br One of the main requirements of successful PDT is
One of the main requirements of successful PDT is a suﬃcient in-tracellular concentration of an applied PS. Unfortunately, analogous to some chemotherapeutic agents, the eﬀect of PDT can be aﬀected by
increased drug eﬄux. Several studies have demonstrated that the ac-tivity of breast cancer resistance protein (BCRP), a member of the G subfamily of ATP-binding cassette (ABC) transporters, reduces the in-tracellular content of diﬀerent PSs and attenuates the sensitivity of cancer Doxorubicin [10–19] and keratinocytes to PDT .
Up to now, it has been demonstrated that HY per se is able to in-crease protein expression of multidrug resistance protein 1 (MDR1) in LS-180V colon adenocarcinoma cells , multidrug resistance-asso-ciated protein 1 (MRP1) and BCRP in HT-29 colon adenocarcinoma cells [22,23], MRP1 in A2780 and A2780cis ovarian adenocarcinoma cells , and BCRP in A549 lung adenocarcinoma cells . How-ever, to the best of our knowledge, none of these studies dealt with a direct link between the ABC transporters mentioned and their impact on HY accumulation and the eﬀectiveness of HY-PDT.
Therefore, in the present study, we investigated whether potential modulations of HY content by some of the well-known ABC transporters could aﬀect the eﬀectiveness of HY-PDT. To prove our hypothesis, we used human leukemia cell lines with diﬀerent levels of MDR1, MRP1 and BCRP. Also, as a negative control, human leukemia cell line HL-60,
E-mail addresses: [email protected] (R. Jendželovský), [email protected] (Z. Jendželovská), [email protected], [email protected] (B. Kuchárová), [email protected] (P. Fedoročko).
Fig. 1. HY content is aﬀected by the overexpression of BCRP protein. HY accumulation in (A) HL-60, (B) cBCRP, (C) cMDR1 and (D) cMRP1 cells was analyzed 2, 4, 8, 12, 16, 24 and 48 h after HY addition. The results are expressed as the mean values ± SD of at least three independent experiments. Experimental groups given HY treatment in cBCRP, cMDR1 and cMRP1 cells were compared to the experimental groups given HY treatment in HL-60 cells (*p < 0.05, ***p < 0.001). (E) Protein levels of BCRP, MDR1 and MRP1 transporters in HL-60, cBCRP, cMDR1 and cMRP1 cells. β-actin served as a loading control. One representative experiment is presented. The results are expressed as the mean values ± SD of three independent experiments. Protein levels of BCRP, MDR1 and MRP1 in cBCRP, cMDR1 and cMRP1 cells were compared with protein levels in HL-60 cells (**p < 0.01, ***p < 0.001).
noted for its basal level of the transport proteins examined, was used.
2. Materials and methods
Human promyelocytic leukemia cell line HL-60 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). MDR1- and MRP1-overexpressing subclones of HL-60 cells (here named as cMDR1 and cMRP1, respectively) and a BCRP-overexpressing deri-vative of HL-60/PLB cells (here named as cBCRP) [26–28] were kindly provided by Balazs Sarkadi, D.Sc. (Membrane Biology Research Group, Hungarian Academy of Sciences, Budapest, Hungary). All these cells were grown in complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biosera,
HY (CAS no.: 548-04-9, HPLC grade; AppliChem GmbH, Darmstadt, Germany), indomethacin (INDO; CAS no.: 53-86-1; Sigma-Aldrich), PSC833 (CAS no.: 121584-18-7; Sigma-Aldrich) and Ko143 (CAS no.: 461054-93-3; Enzo Life Sciences, Inc., Farmingdale, NY, USA) stock solutions were prepared in dimethyl sulfoxide (DMSO) and further di-luted to working solutions which were always freshly prepared im-mediately before addition to the cell culture. The final concentration of DMSO was less than 0.1% and did not influence the cytokinetic para-meters. Because no significant diﬀerences in the response to diluent
H) before HY activation (PDT). Metabolic activity of the cells was evaluated by MTT assay and analyzed 24 and 48 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).
were observed, these data are considered the control.
2.3. Intracellular content of HY
Analysis of HY content was performed to examine the accumulation of HY (10–100 nM) in the cells 2, 4, 8, 12, 16, 24 and 48 h after HY addition. The total cell population was harvested, washed in phosphate-buﬀered saline (PBS) and resuspended in Hank’s balanced salt solution