ABT-737 and erufosine combination against castration-resistant prostate cancer: a promising but cell-type specific response associated with the modulation of anti-apoptotic signaling
Ezgi Avsar Abdika,*, Ferda Kaleagasioglua,b,*, Hüseyin Abdika, Fikrettin Sahina and Martin R. Bergerc
A deeper understanding of the molecular basis of castration-resistant prostate cancer (CRPC) paved the way for the rational design and development of targeted therapies, which yielded promising preclinical results. However, translation of these potentially promising agents into clinics has usually failed, partly because of tumor heterogeneity. In this study, anticancer activities of the Bcl-2 inhibitor ABT-737 and the Akt-inhibitor erufosine (ErPC3) alone and in combination were compared between CRPC (PC-3 and DU-145) and healthy (PNT-1A) cell lines. The combination of ABT-737 and ErPC3 showed synergistic antiproliferative, antimigratory, and apoptotic effects in PC-3 cells. In DU-145 cells, ErPC3 showed a resistant profile, with half-maximal inhibitory concentration (IC50) values more than two-fold of PC-3, and combining ErPC3 with ABT-737 yielded no added benefit for all the incubation periods compared with ErPC3 alone. In PNT-1A cells, ABT-737 and ErPC3 alone and in combination reduced cell survival slightly and only at the highest concentrations. Apoptosis analysis showed that ABT-737 induced increased Akt expression and ErPC3 induced increased Mcl-1 expression in DU-145 cells. In conclusion, the ABT-737 and ErPC3
combination seems to be promising against CRPC, with a favorable safety profile in healthy cells. However, CRPC cell- type-specific resistance may be induced by enhancement of antiapoptotic signaling. Anti-Cancer Drugs 30:383–393 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
Anti-Cancer Drugs 2019, 30:383–393 Keywords: ABT-737, Akt, apoptosis, Bcl-2,
castration-resistant prostate cancer, drug resistance, erufosine
aDepartment of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, bDepartment of Medical Pharmacology, Faculty of Medicine, Near East University, Mersin, Turkey and cToxicology and Chemotherapy Unit, German Cancer Research Center, Heidelberg, Germany
Correspondence to Ezgi Avsar Abdik, PhD, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, 26 August Campus, Kayısdagı Street, 326A, Ataşehir, Istanbul 34755, Turkey
Tel: + 90 216 578 0000/ + 90 216 578 3198; fax: + 90 216 578 0490; e-mail: [email protected]
*Ezgi Avsar Abdik and Ferda Kaleagasioglu contributed equally to the writing of this article.
Received 2 October 2018 Revised form accepted 30 November 2018
Introduction
Prostate cancer (PCa) is an androgen-dependent disease and responds to androgen-deprivation therapy, but it may transform into an aggressive and lethal phenotype [cas- tration-resistant prostate cancer (CRPC)], which grows albeit castrate levels of androgens [1,2]. Currently avail- able treatment methods are deemed to be ineffective in CRPC [3] and there exists an obvious need for the development of novel drugs against various targets [4,5].
PCa has intrinsic molecular heterogeneity. The PI3K/NF- κB/Bcl-2 survival mechanism is considered the dominant pathway, especially in the pathogenesis of PCa progression and CRPC [6–8]. Bcl-2 is not expressed in secretory pros- tate epithelial cells and is proposed as an androgen receptor (AR) bypassing pathway leading to a transition to androgen independency in PCa [9]. The PI3K/Akt/mTOR is another crucial pathway. Aberrant PI3K pathway signaling has been shown to have a huge impact on PCa initiation and pro- gression [10,11]. Therefore, targeting Bcl-2 and PI3K/Akt/
mTOR pathways, alone or in combination, seems to be a promising approach against CRPC [12–14].
ABT-737 is a BH3 mimetic, which interacts with anti- apoptotic Bcl-2, Bcl-xL, and Bcl-w with high affinity, but not with Mcl-1, thus preventing their interaction with pro-apoptotic proteins Bax and Bak and releasing these sequestered proteins [15,16]. By shifting the pro-apoptotic/
anti-apoptotic balance toward apoptosis, ABT-737 has been shown to show remarkable anti-cancer activity in various primary as well as established cell lines including leukemia, lymphoma [16], lung cancer [17], and PCa [18]. In addition, ABT-737 activates Akt by phosphorylation of Ser473, which in turn phosphorylates Bad on Ser136 and Ser112. This inactivation of Bad through Akt activation is another established resistance mechanism against ABT- 737 [19]. Therefore, inhibition of Bcl-2 only by ABT-737 is not sufficient, but other signaling pathways, which are related to androgen resistance, should also be targeted to kill CRPC cells [3].
0959-4973 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/CAD.0000000000000736
Alkylphosphocholines (APCs) are structurally similar to and derived from lysophosphatidylcholine. In contrast to traditional chemotherapeutics, APCs are called membrane-targeted agents as they interact with the cell membrane rather than DNA. Erufosine (erucylphospho- N,N,N-trimethylpropylammonium, ErPC3) is a novel APC that is under development. ErPC3 depho- sphorylates Akt on Ser473 and inhibits its translocation to the plasma membrane. Apart from its inhibitory action on the prosurvival PI3K/Akt pathway, ErPC3 initiates apoptosis by pro-sapoptotic SAPK/JNK and MAPK/ERK pathways [20,21]. ErPC3 can cause endoplasmic reticu- lum and mitochondrial stress, which lead to activation of PERK and IRE-1α pathways, and hence apoptosis, autophagy, and reactive oxygen species production [22]. ErPC3 was shown to induce apoptosis in acute myeloid leukemia [23,24], chronic lymphocytic leukemia [25], acute T-lymphocytic leukemia [26], oral squamous car- cinoma [22,27], human glioblastoma [26,28], and PCa cell lines [29] of human origin.
Therefore, ABT-737 and ErPC3, alone or in combina- tion, should be evaluated further for CRPC treatment. CRPC may respond to the AR signaling axis at least for a short time [30], but ultimate development of resistance and AR independence is inevitable because of its divergent clonal evolution [31]. The present study aimed to investigate this AR-independent stage by comparing two CRPC cell lines: PC-3 and DU-145. PC-3 and DU- 145 are usually considered to be AR negative, although they express low but detectable AR protein levels com- pared with AR-positive cell lines [32]. The comparative effects in a healthy, AR-positive prostate epithelium cell line (PNT-1A) were expected to provide toxicity profil- ing of the investigational drugs for future in vivo studies.
Materials and methods
Reagents
ABT-737 was purchased from Abbott Laboratories (Abbott Park, Illinois, USA), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mmol/l, and stored at
- 20°C. ErPC3 was provided by Prof. H. Eibl (Max Planck-Institute of Biophysical Chemistry, Göttingen, Germany), dissolved in saline at a concentration of 20 mmol/l, and stored at 4°C.
Cell lines and culture condition
Human PCa cell lines, PC-3 and DU-145, and normal prostate epithelium cells PNT-1A were kindly provided by the Biotechnology Department of Yeditepe University (Istanbul, Turkey). Cells were incubated at 37°C in com- plete RPMI-1640 (Invitrogen, Gibco, Paisley, UK) supple- mented with 10% fetal bovine serum (Invitrogen, Gibco) and 1% penicillin/streptomycin/amphotericin (Invitrogen, Gibco) in an incubator with a humidified atmosphere (95% O2/5% CO2).
Cell proliferation assay
Cell proliferation was measured using the MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide) dye reduction assay as described by Mossman [33]. Briefly, MTT (Sigma, Munich, Germany) solution was added (10 µl/well) and incubated at 37°C for 3 h. After 3 h, 100 μl solvent (0.04 N HCl acid in 2-propanol) was added to dissolve formazan crystals. Absorbance at 540 nm (reference 690 nm) was detected using an ELISA plate reader (Biotek, Winooski, Vermont, USA). The cell doubling time (Td) was calculated using the Patterson formula: Td = T × lg 2/(lg N2 – lg N1). Cell growth rates (in h) were calculated using the following formula: Growth rate (μ) = ln (N2/N1)/T2 – T1. For exposure to drugs alone and in combination, cells were seeded into 96-well plates (5000 cells/well) by overnight incubation at 37°C. After 24 h, cells were treated with 3.13–100 µmol/l of ErPC3 and 1–20 µmol/l of ABT-737 alone prepared in complete RPMI-1640 for 24, 48, and 72 h incubation periods. In the combination treatments, 6.25, 12.5, and 25 µmol/l ErPC3 with 5, 10, and 20 µmol/l ABT-737 were added to the cells at the same time. Cell survival rates were expressed as the percentage of control groups at 24, 48, and 72 h. Half-maximal inhibitory concentration (IC50) was calculated using the equation of logarithmic regres- sion trendline.
In vitro wound-healing (scratch) assay
Cells were seeded at a density of 100 000 cells/well into 24-well plates and incubated in an incubator overnight at 37°C in humidified air with 5% CO2. After 24 h, cells were scratched with a sterile 200-µl pipette tip and the medium was immediately changed with culture medium containing different concentrations of drugs alone and in combination. Scratch zones were photographed at 0 and 24 h by the Zeiss PrimoVert light microscope with an AxioCam ERc5s camera (Carl Zeiss Microscopy LLC, Thornwood, New York, USA). ErPC3 and ABT-737 were added at final concentrations of 6.25–50 and 2.5–20 µmol/l/well, respectively, and the effect on wound healing was monitored. Each concentration of ErPC3, or ABT-737, alone and in combination as well as the control groups were tested in triplicate wells. Wound closure area was measured by three randomly selected regions using Zen 2011 software (Carl Zeiss Microscopy GmbH, Göttingen, Germany). All cell lines were studied in parallel and the duration of the microscopic procedure was maintained the same to exclude environ- mental condition-related differences in wound-healing responses.
Annexin V apoptosis assay
Cells were stained with fluorescent annexin V (annexin V-FITC) and propidium iodide (PI) using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences Pharmingen, San Diego, California, USA) according to the manufacturer’s instruction. Briefly, cells (2 × 105cells/
well) were seeded in six-well plates and incubated in an incubator overnight at 37°C and humidified air with 5% CO2. After 24 h, cells were treated with 6.25 µmol/l ErPC3 and 5 µmol/l ABT-737 for PC-3 and PNT-1A, and 50 µmol/l ErPC3 and 5 µmol/l ABT-737 for DU-145. After 24 h, cells were harvested, washed twice with cold PBS, and resuspended in 100 μl Annexin V binding buffer. Then, 5 μl Annexin V-FITC (#556547; BD Biosciences Pharmingen, San Diego, California, USA) and 5 μl PI staining solution were added to each tube. Cell samples were incubated at room temperature (RT) for 20 min in the dark and analyzed using the BD FACS Calibur Cell Sorting System (BD Biosciences Pharmingen).
Western blot analysis
Primary antibodies for Bcl-2 (#2872), Mcl-1 (#4572), Akt (#9272), p-Akt (#9271), and GAPDH (#8884) were pur- chased from Cell Signaling Technology (Beverly, Massachusetts, USA). Briefly, total protein isolated from cells were treated with the drugs by RIPA buffer (#sc- 24948; Santa Cruz Biotechnology, Santa Cruz, California, USA). Protein concentrations were determined using the BCA assay (#23227; Pierce, Rockford, Illinois, USA). Sufficient amounts of protein samples were prepared and denatured for 5 min at 95°C. Any kD Mini-PROTEAN TGX precast gels (#456-9033; Bio-Rad, Hercules, California, USA) were used in electrophoresis of proteins. Proteins were loaded at 20 μg/well and electrophoresed by applying 90 V for 120 min. Then, proteins were transferred from gel to nitrocellulose membranes (#162- 0115; Bio-Rad) using the semi-dry transfer technique at 250 mA for 60 min. Following the transfer step, mem- branes were blocked by incubating for 1 h at RT in a 5% nonfat dry milk powder (#170-6404) prepared in Tris- buffered saline and Tween-20 solution (TBS-T). Membranes were incubated with primary antibodies (dilution 1 : 3000) in blocking solution at 4°C overnight. After the incubation, the membranes were washed in TBS-T three times for 10 min and then incubated with anti-rabbit (#7074, dilution 1 : 5000) IgG secondary antibody prepared in TBS-T for 1 h at RT. Then, the membranes were incubated with the Clarity ECL Western Blotting Substrate (#1705060; Bio-Rad) at RT for 1 min and images were taken using a ChemiDoc MP imaging system (Bio-Rad). Bands were normalized by glyceraldehyde 3-phosphate dehydrogenase band intensities and calculated using the Image Lab software program (Biorad). The results were evaluated as fold change of the control groups.
Statistical analysis
The data are presented as mean ± SD. The data were analyzed statistically using one-way analysis of variance with Tukey’s post-hoc test. P values less than 0.05 were considered statistically significant results.
Results
Growth curves of PC-3, DU-145, and PNT-1A
The doubling times (Td)/growth rates of PC-3, DU-145, and PNT-1A calculated for 96 h were 24.12/0.0287, 29.36/0.0236, and 59.68/0.0116/h, respectively.
Anti-proliferative effects of ABT-737 and ErPC3 alone and in combination in PC-3
Both ABT-737 and ErPC3 exerted time-dependent and concentration-dependent cytotoxic effects over 72 h of the incubation period (Fig. 1a and b, respectively). An anti-proliferative effect was observed after 72 h at the concentrations of at least 5 μmol/l for ABT-737 and of at least 6.25 μmol/l for ErPC3. IC50 (µmol/l) values were 30, 16, and 14 and for ABT-737 as well as 73, 55, and 12.5 for ErPC3 following 24, 48, and 72 h of incubation. With the combination treatment, a ceiling effect was observed after 72 h at concentrations equal to and greater than ABT-737 (5 µmol/l)/ErPC3 (6.25 µmol/l), respectively (Fig. 1c). Following 24, 48, and 72 h, combined treatment with ABT-737 (5 µmol/l)/ErPC3 (6.25 µmol/l) reduced the cell viability to 84 ± 10, 48 ± 5, and 30 ± 4%, respec- tively. The observed cytotoxic effect was lower when ABT-737 and ErPC3 were used as single agents at the same concentrations (C): cell viability decreased to 98 ± 11, 96 ± 10, and 86 ± 12% (C%) and 97 ± 15, 81 ± 9, and 69 ± 12% (C%) following 24, 48, and 72 h of incuba- tion with ABT-737 (5 μmol/l)/ErPC3 (6.25 μmol/l), respectively. Therefore, the ABT-737/ErPC3 combina- tion exerted a synergistic cytotoxic effect in PC-3.
Anti-proliferative effects of ABT-737 and ErPC3 alone and in combination on DU-145
ABT-737 exerted a time-dependent cytotoxic effect (Fig. 1a). After 24 h, no significant cytotoxic effect was found at all concentrations. Following 48 and 72 h, only the highest concentration of ABT-737 (20 µmol/l) reduced cell survival to 89 ± 7 and 70 ± 8% (0.1% DMSO control group), respectively. ErPC3 exerted time-dependent and concentration-dependent cytotoxic effects only at high doses (50 and 100 µmol/l) after all incubation periods (Fig. 1b). An anti-proliferative effect was observed at concentrations of at least 25 μmol/l after 72 h. IC50 (µmol/l) values were 46, 44, and 30 for ABT-737 as well as 89, 49, and 22 for ErPC3 following 24, 48, and 72 h of incubation. Following incubation for 24, 48, and 72 h with ABT-737 (5 µmol/l) and ErPC3 (50 µmol/l) alone, cell viability was modulated to 108 ± 9, 108 ± 14, and 108 ± 9% (0.1% DMSO control group) as well as 86 ± 4, 51 ± 5, and 40 ± 3% (growth medium only), respectively. Survival rates of DU-145 fol- lowing combination exposure to ABT-737 (5, 10, and 20 µmol/l) and ErPC3 (6.25, 12.5, 25, and 50 µmol/l) are shown in Fig. 1c. Following 24, 48, and 72 h, combination treat- ment with ABT-737 (5 µmol/l)/ErPC3 (50 µmol/l) was more effective than other combination treatments and a ceiling effect was reached. Following 24, 48, and 72 h, ABT-737
(5µmol/l)/ErPC3 (50 µmol/l) combination treatment
Fig. 1
Cytotoxic effect of ABT-737 (a) and ErPC3 (b) alone and in combination (c) in PC-3, DU-145, and PNT-1A as determined by the MTT assay at 24, 48, and 72 h after treatment. Cell survival fractions are given as percentage of respective controls (untreated control for ErPC3 and treated only with DMSO 0.1% for ABT-737). The data are mean ± SD values of three independent experiments conducted in triplicate (*P ≤ 0.05 compared with untreated control cells).
decreased cell viability to 92 ± 5, 52 ± 5, and 41 ± 2% (0.1% DMSO control group), respectively. Therefore, combining 5 µmol/l ABT-737/50 μmol/l ErPC3 provided no added value for all the incubation periods compared with ErPC3 alone.
Anti-proliferative effects of ABT-737 and ErPC3 alone and in combination on PNT-1A
Survival rates of PNT-1A following exposure to ABT-737 (1–20 µmol/l) are shown in Fig. 1a. After 24 h, no significant cytotoxic effect was found at all concentrations. Following 48 and 72 h, ABT-737 (5, 10, and 20 µmol/l) exerted cytotoxic effects. After 72 h of exposure to ABT-737 (5, 10, and 20 µmol/l), cell viability decreased to 79 ± 8, 70 ± 6, and 67 ± 5% (0.1% DMSO control group), respectively. Survival rates of the PNT-1A cells following exposure to ErPC3 (3.13–100 µmol/l) are shown in Fig. 1b. After 24 h, no sig- nificant cytotoxic effect was found at all concentrations. Following 72 h, ErPC3 (25, 50, and 100 µmol/l) exerted cytotoxic effects. After 72 h exposure to ErPC3 (25, 50, and 100 µmol/l), cell viability decreased to 82 ± 6, 82 ± 5, and 83 ± 5% (growth medium only), respectively. IC50 (µmol/l) values were 388, 78, and 40 for ABT-737, and – 403, 476,
and 430 for ErPC3 following 24, 48, and 72 h of incubation. Survival rates of the PNT-1A following exposure to com- binations of ABT-737 (5, 10, and 20 µmol/l) and ErPC3 (6.25, 12.5, 25, and 50 µmol/l) are shown in Fig. 1c. Combination treatments, even at the highest concentration (20 µmol/l ABT-737/50 µmol/l ErPC3), did not show sig- nificantly higher cytotoxicity than exposure to a single agent. An anti-proliferative effect was more evident at higher concentrations and after longer incubation times. The cell survival rate after 72 h was higher (77%), even with 20 µmol/l ABT-737/50 µmol/l ErPC3 in PNT-1A, than that in DU-145 (41%) exposed to 5 µmol/l ABT-737/50 µmol/l ErPC3.
Wound-healing assay in PC-3, DU-145, and PNT-1A
The wound-healing assay was performed for 24 h with concentrations that were lower than 24 h IC50 values in all cell lines and that had no or limited action on cell proliferation. IC50 values (µmol/l; 24 h) for ABT-737 and ErPC3 alone were as follows: in PC-3 cells, 30 and 73 µmol/l, respectively; in DU-145 cells, 46 and 89 µmol/l, respectively; and in PNT-1A cells, 388 and – 403 µmol/l, respectively. On the basis of these IC50 values, the
following concentrations (which are much below 24 h IC50 values) were chosen for wound-healing testing: for ErPC3: 6.25, 12.5, and 25 µmol/l in PC-3 and PNT-1A cells and 12.5, 25, and 50 µmol/l in DU-145 cells; for ABT-737: 5, 10, and 20 µmol/l in all cell lines.
All cell lines showed different migratory profiles during wound healing. In PC-3, both agents showed a concentration-dependent inhibitory effect on wound clo- sure. ABT-737 at concentrations of 5, 10, and 20 µmol/l reduced wound closure significantly (Fig. 2a). Following
24h, the gap closure (%) in the 20 µmol/l ABT-737-treated cells was 26 ± 6% compared with 42 ± 4 and 52 ± 6% in 10 and 5 µmol/l ABT-737-treated cells, respectively (vs. 69 ± 4% in the 0.1% DMSO control group; Fig. 2b). ErPC3 at concentrations of 6.25, 12.5, and 25 µmol/l reduced wound closure significantly compared with the control group (Fig. 2c). Following 24 h, the gap closure (%) in the
25µmol/l ErPC3-treated cells was 26 ± 5% compared with 46 ± 5%, and 61 ± 3% in 12.5 and 6.25 µmol/l ErPC3-treated cells, respectively (vs. 70 ± 2% in the growth medium only;
Fig. 2d). In addition, the ABT-737 and ErPC3 combination increased the inhibitory effect on wound closure, which became evident by 24 h compared with single agents (Fig. 2e). The combination of 5 µmol/l ABT-737/6.25 µmol/l ErPC3 induced a gap closure of 26 ± 5% (Fig. 2f).
In DU-145, both agents showed a concentration-dependent inhibitory effect on wound closure. ABT-737 (5, 10, and 20 µmol/l; Fig. 3a) and ErPC3 (12.5, 25, and 50 µmol/l; Fig. 3c) alone inhibited wound-healing capacity, which became evident by 24 h at all concentrations tested compared with the control groups. ABT-737 was more effective at 20 µmol/l than 5 and 10 µmol/l. Following 24 h, the gap closure in the 20 µmol/l ABT-737-treated cells was 7 ± 3% compared with 15 ± 2 and 40 ± 3% in 10 and 5 µmol/l ABT-737-treated cells, respectively (vs. 51 ± 2% in the 0.1% DMSO control group; Fig. 3b). Fifty micromole per liter ErPC3 was significantly more effective than 12.5 and 25 µmol/l ErPC3. After 24 h, gap closure in the 50 µmol/l ErPC3-treated cells was 6 ± 4% compared with 22 ± 6 and 38 ± 6% in 25 and 12.5 µmol/l ErPC3-treated cells, respectively (vs. 53 ± 3% in the growth
Fig. 2
Representative images of ABT-737 (a), ErPC3 (c) alone, and in combination (e) treated PC-3 scratches taken by an inverted light microscope, and wound closure rates of PC-3 cells after ABT-737 (b), ErPC3 (d) alone and in combination [ErPC3 (6.25 µmol/l)/ABT-737 (5 µmol/l)] (f). The data are mean ± SD values of three independent experiments conducted in triplicate (*P ≤ 0.05 compared with untreated control cells). NC, normal control.
Fig. 3
Representative images of ABT-737 (a), ErPC3 (c) alone, and in combination (e) treated DU-145 scratches taken by an inverted light microscope, and wound closure rates of DU-145 cells after ABT-737 (b), ErPC3 (d) alone and in combination [ErPC3 (12.5 µmol/l)/ABT-737 (5 µmol/l)] (f). The data are mean ± SD values of three independent experiments conducted in triplicate (*P ≤ 0.05 compared with untreated control cells). NC, normal control.
medium only; Fig. 3d). In addition, no significant difference was observed between the 12.5 µmol/l ErPC3, 5 µmol/l ABT-737 alone, and the combination group (Fig. 3e). At 24 h, the gap closure in the 5 µmol/l ABT-737/12.5 µmol/l ErPC3 combination-treated cells was 36 ± 5% (Fig. 3f).
PNT-1A cells were treated with 5, 10, and 20 µmol/l ABT-737 for 24 h in the wound-healing assays (Fig. 4a). After 24 h, gap closure in the 20 µmol/l ABT-737-treated cells was 38 ± 1% compared with 39 ± 4 and 44 ± 3% in 10 and 5 µmol/l ABT-737-treated cells (vs. 44 ± 3% in the 0.1% DMSO control group). At the highest concentration of ABT-737 (20 µmol/l), wound closure was not highly significant compared with the control group (Fig. 4b). In PNT-1A cells, gap closure (%) in 6.25, 12.5, and 25 µmol/l ErPC3-treated cells was 45 ± 11, 43 ± 3, and 39 ± 5%, respectively, and it was not significantly different com- pared with the control group (Fig. 4c and d). The gap closure (%) in the cells exposed to 5 µmol/l ABT- 737/6.25 µmol/l ErPC3 combination treatment was 44 ± 10% (Fig. 4f). There was no statistically significant
difference between the combination and single-agent treatments (Fig. 4e).
Apoptosis analysis in PC-3, DU-145, and PNT-1A Apoptotic status of PC-3, DU-145, and PNT-1A was studied by the Annexin V-FITC assay (Fig. 5a). In PC-3, following 24 h of incubation, 5 µmol/l ABT-737 induced an increase in the percentage of total apoptotic cells to 30 ± 6% compared with the 0.1% DMSO control group
(6± 4%). Similarly, following 24 h incubation with ErPC3 (6.25 µmol/l), the apoptotic cell percentage increased to 18 ± 2% compared with the control group (0 ± 3%). The percentage of total apoptotic cells was 75 ± 8% (vs. the growth medium only control group 6 ± 4%) with 6.25 µmol/l ErPC3/5 µmol/l ABT-737. The combination treat- ment significantly increased the percentage of apoptotic cells compared with the single agents and a synergistic effect on apoptosis was observed (Fig. 5b). In DU-145, no significantly different effect on the total apoptotic cell percentage was observed between the 5 µmol/l ABT-
Fig. 4
Representative images of ABT-737 (a), ErPC3 (c) alone and in combination (e) treated PNT-1A scratches taken by an inverted light microscope, and wound closure rates of PNT-1A cells after ABT-737 (b), ErPC3 (d) alone and in combination [ErPC3 (6.25 µmol/l)/ABT-737 (5 µmol/l)] (f). The data are mean ± SD values of three independent experiments conducted in triplicate (*P ≤ 0.05 compared with untreated control cells). NC, normal control.
737-treated (9 ± 3%) and the 0.1% DMSO control group (9 ± 1%) at 24 h. Fifty micromole per liter ErPC3 alone and in combination with 5 µmol/l ABT-737 significantly increased the percentage of total apoptotic cells to 42 ± 2 and 27 ± 4%, respectively (vs. the 0.1% DMSO control group 0 ± 2 and 9 ± 1%, respectively) at 24 h. Fifty micromole per liter ErPC3 alone was more effective than the combination treatment and the addition of ABT-737 to ErPC3 decreased the apoptotic cell ratio (Fig. 5c). In PNT-1A, 5 µmol/l ABT-737 and 6.25 µmol/l ErPC3 alone and in combination increased the percentage of total apoptotic cells to 5 ± 1, 1 ± 0.3, and 5 ± 1%, respectively (vs. 0.01 % DMSO control group 0 ± 0.5%, and growth medium only control group 0 ± 0.5%, respectively) at 24 h. ABT-737 treatment and the ABT-737/ErPC3 combina- tion showed similar effects and their apoptotic effects were greater than 6.25 µmol/l ErPC3 (Fig. 5d).
Western blot analysis in PC-3, DU-145, and PNT-1A
Bcl-2, Mcl-1, Akt, and p-Akt protein levels were deter- mined for western blot analysis. PC-3 and PNT-1A were
exposed for 12 h–6.25 µmol/l ErPC3 or 5 µmol/l ABT- 737, alone and in combination, and DU-145 were treated with 50 µmol/l ErPC3 or 5 µmol/l ABT-737, alone and in combination. In PC-3, Bcl-2 expression decreased sig- nificantly following exposure to 6.25 µmol/l ErPC3 (∼20%), 5 µmol/l ABT-737 (∼50%), and 6.25 µmol/l ErPC3/5 µmol/l ABT-737 (∼70%). The latter decrease was significant versus all other groups. Similarly, the Mcl- 1 level was significantly reduced by 6.25 µmol/l ErPC3 (∼30%) and 6.25 µmol/l ErPC3/5 µmol/l ABT-737 (∼40%) in treated cells compared with 5 µmol/l ABT-737 and the control groups. Akt and p-Akt levels were also sig- nificantly reduced in 6.25 µmol/l ErPC3 and 6.25 µmol/l ErPC3/5 µmol/l ABT-737-treated cells compared with the control groups. The Akt level decreased by ∼ 30 and ∼ 50% after 6.25 µmol/l ErPC3 and 6.25 µmol/l ErPC3/5 µmol/l ABT-737 treatments, respectively, compared with the control groups, respectively. The p-Akt level was significantly decreased in 6.25 µmol/l ErPC3 (∼40%) and 6.25 µmol/l ErPC3/5 µmol/l ABT-737 (∼30%)-treated cells, respectively, compared with the control groups.
Fig. 5
Annexin V-FITC/PI-staining assay. Representative FACS analysis of apoptosis by ErPC3 (6.25 for PC-3 and PNT-1A; 50 µmol/l for DU-145) and ABT- 737 (5 µmol/l), alone and in combination for 24 h (a). Graphical representation of the percentage of apoptotic, PC-3 (b), DU-145 (c) and PNT-1A (d) cells. The data are mean ± SD values of three independent experiments conducted in triplicate (*P ≤ 0.05 compared with untreated control cells). NC, normal control.
Interestingly, 6.25 µmol/l ErPC3 significantly reduced the p-Akt level more than 6.25 µmol/l ErPC3/5 µmol/l ABT-737, whereas 5 µmol/l ABT-737 alone did not alter Akt and p-Akt levels (Fig. 6a).
In DU-145, following 12 h of incubation, Bcl-2 levels were significantly decreased following 50 µmol/l ErPC3/5 µmol/l ABT-737 (∼80%) treatment compared with the 0.1% DMSO control group. The Bcl-2 level did not change in 50 µmol/l ErPC3 or 5 µmol/l ABT- 737-treated cells. The Mcl-1 level increased significantly in 50 µmol/l ErPC3 (∼40%) and 50 µmol/l ErPC3/5 µmol/l ABT-737 (∼40%)-treated cells, but no significant change was observed by 5 µmol/l ABT-737 compared with the control groups. Akt levels significantly decreased by ∼ 80 and ∼ 30% following 50 µmol/l ErPC3 and 50 µmol/l ErPC3/5 µmol/l ABT-737 treatments, respectively, but increased significantly in 5 µmol/l ABT-737 (∼40%) compared with the control groups. The p-Akt level was significantly reduced in 50 µmol/l ErPC3 (∼60%)-treated
cells compared with the growth medium-only group. Five micromole per liter ABT-737 and 50 µmol/l ErPC3/5 µmol/l ABT-737 did not induce significant changes in p-Akt expression (Fig. 6b).
In PNT-1A, following 12 h of incubation, the Bcl-2 levels did not change in 50 µmol/l ErPC3, 5 µmol/l ABT-737, and combined drug-treated cells. The Mcl-1 level decreased significantly in 6.25 µmol/l ErPC3 (∼30%) and 5 µmol/l ABT-737 (∼40%)-treated cells and did not change significantly at the same concentrations of both agents in combination compared with the control groups. Akt levels in 6.25 µmol/l ErPC3, 5 µmol/l ABT-737, and 6.25 µmol/l ErPC3/5 µmol/l ABT-737-treated cells were not significantly different from the control groups. p-Akt levels decreased significantly and p-Akt levels did not change significantly in 6.25 µmol/l ErPC3/5 µmol/l ABT- 737 (∼30%)-treated cells compared with the 0.1% DMSO control group (Fig. 6c).
Fig. 6
Effects of various concentrations of ErPC3 (6.25 µmol/l for PC-3 and PNT-1A; 50 µmol/l for DU-145) and ABT-737 (5 µmol/l) on the western blot analysis of PC-3 (a), DU-145 (b) and PNT-1A (c) at 12 h. The numbers below the western blots represent the relative expression levels of the indicated proteins. Results shown are representative of three independent experiments. The values were derived by dividing the densitometric output for each band by the densitometric output for the corresponding GAPDH band and subsequent normalization.
Discussion
This is the first report to show that targeting two aberrant pathways in CRPC cell lines PC-3 and DU-145, by the Bcl-2 inhibitor ABT-737 and the Akt-inhibitor ErPC3, induced differential effects on proliferation, migration, and apoptosis. ABT-737 and ErPC3 synergistically induced growth inhibition and apoptosis in PC-3 cells, whereas DU-145 cells showed a more resistant pheno- type, which was associated with the specific modulation of antiapoptotic signaling. Both ABT-737 and ErPC3 alone and in combination showed lower toxicity in PNT- 1A cells. The favorable in vivo safety profile of ErPC3 has been confirmed in a recent study in which ErPC3 inhibited tumor growth of head and neck squamous carcinoma cell xenografts over a period of 4 weeks without causing significant toxicity [34].
Similar results were also reported by the previous studies in PC-3 [29,35–40]. The anti-proliferative effect of ABT-737 was enhanced when combined with hydroxychloroquine, docetaxel, gemcitabine, and Pim serine/threonine kinase inhibitor, SMI-4a, in PC-3 cells [36–40]. In these cells, the combination of ErPC3 (12.5 μmol/l) with irradiation did not significantly increase the antineoplastic effects compared with ErPC3 alone [29].
This is also the first study to show the effects of ABT-737 and ErPC3 alone and in combination on the migration of CRPC cells. The ABT-737/ErPC3 combination sig- nificantly inhibited in vitro wound healing more than the correspondent concentrations of single agents in PC-3 and DU-145 cells, whereas no significant difference was observed in PNT-1A cells (Figs 2–4). PI3K/NF-κB/Bcl-2
is a well-known survival pathway in PCa, but its impact on metastasis has only recently been understood. Bcl-2 proteins are highly expressed in PC-3 and DU-145 cells, but normally not in secretory prostate epithelial cells [9, 39]. However, Bcl-2 is also expressed in the immortalized human prostate cell line PNT-1A [41]. The fact that ABT-737 and ErPC3 alone and in combination showed nearly undetectable anti-migratory effects in PNT-1A indicates the need to consider mechanisms in addition to Bcl-2.
In PC-3, this study showed a synergistic apoptotic effect (75%) with the ABT-737 (5 µmol/l)/ErPC3 (6.25 µmol/l) combination following 24 h. Parrondo et al. [18] utilized a lower dose of ABT-737 (1 µmol/l) for 72 h, which increased the percentage of apoptotic cell death ∼ 30%, whereas combination with docetaxel increased apoptotic cell death by two-fold. Forty-eight hour incubation with 2.5 µmol/l ABT-737 was associated with a higher rate of apoptotic cell death (34%). A significant increase in apoptotic cell death (∼77%) after 48 h was also observed with the combination of ABT-737 (5 µmol/l)/pseudolaric acid B (2 µmol/l) compared with single agents [42].
In DU-145 cells, ErPC3 treatment alone had a greater effect on apoptosis induction than its combination with ABT-737 in this study. Bax-deficient DU-145 usually show a resistant phenotype requiring higher concentrations and longer exposure times to antineoplastic agents [19]. A longer incubation period than that used in our study (48 h) was associated with significantly higher percentages of apoptotic cell death (15–8% with 5 –15 µmol/l ABT-737, respectively). The combination of ABT-737 (5 µmol/l)/
pseudolaric acid B (2 µmol/l) and ABT-737 (15 µmol/l)/
methylseleninic acid (3 µmol/l) increased the percentage of cell death (∼38 and 40.6%, respectively) compared with single agents in DU-145 cells [19,42]. Pandit and Gartel also investigated the apoptotic effect of a much lower dose (0.35 µmol/l) of ABT-737 for 48 h and showed that there was no significant difference from the control group. The percentage of DU-145 cells undergoing apoptosis after treatment for 48 h in response to a combination of the global transcription inhibitor ARC/ABT-737 increased significantly, as evidenced by the nearly nine-fold increase to 27% compared with the control group [43]. Wnętrzak et al. [44] incubated DU-145 cells with ErPC3 at con- centrations ranging between 12.5 and 50 µmol/l for 24 and 48 h, and showed that the therapeutic effect was related to prolonged exposure time and the higher drug concentration.
In this study, 12 h exposure to investigational agents induced markedly different protein expression patterns in PC-3 and DU-145 cells. In PC-3 cells, ErPC3 and the combination treatment reduced the expression of Bcl-2, Mcl-1, Akt, and p-Akt, whereas ABT-737 reduced only Bcl-2 expression and the addition of ErPC3 potentiated this effect. Compared with the current study, Rudner et al. [29] applied higher concentrations (12.5–25 μmol/l) of ErPC3 for 48 h in PC-3 cells and found a decrease in p-Akt expression, but no change in Bcl-2 and Mcl-1 levels. Parrondo et al. [18] demonstrated that following 72 h of incubation, 1 µmol/l ABT-737 significantly increased Bcl-2 and Mcl-1 protein levels, but combina- tion with docetaxel counteracted the ABT-737-mediated increase in Mcl-1. Similar to the findings by Parrondo and colleagues, Hao et al. [36] reported that the ABT-737 (0.4 µmol/l)/docetaxel combination for 48 h decreased Bcl-2 and Mcl-1 levels.
In DU-145 cells, ErPC3 alone reduced Akt and p-Akt inhibition at much higher concentrations than in PC-3. The resistance against ABT-737 in DU-145 has been associated with its low affinity to the anti-apoptotic Mcl-1 protein and the lack of proapoptotic protein Bax [18,19]. ABT-737-induced increase in Akt expression in DU-145 cells may have reduced the anti-proliferative and apop- totic effects. A similar finding was reported by Yin et al. [19], who showed that 15 µmol/l ABT-737 treatment for 48 h slightly increased the p-Akt level. In addition, the ErPC3-induced increase in Mcl-1 expression may have contributed toward the high resistance of DU-145 cells. Combination treatment also provided no further benefit in terms of anti-proliferative and apoptotic effects, as supported by analysis of protein expression in DU- 145 cells.
Because of the high intrinsic heterogeneity of CRPC, the combined inhibition of Bcl-2 and Akt may provide new avenues. The anticancer effects of ErPC3 and ABT-737 in CRPC were shown to be promising, but at the same
time cell line specific. The high resistance of DU-145 cells could be associated with ABT-737-induced increase in Akt expression and ErPC3-induced increase in Mcl-1 expression. Therefore, resistance against Bcl-2 and Akt inhibition seems to be associated with the differential modulation of antiapoptotic signaling in CRPC cell lines. Future prospects for improving the therapeutic outcome in CRPC with targeted antineoplastic agents may include greater awareness of drug-induced antiapoptotic pathway aberration, drug susceptibility testing for each specimen, and biomarker-based selection of drug combinations.
The decreased toxic effects in PNT-1A are indicative of a better safety profile. Accordingly, already published favorable data on in vitro safety of both agents seem to be promising for the planned in vivo studies in the future.
Acknowledgements
The authors thank Burcin Asutay for her help in flow cytometry analysis.
This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK), Project no: 315S039.
Authors’ contributions: E.A.A. and F.K. conceived and designed the experiments, and E.A.A. and H.A. con- ducted the experiments and analyzed the data. All authors have read and approved the final manuscript.
Conflicts of interest
There are no conflicts of interest.
References
1Scher HI, Fizazi K, Saad F, Taplin M-E, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 2012; 367:1187–1197.
2Wise HM, Hermida MA, Leslie NR. Prostate cancer, PI3K, PTEN and prognosis. Clin Sci 2017; 131:197–210.
3Kim J-H, Lee H, Shin EA, Kim DH, Choi JB, Kim S-H. Implications of Bcl-2 and its interplay with other molecules and signaling pathways in prostate cancer progression. Expert Opin Ther Targets 2017; 21:911–920.
4Clarke J, Armstrong A. Novel therapies for the treatment of advanced prostate cancer. Curr Treat Options Oncol 2013; 14:109–126.
5Wang G, Reed E, Li QQ. Apoptosis in prostate cancer: progressive and therapeutic implications. Int J Mol Med 2004; 14:23–34.
6Catz S, Johnson J. BCL-2 in prostate cancer: a minireview. Apoptosis 2003; 8:29–37.
7Heath EI, Carducci MA. Targeted therapy trials for prostate cancer. Prostate Cancer 2008. pp. 383–400.
8Zielinski RR, Eigl BJ, Chi KN. Targeting the apoptosis pathway in prostate cancer. Cancer J 2013; 19:79–89.
9Hu R, Denmeade SR, Luo J. Molecular processes leading to aberrant androgen receptor signaling and castration resistance in prostate cancer. Expert Rev Endocrinol Metab 2010; 5:753–764.
10Bitting RL, Armstrong AJ. Targeting the PI3K/Akt/mTOR pathway in castration-resistant prostate cancer. Endocr Relat Cancer 2013; 20: R83–R99.
11Edlind MP, Hsieh AC. PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance. Asian J Androl 2014; 16:378.
12LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/
Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 2008; 11:32–50.
13Polivka J Jr, Janku F. Molecular targets for cancer therapy in the PI3K/AKT/
mTOR pathway. Pharmacol Ther 2014; 142:164–175.
14Toren P, Zoubeidi A. Targeting the PI3K/Akt pathway in prostate cancer: challenges and opportunities. Int J Oncol 2014; 45:1793–1801.
15Kline M, Rajkumar SV, Timm M, Kimlinger T, Haug J, Lust J, et al. ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells. Leukemia 2007; 21:1549.
16Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT- 737 in acute myeloid leukemia. Cancer Cell 2006; 10:375–388.
17Hauck P, Chao BH, Litz J, Krystal GW. Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737. Mol Cancer Ther 2009; 8:883–892.
18Parrondo R, de las Pozas A, Reiner T, Perez-Stable C. ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells. PeerJ 2013; 1:e144.
19Yin S, Dong Y, Li J, Fan L, Wang L, Lu J, et al. Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad. Apoptosis 2012; 17:388–399.
20Chometon G, Cappuccini F, Raducanu A, Aumailley M, Jendrossek V. The membrane-targeted alkylphosphocholine erufosine interferes with survival signals from the extracellular matrix. Anticancer Agents Med Chem 2014; 14:578–591.
21van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta 2013; 1831:663–674.
22Ansari SS, Sharma AK, Soni H, Ali DM, Tews B, König R, et al. Induction of ER and mitochondrial stress by the alkylphosphocholine erufosine in oral squamous cell carcinoma cells. Cell Death Dis 2018; 9:296.
23Fiegl M, Lindner LH, Juergens M, Eibl H, Hiddemann W, Braess J. Erufosine, a novel alkylphosphocholine, in acute myeloid leukemia: single activity and combination with other antileukemic drugs. Cancer Chemother Pharmacol 2008; 62:321–329.
24Martelli A, Papa V, Tazzari P, Ricci F, Evangelisti C, Chiarini F, et al. Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK-and PP2A-dependent mechanisms. Leukemia 2010; 24:687.
25Königs SK, Pallasch CP, Lindner LH, Schwamb J, Schulz A, Brinker R, et al. Erufosine, a novel alkylphosphocholine, induces apoptosis in CLL through a caspase-dependent pathway. Leuk Res 2010; 34:1064–1069.
26Lemeshko VV, Kugler W. Synergistic inhibition of mitochondrial respiration by anticancer agent erucylphosphohomocholine and cyclosporin A. J Biol Chem 2007; 282:37303–37307.
27Kapoor V, Zaharieva MM, Das SN, Berger MR. Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt–mTOR signaling pathway in oral squamous cell carcinoma. Cancer Lett 2012; 319:39–48.
28Veenman L, Alten J, Linnemannstöns K, Shandalov Y, Zeno S, Lakomek M, et al. Potential involvement of F0F1-ATP (synth) ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines. Apoptosis 2010; 15:753–768.
29Rudner J, Ruiner C-E, Handrick R, Eibl H-J, Belka C, Jendrossek V. The Akt- inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and
increases the short term effects of ionizing radiation. Radiat Oncol 2010; 5:108.
30Wyatt AW, Gleave ME. Targeting the adaptive molecular landscape of castration‐resistant prostate cancer. EMBO Mol Med 2015; 7:878–894.
31Beltran H, Prandi D, Mosquera JM, Benelli M, Puca L, Cyrta J, et al. Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer. Nat Med 2016; 22:298.
32Alimirah F, Chen J, Basrawala Z, Xin H, Choubey D. DU‐145 and PC‐3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation. FEBS Lett 2006; 580:2294–2300.
33Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55–63.
34Ansari SS, Sharma AK, Zepp M, Ivanova E, Bergmann F, König R, et al. Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine’s down regulation of cell cycle processes in OSCC cells. Oncotarget 2018; 9:5797.
35Dash R, Azab B, Quinn BA, Shen X, Wang X-Y, Das SK, et al. Apogossypol derivative BI-97C1 (Sabutoclax) targeting Mcl-1 sensitizes prostate cancer cells to mda-7/IL-24–mediated toxicity. Proc Natl Acad Sci 2011; 108:8785–8790.
36Hao J, Mao X, Ding D, Du G, Liu Z. The effect of cell killing by ABT-737 synergized with docetaxel in human prostate cancer PC-3 cells [Article in Chinese]. Zhonghua Wai Ke Za Zhi 2012; 50:161–165.
37Saleem A, Dvorzhinski D, Santanam U, Mathew R, Bray K, Stein M, et al. Effect of dual inhibition of apoptosis and autophagy in prostate cancer. Prostate 2012; 72:1374–1381.
38Song JH, Kandasamy K, Kraft AS. ABT-737
induces expression of the death receptor 5 and sensitizes human cancer cells to TRAIL-induced apoptosis. J Biol Chem 2008; 283:25003–25013.
39Tamaki H, Harashima N, Hiraki M, Arichi N, Nishimura N, Shiina H, et al. Bcl-2 family inhibition sensitizes human prostate cancer cells to docetaxel and promotes unexpected apoptosis under caspase-9 inhibition. Oncotarget 2014; 5:11399.
40Zhang C, Cai T-Y, Zhu H, Yang L-Q, Jiang H, Dong X-W, et al. Synergistic antitumor activity of gemcitabine and ABT-737 in vitro and in vivo through disrupting the interaction of USP9X and Mcl-1. Mol Cancer Ther 2011; 10:1264–1275.
41Sztalmachova M, Hlavna M, Gumulec J, Holubova M, Babula P, Balvan J, et al. Effect of zinc (II) ions on the expression of pro-and anti-apoptotic factors in high-grade prostate carcinoma cells. Oncol Rep 2012; 28:806–814.
42Tong J, Yin S, Dong Y, Guo X, Fan L, Ye M, et al. Pseudolaric acid B induces caspase‐dependent apoptosis and autophagic cell death in prostate
cancer cells. Phytother Res 2013; 27:885–891.
43Pandit B, Gartel AL. New potential anti‐cancer agents synergize with bortezomib and ABT‐737 against prostate cancer. Prostate 2010; 70:825–833.
44Wnętrzak A, Lipiec E, Łątka K, Kwiatek W, Dynarowicz-Łątka P. Affinity of alkylphosphocholines to biological membrane of prostate cancer: studies in natural and model systems. J Membr Biol 2014; 247:581–589.