toxicity of HY-PDT without direct evidence for participation of ABC transporters was also reported after pretreatment with a phloroglucin derivative, hyperforin, its syntheti-cally prepared derivative, aristoforin [32,33], and MK-886, an eﬃcient 5-lipoxygenase pathway inhibitor .
Therefore, two focal points raised in the present study concern the impact of the most frequently discussed drug eﬄux transporters on HY accumulation and the eﬀectiveness of HY-PDT. To provide a specific proof about HY as a preferential substrate of some ABC transporters and about their impact on the toxicity of HY-PDT, promyelocytic leukemia cells diﬀering in their expression levels of the main drug eﬄux trans-porters P-glycoprotein (P-gp), MRP1 and BCRP were used. The ex-perimental model applied has been used in several former studies [26–28]. Moreover, Prochazkova et al. analyzed whether the determi-nation of intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters in these cell lines. Using qRT-PCR and Western blot analyses, they confirmed high mRNA and protein levels of MDR1, MRP1 and BCRP in cMDR1, cMRP1 and cBCRP subclones, respectively. The levels of non-over-expressed ABC transporters in subclones were inconsiderable . This observation is consistent with our Western blot analysis of particular ABC transporters and supports the suitability of appropriate cells for the present study.
In our previous studies, we hypothesized that HY is a presumable substrate of the ABC transporter BCRP [22,24,25]. However, based on drug accumulation studies performed in cancer cells of diﬀerent tissue origin, we speculated also that MRP1 could modulate the intracellular level of HY [22–24,32]. Drug accumulation studies are based on the use of specific ABC transporter inhibitors and fluorescent substrates. The accumulation of substrates, or HY in our case, was measured for short periods of incubation (0.5–2 h) in a nonspecific experimental model
R. Jendželovský et al.
Fig. 5. BCRP inhibition reverses resistance to HY-PDT in cBCRP cells. Eﬀect of the BCRP inhibitor Ko143, MDR1 inhibitor PSC833 and MRP1 inhibitor INDO on phosphatidylserine externalization and via-bility in (A) cBCRP, (B) cMDR1, (C) cMRP1 and (D) HL-60 cells. An annexin V/PI assay was performed 24 h after HY-PDT. Cells were in-cubated with 50 nM HY for 16 h before HY activation (PDT). Appropriate inhibitors were added to the cells 30 min before HY treatment. The results are expressed as the mean values ± SD of at least three independent experiments. The groups given combined treatment were compared to groups exposed to HY-PDT alone and appropriate inhibitor alone (***p < 0.001). Right side – a re-presentative set of data from one experiment is presented.