Testing of the Survivin Suppressant YM155 in a Large Panel of Drug-Resistant Neuroblastoma Cell Lines

Abstract

The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC₅₀s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.

Keywords: ABCB1; ABCC1; YM155; drug resistance; neuroblastoma; survivin.

Figure 1

Anti-neuroblastoma effects of YM155 in a panel of 17 neuroblastoma cell lines. (A) YM155 concentrations that reduce the viability of neuroblastoma cell lines by 50% (IC₅₀, mean ± S.D., n=3) as determined by MTT assay after a 5-day treatment period. Numerical values are presented in Table S1. Information on the ABCB1 status of the cell lines is provided in Table S2. (B) Effects of the ABCB1 inhibitors verapamil (5 µM) and zosuquidar (1.25 µM) on the YM155 IC₅₀ values in neuroblastoma cell lines characterised by high or low ABCB1 levels displayed as fold change YM155 IC₅₀/ YM155 IC₅₀ in the presence of ABCB1 inhibitor. Numerical data and the effects of the ABCB1 inhibitors alone on cell viability are presented in Table S6.

Figure 1

Anti-neuroblastoma effects of YM155 in a panel of 17 neuroblastoma cell lines. (A) YM155 concentrations that reduce the viability of neuroblastoma cell lines by 50% (IC₅₀, mean ± S.D., n=3) as determined by MTT assay after a 5-day treatment period. Numerical values are presented in Table S1. Information on the ABCB1 status of the cell lines is provided in Table S2. (B) Effects of the ABCB1 inhibitors verapamil (5 µM) and zosuquidar (1.25 µM) on the YM155 IC₅₀ values in neuroblastoma cell lines characterised by high or low ABCB1 levels displayed as fold change YM155 IC₅₀/ YM155 IC₅₀ in the presence of ABCB1 inhibitor. Numerical data and the effects of the ABCB1 inhibitors alone on cell viability are presented in Table S6.

Figure 2

Effects of YM155 on the viability of neuroblastoma cells in dependence on the MYCN status. (A) YM155 concentrations that reduce the viability of neuroblastoma cell lines by 50% (IC₅₀) were determined by MTT assay after a 5-day treatment period in the presence of the ABCB1 inhibitors verapamil (5 µM) or zosuquidar (1.25 µM) to avoid interference of ABCB1-mediated effects with MYCN-mediated effects. Numerical data are presented in Table S3. (B) YM155 IC₅₀ values in SH-EP-MYCN (TET21N) cells in the absence or presence of doxycycline as determined by MTT assay after a 120h of treatment. All values are presented as mean ± S.D. (n = 3).

Figure 3

Effects of YM155 on the viability of parental p53 wild-type neuroblastoma cell lines and their p53 mutant nutlin-3-adapted sub-lines. YM155 concentrations that reduce neuroblastoma cell viability by 50% (IC₅₀, mean ± S.D., n = 3) as determined by MTT assay after a 5-day treatment period. Numerical data are presented in Table S4.

Figure 4

Effects of YM155 on the viability of neuroblastoma cell lines adapted to particular drug classes. Distribution of the YM155 IC₅₀ values (mean ± S.D., n = 3) within the groups of drug-adapted cancer cell lines. Numerical data are presented in Table S1. ¹UKF-NB-3^rDOX²⁰ (YM155 IC₅₀ 15,700 ± 1,019 nM); ²IMR-5^rDOCE²⁰ (YM155 IC₅₀ 21,549 ± 638 nM); ³UKF-NB-2^rDOX²⁰ (YM155 IC₅₀ 1,108 ± 179 nM); ⁴NGP^rVCR²⁰ (YM155 IC₅₀ 6,986 ± 716 nM); ⁵UKF-NB-2^rVCR¹⁰ (YM155 IC₅₀ 5,940 ± 247 nM); ⁶IMR-5^rVINOR²⁰ (YM155 IC₅₀ 4,978 ± 147 nM); ⁷IMR-5^rVINB²⁰ (YM155 IC₅₀ 1,608 ± 212 nM).

Figure 5

Effects of YM155 on the viability of neuroblastoma cell lines adapted to particular drugs. (A) YM155 concentrations that reduce the viability of neuroblastoma cell lines adapted to the topoisomerase II inhibitors doxorubicin or etoposide by 50% (IC₅₀) as determined by MTT assay after a 5-day treatment period. Values are presented as mean ± S.D. over all cell lines from the individual groups. The cell line UKF-NB-3^rDOX²⁰ was not included into this analysis because it was regarded as outliers (please refer to the text). (B) Distribution of the YM155 IC₅₀ values (mean ± S.D., n = 3) in doxorubicin- and etoposide-adapted cells. (C) YM155 IC₅₀ concentrations in neuroblastoma cell lines adapted to the platinum drugs carboplatin, cisplatin, or oxaliplatin (mean ± S.D)) as determined by MTT assay after a 5-day treatment period. (D) Distribution of the YM155 IC₅₀ values (mean ± S.D., n = 3) in carboplatin-, cisplatin- and oxaliplatin-adapted cells. Numerical data are presented in Tables S1 and S6. ¹UKF-NB-3^rDOX²⁰ (YM155 IC₅₀ 15,700 ± 1,019 nM).

Figure 6

Effects of YM155 in ABCB1-transduced cells. (A) Representative western blots and flow cytometry histograms indicating ABCB1 levels in UKF-NB-3 cells and in UKF-NB-3 transduced with a lentiviral vector encoding ABCB1 (UKF-NB-3^ABCB1). (B) Effect of siRNA directed against ABCB1 on cellular ABCB1 levels in UKF-NB-3^ABCB1 cells. (C) Concentrations of YM155 and doxorubicin (alternative ABCB1 substrate used as control) that reduce the viability of UKF-NB-3^ABCB1 cells by 50% (IC₅₀, mean ± S.D., n = 3) as determined by MTT assay after 120h of incubation. (D) Effects of YM155 (100 nM) on survivin levels in UKF-NB-3ABCB1 cells after 24h of incubation in the presence or absence of verapamil (5 µM) or zosuquidar (1.25 µM). Uncropped Western blots are presented in Figure S2.

Figure 7

ABCB1 expression and YM155 activity in drug-adapted neuroblastoma cells. (A) Representative western blots indicating ABCB1 levels in IMR-5, IMR-5^rDOCE²⁰, IMR-32, and IMR-32^rDOX²⁰. (B) Effects of YM155 (500 nM) on survivin levels and PARP cleavage in IMR-5^rDOCE²⁰ cells in the presence or absence of verapamil (5 µM) or zosuquidar (1.25 µM) after 24h of incubation. Uncropped Western blots are presented in Figure S3.

Figure 8

Comparison of verapamil- and zosuquidar-induced neuroblastoma cell sensitisation to YM155 in a panel of 74 neuroblastoma cell lines. The fold sensitisation (YM155 IC₅₀/ YM155 IC₅₀ in the presence of ABCB1 inhibitor) was determined by MTT assay after neuroblastoma cell incubation with YM155 for 120 h in the absence or presence of verapamil (5 µM) or zosuquidar (1.25 µM). Numerical data are presented in Table S6. ¹UKF-NB-3^rDOX²⁰, fold change 9235; ²IMR-5^rDOCE²⁰, fold change 1581; ³ UKF-NB-3^rDOCE¹⁰, fold change 939.