br BCRP overexpression attenuates HY accumulation in
3.2. BCRP overexpression attenuates HY accumulation in cBCRP cells
To define the extent of the impact of particular ABC transporter overexpression on intracellular HY content, we measured the kinetics of HY incorporation into the Butyrolactone I by flow cytometry. Analyses of HY content were performed 2, 4, 8, 12, 16, 24 and 48 h after HY addition. In all cell lines, time- and concentration-dependent accumulation of HY was observed. As expected, the lowest accumulation of HY was found in cBCRP cells. Surprisingly, cMDR1 and cMRP1 cells incorporated much more HY in comparison to HL-60 cells (Fig. 1).
Fig. 3. No eﬀect of HY-PDT on phosphatidylserine externalization, viability, mitochondrial membrane depolarization and caspase 3 activation in cBCRP cells. HL-60, cBCRP, cMDR1 and cMRP1 cells were incubated with HY for 16 h before HY activation (PDT). Annexin V/PI (A, C, E, G) and caspase 3/TMRE assays (B, D, F, H) were performed 6 and 24 h after HY-PDT. The results are expressed as the mean values ± SD of at least three independent experiments. Experimental groups given HY-PDT treatment in cBCRP, cMDR1 and cMRP1 cells were compared to the experimental groups given HY-PDT treatment in HL-60 cells (*p < 0.05, **p < 0.01, ***p < 0.001).
3.3. Longer incubation of cells with HY does not influence the eﬀect of HY-PDT
To determine the duration of HY incorporation into cells that would be optimal for maximal PDT eﬃciency, we used a simple and well-established MTT assay. Based on the data from HY kinetics, 16- and 48-h time intervals required for HY accumulation were selected and
compared. The MTT assay was performed 24 and 48 h after exposure of cells to 10–100 nM HY activated by light at a total dose of 3.15 J cm−2. HY-PDT showed a concentration-dependent eﬀect on the metabolic activity of HL-60, cMDR1 and cMRP1 cells. On the contrary, no eﬀect of HY-PDT on the metabolic activity of cBCRP cells was observed (Fig. 2). Estimated IC50 values derived from mean metabolic activity are shown in Table 1. As no considerable diﬀerences in the eﬀect of the compared
Fig. 4. BCRP inhibition increases HY content in cBCRP cells. Eﬀect of the BCRP inhibitor Ko143, MDR1 inhibitor PSC833 and MRP1 inhibitor INDO on HY accumulation in HL-60, cBCRP, cMDR1 and cMRP1 cells. Appropriate in-hibitors were added to the cells 30 min before HY treatment. HY accumulation was analyzed 16 h after addition of 50 nM HY. The results are expressed as the mean values ± SD of at least three independent experiments. The groups given combined treatment were compared to groups treated with HY alone (***p < 0.001).
time intervals of HY incubation with cells on PDT were observed, a 16-h time interval for HY accumulation before its light activation was chosen for subsequent experiments.
3.4. BCRP overexpression protects cells from HY-PDT-induced phosphatidylserine externalization, mitochondrial membrane depolarization and caspase 3 activation
Even though the MTT assay is a generally accepted method, con-cerning our own experiences, it cannot actually be used to evaluate the eﬀect of substances on the onset of cell death [30,31]. Therefore, an-nexin V/PI and caspase 3/TMRE double staining methods were used to establish the influence of MDR1, MRP1 and BCRP overexpression on the cytotoxic action of HY-PDT. The detection of phosphatidylserine ex-ternalization and cell viability by annexin V/PI double staining re-vealed a concentration- and time-dependent eﬀect of HY-PDT in HL-60, cMDR1 and cMRP1 cells (Fig. 3A, E, G). Moreover, HY-PDT induced depolarization of mitochondrial membranes which was accompanied by the activation of apoptotic execution caspase 3 in HL-60, cMDR1 and cMRP1 cells (Fig. 3B, F, H). Estimated IC50 values derived from an-nexin V+/PI+ cells and caspase 3+/TMRE- cells are shown in Table 1. Consistent with the results of the MTT assay, no changes in phospha-tidylserine externalization, cell viability, depolarization of mitochon-drial membranes and activation of caspase 3 were observed in cBCRP cells after HY-PDT (Fig. 3C and D). Simultaneously, in comparison with parental HL-60 cells, a decrease in the population of annexin V+/PI+ and caspase 3+/TMRE- cells was observed in cMDR1 and cMRP1 cells exposed to 25 nM and/or 50 nM HY activated by light at a total dose of 3.15 J cm−2 (Fig. 3).
3.5. Inhibition of BCRP by Ko143 increases HY content in cBCRP cells
To confirm the above-mentioned results and to prove our previous hypotheses about HY as a possible substrate of BCRP, flow cytometric analysis of HY content in cells pretreated with inhibitors of particular ABC transporters was performed. Appropriate inhibitors, PSC833 as an MDR1 inhibitor, INDO as an MRP1 inhibitor and Ko143 as a BCRP inhibitor, were added to the equivalent cells 30 min before HY treat-ment. HY accumulation in HL-60, cMDR1, cMRP1 and cBCRP cells was analyzed 16 h after addition of HY (i.e. at the time of HY light activa-tion). No eﬀect on HY content was observed in HL-60 cells treated with