HBSS The analysis was performed cells per sample using a
(HBSS). The analysis was performed (1 × 104 314-19-2 per sample) using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) with a 488-nm argon-ion excitation laser, and fluorescence was de-tected via a 585/42-nm band-pass filter (FL-2). The intensity of HY fluorescence in the cells was quantified using FlowJo software (Tree Star Inc., Ashland, OR, USA).
To assess the impact of ABC transporter inhibition on HY accumu-lation, cells were incubated in the presence or absence of the
IC50 values of HY-PDT in HL-60, cBCRP, cMDR1 and cMRP1 cells. Estimated IC50 values (nM) are derived from mean metabolic activity (MTT assay), an-nexin V+/PI+ cells (annexin V/PI assay) and caspase 3+/TMRE- cells (cas-pase 3/TMRE assay).
Assay HY Time of HL-60 cBCRP cMDR1 cMRP1
N/A - Not Available.
appropriate inhibitor (BCRP inhibitor Ko143, 5 μM; MDR1 inhibitor PSC833, 2.5 μM; MRP1 inhibitor INDO, 20 μM) for 30 min at 37 °C, 95% humidity and 5% CO2. Subsequently, cells were incubated with 50 nM HY for 16 h. Afterwards, cells were harvested, washed in PBS, re-suspended in HBSS and analyzed (1 × 104 cells per sample) using a BD FACSCalibur flow cytometer. Accumulation of HY was calculated as the ratio of relative fluorescence (RFU) of combined therapy compared to the RFU of inhibitor and HY by themselves, as previously described .
2.4. Western blot analysis
Culture plates containing cells treated with HY as well as untreated controls were placed on a plastic diﬀuser sheet of the irradiating device (a set of twelve L18W/30 white tubes, Osram, Berlin, Germany) with a maximum emission range of 530–620 nm (fluency rate 3.15 mW cm−2). Incorporated HY was activated by light at a total dose of 3.15 J cm−2.
The MTT assays were performed as previously reported by , to evaluate changes in the metabolic activity of cells that occurred as a consequence of HY-PDT treatment. The results were evaluated 24 and 48 h after HY-PDT as a percentage of the absorbance (λ = 584 nm) of the untreated control. HY-PDT IC50 values were extrapolated from a dose–response fit to the metabolic activity data using OriginPro 8.5.0 SR1 (OriginLab Corp., Northampton, MA, USA).
2.7. Phosphatidylserine externalization analysis
For phosphatidylserine externalization and viability analysis, a BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s
instructions. The total cell population was harvested 6 and 24 h after HY-PDT, centrifuged, washed with HBSS, stained with annexin V-FITC in 1× binding buﬀer for 20 min at room temperature (RT) in the dark, stained with propidium iodide (PI) for 5 min and then analyzed (1 × 104 cells per sample) using a BD FACSCalibur flow cytometer. Fluorescence was detected via a 530/30-nm band-pass filter (FL-1; annexin V/FITC) and via a 670-nm long-pass filter (FL-3; PI). The re-sults were analyzed using FlowJo software (Tree Star Inc.). HY-PDT IC50 values were extrapolated from a dose-response fit to the frequency of annexin V+/PI+ cells using OriginPro 8.5.0 SR1 (OriginLab Corp.).
2.8. Detection of mitochondrial membrane depolarization and caspase 3 activation
Simultaneous staining with tetramethylrhodamine ethyl ester (TMRE; Molecular Probes, Eugene, OR, USA) and NucView 488 caspase 3 substrate (Biotium, Hayward, CA, USA) was used to assess the impact of HY-PDT on mitochondrial membrane depolarization and caspase 3 activation, as previously reported by . The total cell population was harvested 6 and 24 h after HY-PDT, centrifuged, washed with PBS and stained with 0.1 μmol.dm−3 TMRE and 4 μmol dm−3 NucView 488 in HBSS for 20 min at 37 °C. Cells (1 × 104 cells per sample) were sub-sequently analyzed using a BD FACSCalibur flow cytometer. Fluores-cence was detected via a 530/30-nm band-pass filter (FL-1; NucView
488) and via a 585/42-nm band-pass filter (FL-2; TMRE). The results were analyzed using FlowJo software (Tree Star Inc.). HY-PDT IC50 values were extrapolated from a dose–response fit to the frequency of caspase 3+/TMRE- cells using OriginPro 8.5.0 SR1 (OriginLab Corp.).
2.9. Statistical analysis
Data were analyzed using one-way ANOVA with Tukey’s post-test and are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Significance levels are indicated in the legend for each particular figure.
3.1. Overexpression of particular ABC transporters in appropriate cell lines
Western blot analysis was used to prove MDR1, MRP1 and BCRP overexpression in appropriate promyelocytic leukemia cell lines. As shown in Fig. 1E, we confirmed the expected high levels of MDR1, MRP1 and BCRP in cMDR1, cMRP1 and cBCRP cells, respectively. Low but insignificant expression of MDR1 was also found in parental HL-60 cells and cMRP1 and cBCRP subclones. Similarly, insignificant expres-sion of MRP1 was found in cMDR1 and cBCRP subclones.