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Apoptosis of N-type neuroblastoma cells after differentiation with 9- cis-retinoic acid and subsequent washout

Lookup NU author(s): Professor Penny Lovat, Professor Archibald Malcolm, Professor Andrew Pearson, Dr Chris RedfernORCiD


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The overall survival rate for patients with neuroblastoma has improved over the past two decades, but long-term survival for the subgroup of patients with high-risk disease remains low. In recent years, there has been interest in the potential clinical use of drugs able to induce differentiation of neuroblastoma cells. Since 9-cis-retinoic acid induces better and more sustained differentiation of neuroblastoma in vitro than other retinoic acid isomers, this may be a more appropriate retinoid for use in neuroblastoma therapy. Purpose: The purpose of this work was to compare the long-term effects of all-trans- and 9-cis-retinoic acid on neuroblastoma differentiation using an N-type (neuroblastic) cell line, SH SY 5Y, as an in vitro model. In addition, we wanted to find out whether 9-cis-retinoic acid would induce programmed cell death (apoptosis) in these N-type neuroblastoma cells and to determine whether the effects of either 9-cis- or all-trans- retinoic acid are dependent on their continued presence in the culture medium. Methods: SH SY 5Y cells were incubated in either the continued presence of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic acid followed by culture in the absence of retinoid for up to 13 days. Morphologic changes were observed using phase-contrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodidestained cells and by using terminal deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells. Results: Culture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. These effects were maintained in the continuous presence of each retinoic acid isomer but were more profound in cells treated with 9-cis-retinoic acid. The differentiation of cells treated with all trans-retinoic acid was reversible once retinoic acid was removed from the medium. Conversely, apoptosis was induced in cells treated with 9-cis-retinoic acid for 5 days and cultured for 9 days (4 days after washout) but not in cells cultured in the continuous presence of 9- cis-retinoic acid. This effect was specific to 9-cis-retinoic acid. Conclusions: Previous studies have demonstrated differential responses to all-trans-retinoic acid in N- and S-type (substrate-adherent or Schwann- like) neuroblastoma cells: Apoptosis is induced in S-type cells, whereas differentiation occurs in N-type cells. The present results show that, unlike all-trans-retinoic acid, 9-cis-retinoic acid induces both differentiation and apoptosis in N-type SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9-cis-retinoic acid from the culture medium. Implications: Since both differentiation and apoptosis are involved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apoptosis on treatment and subsequent removal of 9-cis-retinoic acid implies that drug scheduling may be an important parameter affecting therapeutic efficacy.

Publication metadata

Author(s): Lovat, P.E., Irving, H., Annicchiarico-Petruzzelli, M., Bernassola, F., Malcolm, A.J., Pearson, A.D.J., Melino, G., Redfern, C.P.F.

Publication type: Article

Publication status: Published

Journal: Journal of the National Cancer Institute

Year: 1997

Volume: 89

Issue: 6

Pages: 446-452

Print publication date: 19/03/1997

ISSN (print): 0027-8874

ISSN (electronic): 1460-2105

PubMed id: 9091647