Study on 13-cis retinoic acid inducing differentiation of three types of human neuroblastoma cell lines in vitro

Abstract

Objective To investigate the effects of 13-cis retinoic acid (13-cis RA) in inducing differen-tiation of 3 types of human neuroblastoma (NB) cells in vitro. Methods The status of MYCN gene amplification of cultured SH-SY5Y, SK-N-SH and SK-N-BE2 cells was detected by fluorescence in situ hybridization.After treatment with different concentrations of 13-cis RA, morphological changes were observed by phase-contrast microscope, and neuron-specific enolase (NSE) concentrations were determined by enzyme linked immunosorbent assay.The cell viability was measured through cell counting kit-8 assay, and the cell apoptosis was assayed with flow cytometry (FCM). Results The morphological changes in differentiation were observed in all 3 types of NB cells after 13-cis RA treatment.MYCN amplification was detected in SK-N-BE2 cells even after 13-cis RA treatment, while the other 2 cell lines were amplification-null.After different concentrations of 13-cis RA treatment, NSE concentration increased with prolonged time, especially for SK-N-BE2 cell(F=27.00, P<0.000 1).13-cis RA stimulated cell proliferation within 48 hours of treatment, and then inhibited cell growth.FCM indicated that the degree of apoptosis in SH-SY5Y cell was significantly higher after 13-cis RA treatment of 10 μmol/L concentration for continuous 96 h and 120 h as compared to the control group (F=16.21, P=0.011; F=16.04, P=0.016). Cell apoptosis of SK-N-SH cell after 13-cis RA treatment of 1 μmol/L and 10 μmol/L concentration for 48 h, were significantly higher than those of the control group(F=15.05, P=0.012; F=31.18, P=0.005); while SK-N-BE2 cell with different concentrations of 13-cis RA(1 μmol/L, 5 μmol/L, 10 μmol/L) for 120 h were significantly higher than those of the control group(F=9.05, P=0.030; F=11.38, P=0.028; F=7.88, P=0.041). Conclusions The present study showed that 13-cis RA could induce differentiation of human NB cells in vitro.It induces cell proliferation within 48 hours of 13-cis RA, and thereafter suppresses cell growth.No improvement was found in MYCN amplification cells with the detection of DNA level after 13-cis RA treatment, which suggests that combined treatment is possibly needed. Key words: Neuroblastoma; Differentiation; 13-cis retinoic acid; MYCN gene