Multidrug resistance caused by the overexpression of ABC transporter proteins in cancer cells remains a major obstacle limiting chemotherapy efficacy. Drugs inhibiting these transporters have been shown to increase the anti-proliferative properties of chemotherapeutics. As we previously described, proadifen, a P450 monooxygenase inhibitor, might also be able to inhibit some ABC transporters, including breast cancer resistance protein (BCRP). Because mitoxantrone (MTX) is a strong BCRP substrate and is often used in the treatment of leukemia, we investigated the effect of 24 h proadifen pre-treatment on the cytotoxicity of MTX in leukemic cell lines that are sensitive to MTX (HL-60) and MTX-resistant ABCG2-overexpressing subclone (cBCRP). We show for the first time that proadifen is able to enhance the cytotoxic properties of MTX in cBCRP cells, particularly through the inhibition of BCRP expression and activity. This proadifen-MTX synergism was also mediated by the inhibition of various cellular proteins engaged in apoptosis, including Mc-1, Bcl-xL, survivin and activation of procaspase-3. Proadifen also decreased the expression of γH2AX, which is involved in the recruitment of reparation proteins. Moreover, the inhibition of DNA damage repair proteins Ku86 and B23 after proadifen treatment indicate a possible role of proadifen in DNA repair blockage, thus suppressing the reparation rate of MTX-induced DSBs.
Chemotherapy is one of the most common strategies in the treatment of cancer, although it is frequently unsuccessful due to the development of chemoresistance, and especially multi-drug resistance (MDR). A number of mechanisms involved in the occurrence of MDR have been described, including the overexpression of one or more ATP binding cassette (ABC) transporter proteins that mediate the efflux of many clinically relevant drugs. More than 40 ABC transporters have been identified so far, but just a few play a role in MDR, including the breast cancer resistance protein (BCRP/ABCG2). BCRP expression was detected in several different solid tumours (
Several compounds have been shown to inhibit the efflux activity of ABC transporters, thereby increasing intracellular drug accumulation and sensitising cancer cells to therapy. It was previously reported that proadifen (SKF-525A), a well-known cytochrome P450 monooxygenase inhibitor, not only has anti-proliferative potential in some cancer cell lines, but it is also able to inhibit BCRP and MRP1 transporter proteins (
Human promyelocytic leukemia cell line HL-60 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and its ABCG2-overexpressing subclone (here referred to as cBCRP) was kindly provided by Dr Balazs Sarkadi (Membrane Research Group, Hungarian Academy of Sciences, Budapest, Hungary) (
Proadifen (SKF-525A; α-phenyl-α-propylbenzene-acetic acid 2-(diethylamino)ethylester; CAS no. 62-68-0; Sigma-Aldrich, St. Louis, MO, USA) and mitoxantrone dihydrochloride (MTX; 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl) amino]-9,10-anthracenedione dihydrochloride; CAS no. 70476-82-3; HPLC grade, Sigma-Aldrich) stock solutions were prepared in distilled water and stored at −20°C). The concentrations of prepared stock solutions were 10 and 1 mM for proadifen and MTX, respectively. Working solutions of both reagents were always freshly prepared before being added to the cultures. The final concentrations of distilled water did not influence the cytokinetic parameters. Because no significant differences in the response to distilled water were observed, these data are referred to as a control.
The cells were seeded in 96-well plates (MTT assay), in 6-well plates (flow cytometry analyses) or in 60-mm Petri dishes (western blotting, membrane-bound BCRP expression, RNA isolation) (all TPP, Trasadingen, Switzerland). Both cell lines were treated immediately after seeding (
For the determination of the IC20 values (20% inhibitory concentration) of proadifen and MTX, MTT assays were performed 24, 48 and 72 h after proadifen and MTX addition (0 in the time schedule).
Cells were pre-treated with proadifen for 24 h (−24 h in the time schedule) prior to MTX addition (0 in the time schedule). Changes in metabolic activity, mitochondrial membrane potential, cell death and cell cycle distribution were analysed 24 and 48 h after MTX treatment. Changes in the phosphorylation of the H2AX histone were analysed 2 and 24 h after MTX addition. Membrane-bound BCRP expression was examined 30 min, 1 h and 2 h after MTX treatment. BCRP mRNA level and the expression of selected proteins were detected 30 min, 1, 2 and 6 h after MTX addition.
To analyse the changes in the cell metabolic activity that occurred as the consequence of single and combined drug treatment, MTT assays were carried out as previously described (
Cells were treated according to the experimental design (
Analysis of cell viability and phosphatidylserine externalisation was performed using the Apoptest™-FITC (Dako Denmark A/S, Glostrup, Denmark) according to the manufacturer's instructions. The cells were harvested at scheduled times (
For the flow cytometric analysis of cell cycle distribution, cells were harvested at scheduled times (
The cells were pre-treated with proadifen for 24 h. Immediately after incubation, the cells were harvested and resuspended in HBSS. Afterwards, MTX was added to the cells and its intracellular accumulation was detected continuously for the duration of 45 min. The measurement was performed by BD FACSAria II SORP (Becton-Dickinson) using excitation with a 640 nm laser and emission acquisition in an Alexa Fluor 700 (DM685LP-710/50) channel.
For the flow cytometry assay that was intended to detect cell surface expression of BCRP, cells (3×105) were harvested at scheduled time-points (
Flow cytometric analysis of the histone H2AX phosphorylation on Ser139 (γH2AX) was performed to examine possible changes in MTX-mediated DNA double strand breaks (DSBs), as previously described (
For detection of the expression of selected proteins, cells were treated in line with the experimental design (
In order to evaluate
The results were analysed using a one-way ANOVA with Tukey's post-test or t-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 figure.
The metabolic activity was assessed after exposure of HL-60 and cBCRP cells to 0–50 μM proadifen and 0–20 μM MTX. Proadifen and MTX showed a time- and dose-dependent inhibitory effect on both cell lines, with a more pronounced effect on sensitive HL-60 cells (data not shown). IC20 values for proadifen, as well as for MTX (
To determine the impact of proadifen on MTX action, cells were pre-treated with proadifen for 24 h. The inhibitory effect of MTX on cell metabolic activity was potentiated by proadifen in both cell lines. A much stronger effect was observed in resistant cBCRP cells, where the metabolic activity significantly decreased in the experimental group administered the combined treatment compared to the group treated with MTX alone (
Based on these results, proadifen-MTX combination index (CI) values were evaluated using CalcuSyn software (
To determine whether proadifen-MTX synergism arising from the MTT assay may reflect the impact of proadifen on MTX cytotoxicity, various cell death parameters were analysed. Changes in mitochondrial membrane depolarisation revealed different effects in sensitive HL-60 and resistant cBCRP cells. In the resistant cell line proadifen alone and MTX alone significantly increased the percentage of cells with dissipated mitochondrial membrane potential (MMP) compared to the untreated control. Additionally, the cytotoxic effect of MTX was more pronounced after proadifen pre-treatment, particularly at 48 h after MTX addition (
Consistent with the results of the MMP analysis, Annexin V-FITC/PI double staining revealed no effect of proadifen on MTX cytotoxicity in HL-60 cells (
Cell cycle distribution was affected by both drugs (
To determine whether the cytotoxic effect of MTX was correlated with its ability to cause DSBs, the phosphorylation of histone H2AX on Ser139 (γH2AX) was analysed. MTX alone elevated the expression of γH2AX, especially in the sensitive cell line. The effect of proadifen on the γH2AX level was cell line-dependent, leading to a slight decrease in γH2AX expression in cBCRP cells. However, no changes in the phosphorylation of H2AX in the HL-60 cells were detected. Interestingly, pre-treatment of cBCRP cells with proadifen markedly decreased the MTX-induced γH2AX expression, with a more significant effect 24 h after MTX addition (
The analysis of MTX accumulation in HL-60 and cBCRP cells was performed to verify the possible inhibitory effect of proadifen on the BCRP transporter (
To determine whether the changes in the increased intracellular accumulation of MTX after combined drug treatment were associated with decreased expression of BCRP, whole cell protein levels, as well as cell surface expression of BCRP, were detected. In the case of the BCRP transporter, no protein levels were detected in the HL-60 cells (
To verify whether proadifen-mediated reduction of the BCRP protein level correlated with its mRNA content, semi-quantitative RT-PCR was performed (
To determine whether the changes in cytotoxicity of the combined drug treatment were associated with altered expression of anti-apoptotic proteins, proteins engaged in DNA damage repair or proteins regulating the expression of BCRP transporter, western blot analysis was performed at early time-points after the MTX addition (30 min, 1, 2 and 6 h) (
In cBCRP cells, proadifen alone and combined with MTX markedly downregulated several anti-apoptotic proteins, especially Mcl-1, Bcl-xL and survivin (
The reduction of transcription factor E2F1 that is potentially involved in the regulation of BCRP expression was observed, except for one time-point (2 h) (
Proteins involved in DSB repair were also investigated (
Cancer cell chemoresistance remains a major obstacle to successful therapy. Great effort has been made to identify various agents that could sensitise cancer cells to treatment, thereby improving chemotherapy outcome. Proadifen (SKF-525A), an inhibitor of cytochrome P450 monooxygenases (P450), is a drug approved by the US Food and Drug Administration (
Proadifen showed a time- and dose-dependent inhibitory effect on HL-60 and cBCRP cells, with markedly lower concentrations inhibiting the sensitive HL-60 cells. On the contrary, such a low proadifen concentration stimulated metabolic activity in resistant cBCRP cells and much higher concentrations were required for significant decreases in this parameter (data not shown). A similar effect of different proadifen concentrations in various cancer cell models was previously reported by other authors (
Proadifen as an inhibitor of cytochrome P450 monooxygenases, along with cyclooxygenase and lipoxygenase inhibitors, could suppress the conversion of free arachidonic acid. Extensive research has focused on the role of cyclo-oxygenase-2 (COX-2) inhibitors in sensitising cancer cells to chemotherapy, mainly through non-specific inhibition of one or more ABC transporter proteins. Our previous study indicates that proadifen represents a potential agent capable of sensitising cancer cells to HY-PDT, most likely via inhibition of more than one transporter protein (
The underlying mechanism by which proadifen may affect BCRP expression and function is not yet fully understood. Several transcription factors and signalling pathways have been shown to regulate BCRP expression or activity (
Subsequently, we focused on the expression of proteins involved in the induction of apoptosis, including Bcl-2, Bcl-xL, Mcl-1 and the inhibitor of apoptosis, survivin, as well as caspase-3, a protease engaged in the execution phase of apoptosis. Interestingly, we detected slightly elevated Bcl-2 protein levels after treatment with both drugs alone and in combination, indicating Bcl-2 independent induction of apoptosis in cBCRP cells. However, proadifen alone and in combined treatment downregulated the expression of survivin, Mcl-1, and Bcl-xL and led to the activation of procaspase-3. These results are consistent with our previous study (
Since proadifen sensitised cBCRP cells to MTX-induced cell death and even affected the cytostatic action of MTX by cell cycle redistribution, we investigated the impact of proadifen on certain proteins involved in DNA damage repair. MTX, a topoisomerase II inhibitor and DNA intercalator, induces DSBs due to the collision of the progressing RNA polymerase with the stabilised topoisomerase II-DNA complex. Therefore, we focused on several proteins engaged in the DSBs repair. The generation of DSBs causes rapid phosphorylation of histone H2AX on Ser139 (γH2AX). γH2AX represents an important factor in DNA damage signalling pathways (
In conclusion, our results revealed anti-proliferative properties of proadifen in MTX-resistant human promyelocytic leukemia cells. Moreover, we demonstrated the ability of proadifen to increase the intracellular accumulation of MTX, thereby enhancing its cytotoxicity in the MTX-resistant cBCRP cell line. This effect was mediated through proadifen-mediated inhibition of BCRP activity and expression. Furthermore, our study indicates that proadifen also affects the expression levels of various anti-apoptotic proteins, as well as proteins engaged in the DNA damage repair pathways, thus enhancing chemotherapy efficacy. Taken together, based on our recent findings, as well as on previous results, we suggest that proadifen may be beneficial in combination with chemotherapy for overcoming multidrug resitance, enhancing the cytotoxicity of these drugs.
This study was supported by the Slovak Research and Development Agency under the contract no. APVV-0040-10, the Scientific Grant Agency of the Ministry of Education of the Slovak Republic under the contract no. VEGA 1/0626/11 and the Cancer Research Foundation under the contract no. 0-12-102/0001-00. The authors are very grateful to Viera Balážová for the assistance with technical procedures.
Experimental design. (A) Analyses of single drug treatment; (B) Analyses of combined drug treatment.
Changes in cell metabolic activity. Effects of proadifen, MTX and their combination on the metabolic activity of HL-60 and cBCRP cells. The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone (IC20 in HL-60 equals 6.95 μM/24 h and 5.51 μM/48 h; IC20 in cBCRP equals 37.19 μM/24 h and 30.74 μM/48 h) and MTX alone (IC20 in HL-60 equals 0.029 μM/24 h and 0.011 μM/48 h; IC20 in cBCRP equals 3.68 μM/24 h and 1.53 μM/48 h) were compared to the untreated control (**p<0.01, ***p<0.001). The groups given combined treatment were compared to proadifen alone (□□□p<0.001) and MTX alone (■p<0.05, ■■■p<0.001).
Changes in the percentage of cells with dissipated mitochondrial membrane potential (MMP). Effects of proadifen, MTX and their combination. The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone (IC20 in HL-60 equals 6.95 μM/24 h and 5.51 μM/48 h; IC20 in cBCRP equals 37.19 μM/24 h and 30.74 μM/48 h) and MTX alone (IC20 in HL-60 equals 0.029 μM/24 h and 0.011 μM/48 h; IC20 in cBCRP equals 3.68 μM/24 h and 1.53 μM/48 h) were compared to the untreated control (*p<0.05, **p<0.01, ***p<0.001). The groups given combined treatment were compared to proadifen alone (□□p<0.01, □□□p<0.001) and MTX alone (■■p<0.01, ■■■p<0.001).
Changes in cell cycle distribution. Effects of proadifen, MTX and their combination on the distribution of the cell cycle. The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone (IC20 in HL-60 equals 6.95 μM/24 h and 5.51 μM/48 h; IC20 in cBCRP equals 37.19 μM/24 h and 30.74 μM/48 h) and MTX alone (IC20 in HL-60 equals 0.029 μM/24 h and 0.011 μM/48 h; IC20 in cBCRP equals 3.68 μM/24 h and 1.53 μM/48 h) were compared to the untreated control (*p<0.05, **p<0.01, ***p<0.001). The groups given the combined treatment were compared to proadifen alone (□p<0.05, □□p<0.01, □□□p<0.001) and MTX alone (■p<0.05, ■■■p<0.001).
Changes in DNA damage and DNA repair proteins. (A) Effects of proadifen, MTX and their combination on the phosphorylation of histone H2AX (Ser 139). The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone (IC20 in HL-60 cells equals 11.28 μM/2 h and 6.95 μM/24 h; IC20 in cBCRP equals 40.49 μM/2 h and 37.19 μM/24 h) and MTX alone (IC20 in HL-60 equals 0.029 μM/2 and 24 h; IC20 in cBCRP equals 3.68 μM/2 and 24 h) were compared to the untreated control (*p<0.05, **p<0.01, ***p<0.001). The groups given combined treatment were compared to proadifen alone (□p<0.05, □□p<0.01, □□□p<0.001) and MTX alone (■■p<0.01). (B) Effects of proadifen, MTX and their combination on protein levels of Ku86 and B23. One representative experiment of two is presented. HL-60 cells were pre-treated with IC20 of proadifen equalling 11.28 and 40.49 μM in cBCRP. IC20 of MTX in HL-60 equals 0.029 μM; IC20 in cBCRP equals 3.68 μM at all time-points.
Effect of proadifen on BCRP expression and activity. (A) Effect of proadifen on MTX intracellular accumulation. (B) Effects of proadifen, MTX and their combination on BCRP whole cell protein levels. One representative experiment of two is presented. (C) Effects of proadifen, MTX and their combination on cell surface expression of BCRP. The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone and MTX alone were compared to the untreated control (*p<0.05, **p<0.01). The groups given combined treatment were compared to proadifen alone and MTX alone (■p<0.05, ■■p<0.01). (D) Effects of proadifen, MTX and their combination on mRNA levels of BCRP. One representative experiment of two is presented. In all experiments, HL-60 cells were pre-treated with IC20 of proadifen equalling 11.28 and 40.49 μM in cBCRP. IC20 of MTX in HL-60 equals 0.029 μM; IC20 in cBCRP equals 3.68 μM.
Effects of proadifen, MTX and their combination on the expression of anti-apoptotic proteins and activation of procaspase-3. One representative experiment of two is presented. HL-60 cells were pre-treated with IC20 of proadifen equalling 11.28 and 40.49 μM in cBCRP. IC20 of MTX in HL-60 equals 0.029 μM; IC20 in cBCRP equals 3.68 μM at all time-points.
Effects of proadifen, MTX and their combination on the expression of proteins involved in the regulation of BCRP activity and expression. One representative experiment of two is presented. HL-60 cells were pre-treated with IC20 of proadifen equalling 11.28 and 40.49 μM in cBCRP. IC20 of MTX in HL-60 equals 0.029 μM; IC20 in cBCRP equals 3.68 μM at all time-points.
The IC20 of proadifen and MTX in HL-60 and cBCRP cells.
HL-60 | cBCRP | |||||
---|---|---|---|---|---|---|
|
| |||||
24 h | 48 h | 72 h | 24 h | 48 h | 72 h | |
Proadifen | ||||||
IC20 (μM) | 11.28 | 6.95 | 5.51 | 40.49 | 37.19 | 30.74 |
MTX | ||||||
IC20 (μM) | 0.029 | 0.011 | 0.002 | 3.68 | 1.53 | 0.36 |
Estimated IC20 values were derived from mean metabolic activity values of at least three independent experiments.
Combination index (CI) values of proadifen-MTX mutual combinations.
Proadifen (μM) | Mitoxantrone (μM) | CI | CalcuSyn effect | |
---|---|---|---|---|
HL-60 | ||||
24 h | 5 | 0.05 | 0.630 | Synergism |
5 | 0.01 | 0.965 | Nearly additive | |
48 h | 5 | 0.01 | 0.744 | Moderate synergism |
BCRP | ||||
24 h | 35 | 5 | 0.780 | Moderate synergism |
40 | 5 | 0.855 | Slight synergism | |
48 h | 30 | 1 | 0.886 | Slight synergism |
CI values were calculated from mean cell metabolic activity of at least three independent experiments using the CalcuSyn program.
Analysis of cell death.
Annexin V+/PI− | Annexin V−/PI+ | Annexin V+/PI+ | |
---|---|---|---|
HL-60 | |||
24 h | |||
Control | 0.47±0.16 | 1.54±0.49 | 2.04±0.44 |
Proadifen | 0.64±0.21 | 1.15±0.27 | 2.96±0.53 |
MTX | 1.21±0.59 | 1.98±0.99 | 6.02±2.12b |
Proadifen + MTX | 0.89±0.35 | 1.43±0.69 | 6.31±1.52b,e |
48 h | |||
Control | 0.44±0.17 | 1.61±0.78 | 3.42±1.16 |
Proadifen | 0.57±0.33 | 1.04±0.57 | 3.12±0.85 |
MTX | 0.56±0.31 | 4.95±2.08 | 14.36±6.09b |
Proadifen + MTX | 0.42±0.22 | 4.70±2.52 | 12.94±5.57 |
cBCRP | |||
24 h | |||
Control | 0.36±0.04 | 0.84±0.13 | 2.17±0.14 |
Proadifen | 0.53±0.26 | 2.86±0.52 | 7.00±3.62 |
MTX | 7.89±0.76d | 7.11±0.48d | 14.27±2.85c |
Proadifen + MTX | 1.79±0.13b,i | 4.82±1.41c,h | 22.68±2.42d,f,h |
48 h | |||
Control | 0.74±0.18 | 0.72±0.08 | 3.11±0.71 |
Proadifen | 0.79±0.05 | 1.15±0.05 | 5.37±1.70 |
MTX | 0.71±0.20 | 6.30±0.35c | 12.97±3.63b |
Proadifen + MTX | 14.26±2.23d,g,i | 6.41±1.99c,f | 31.39±3.87d,g,i |
Effects of proadifen, MTX and their mutual combination on phosphatidylserine externalisation (Annexin V) and viability (PI). The results are expressed as the mean values ± SD of at least three independent experiments. The groups treated with proadifen alone (IC20 in HL-60 equals 6.95 μM/24 h and 5.51 μM/48 h; IC20 in cBCRP equals 37.19 μM/24 h and 30.74 μM/48 h) and MTX alone (IC20 in HL-60 equals 0.029 μM/24 h and 0.011 μM/48 h; IC20 in cBCRP equals 3.68 μM/24 h and 1.53 μM/48 h) were compared to the untreated control (bp<0.05, cp<0.01, dp<0.001). The groups given combined treatment were compared to proadifen alone (ep<0.05, fp<0.01, gp<0.001) and MTX alone (hp<0.05, ip<0.001).