The effects of retinoic acid on the In Vitro and In Vivo growth of neuroblastoma cells

Abstract In large part, malignancy is the end result of aberrant cell growth and differentiation. Control of these processes is anticipated to result in a suppression of oncogenicity. Retinoic acid (RA), a derivative of vitamin A, has been shown to inhibit proliferation, induce cell differentiation and reverse the malignant phenotype of a variety of tumor cell types. In order to further characterize the antitumor potential of RA, this study examined the in vitro and in vivo effects of this retinoid on cell lines derived from human neuroblastoma (NB). The in vitro phase of this study tested the ability of various compounds to raise intracellular cyclic adenosine 3′:5′‐monophosphate (cAMP) levels and either alone or in combination with RA, to promote differentiation of two relatively RA‐resistant cell lines. Direct activation of the synthetic enzyme adenylate cyclase by forskolin or cholera toxin increased intracellular cAMP levels over 10‐fold after 1 hour of treatment, declining over the next 16 to 24 hours. After 5 days of continuous growth in the presence of these agents, cAMP levels remained elevated 2‐ to 7‐fold above control values and were accompanied by a decrease in cell proliferation and an increase in cell differentiation. All these effects were exaggerated in the presence of phosphodiesterase inhibitors. Isoproterenol and epinephrine did not alter cAMP levels and had no discernible biological effects. RA promoted differentiation with little effect on cAMP levels. Combination treatment of cells with RA plus agents that raised cAMP levels resulted in greater degrees of differentiation than seen with single‐agent treatment. From these data, it was concluded that: 1. the cAMP synthetic and degradative pathways are functional in the NB cell lines studied; 2. elevation of cAMP is a sufficient but not necessary condition for inhibiting proliferation and promoting differentiation in these cells; 3. elevation of intracellular cAMP potentiates the differentiation‐inducing activity of RA; and 4. overcoming retinoid resistance in some tumor cell lines may be feasible by alterations in the cAMP system. This would be of particular value in treating tumors that have lost retinoid responsiveness. The in vivo phase of this study examined the effects of single‐agent treatment using RA on the development and growth in nude mice of tumors derived from a NB cell line. When cells were treated in culture with 4 × 10 −6 M retinoic acid and subsequently injected into nude mice, there was a marked reduction in the number of mice developing tumors when compared to solvent‐treated controls. Intragastric administration of RA reduced tumor formation when the retinoid was given for 5 days before tumor injection and continued for 14 days thereafter. In established tumors, RA treatment for 32 days inhibited progressive tumor growth. With the RA regimens used, there was no discernible effect on final morphologic phenotype. These data suggest that oral retinoid treatment may be of value in inhibiting or arresting NB growth, particularly in the face of an initial small tumor burden. In vitro and in vivo studies such as reported here may serve as useful models for understanding the potential use of retinoids in the prevention and treatment of malignant disease.