Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

Abstract

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.

Figure 1

SH-SY5Y cells were incubated with acitretin (0–50 μM) for 24 h prior to the addition of 10 μM ATRA (A) or 10 μM 13cisRA (B) for a further 24 h. Values are expressed as intracellular retinoic acid (RA) concentrations based on cell counts and cell volume calculations. Data are mean values±s.e.m. from at least three separate experiments. Statistical significance relative to controls without acitretin is indicated by ^*(P<0.05). For the ANOVA with intracellular ATRA levels in (B) (F_3,13=5.078, P=0.015), one outlier for the 10 μM acitretin dose was removed (studentised residual −2.682).

Figure 2

CRABP I/II expression determined by Western blot analysis in SH-SY5Y, SH-EP and IMR-32 cells after 0–72 h incubations with ATRA or 13cisRA (10 μM) (A) and after 48 h incubation with acitretin (Ac, 50 μM) (B). β-Actin was used as a reference to correct for protein loading. (C) Graph plotting CRABP I induction after 48 h incubation with ATRA or 13cisRA (10 μM) against the mean increase in intracellular ATRA concentration for each cell line from Table 1 (control compared to acitretin treated cells).

Figure 3

SH-SY5Y cells were coincubated with R116010 (0–10 μM) and either ATRA (A) or 13cisRA (B) (10 μM) for 24 h. Values are expressed as intracellular retinoic acid (RA) concentrations based on cell counts and cell volume calculations. Data plotted are mean values±s.e.m. from at least four separate experiments. Statistical significance relative to controls without R116010 is indicated by ^*** for P<0.001 and ^** for P<0.01.

Figure 4

Induction of SEAP expression. SH-SY5Y cells were incubated with ATRA or 13cisRA (0.1 μM) for 48 h in the absence or presence of acitretin (Ac, 1 μM) or R116010 (R, 1 μM). Values are expressed as fold increase relative to expression in untreated control cells. Data are mean values±s.e.m. from at least three independent experiments. Statistical significance from hypothesis tests in ANOVA is indicated by ^*** for P<0.001, ^** for P<0.01 and ^* for P<0.05.

Figure 5

Induction of CYP26A1 mRNA expression determined by RT–PCR and real-time PCR. SH-SY5Y cells were treated with ATRA or 13cisRA for 24 h at the concentrations indicated (A) or with a fixed concentration of 1 μM ATRA or 13cisRA for up to 72 h (B). SH-SY5Y cells were treated with ATRA or 13cisRA (0.01 μM) for 24 h in the absence or presence of acitretin (Ac, 1 μM) or R116010 (R, 1 μM) (C). β-actin mRNA expression was used to normalise the reverse transcription reaction. Amplified products were analysed by agarose gel electrophoresis with ethidium bromide staining. Reverse-transcribed RNA from SH-SY5Y cells treated with ATRA or 13cisRA (0.01 or 0.1 μM) for 24 h in the absence or presence R116010 (R, 1 μM) was subjected to real-time PCR using TaqMan probes for CYP26A1 and β-actin (D). Values are normalised for β-actin levels and expressed as fold increase relative to expression of CYP26A1 in cells treated with 0.01 μM ATRA. Data are mean values±s.e.m. from at least three independent experiments. Statistical significance from hypothesis tests in ANOVA is indicated by ^* where F_1,32=5.714, P<0.05, and ^*** where F_1,32=15.496, P<0.001.