Differential Effects of N-(4-Hydroxyphenyl)retinamide and Retinoic Acid on Neuroblastoma Cells: Apoptosis versus Differentiation

Abstract

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 × 10^-6 m induces a very rapid (24–72 h) fall in thymidine uptake (>90%), whereas at 3 × 10^-7 m it exhibits cytostatic effects.

In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation.

DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of “DNA ladders” on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3–6 h after treatment of NB cells with 10^-5 m HPR, while more than 50% of cells were apoptotic by 24–72 h following exposure to 3 × 10^-6 m HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3–5 days.

Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells.

In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramatically suppresses NB cell growth by inducing programmed cell death. Since the peak HPR plasma concentration in patients treated with the drug is superimposable to the dose inducing apoptosis in vitro (i.e., 1–3 × 10^-6 m), our results may support the hypothesis of the use of HPR in the treatment of advanced neuroblastoma.