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Antiproliferative activity and apoptosis induced by retinoic acid receptor-gamma selectively binding retinoids in
B Meister1, F M Fink, A Hittmair
1Department of Pediatrics, University of Innsbruck, Austria. b.meister@tirol.com
Abstract:
Retinoids modulate several cell functions and especially inhibit the growth of tumor cells. Their biological activity is mediated by retinoic acid receptors (RARs), of which three subtypes (alpha, beta, gamma) have been identified. In human neuroblastoma (NB) reduced endogenous RAR-gamma expression was suggested to diminish the sensitivity for retinoids, to promote proliferation, and to contribute to the malignant phenotype. To correlate receptor selectivity with in vitro activity, we analysed the effect of six synthetic retinoids with selectivity for human RAR-alpha/beta/gamma on the human LAN-5 NB cell line and compared it with the natural compound all-trans-retinoic acid (ATRA). Apoptosis was determined by flow-cytometry using terminal-deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells. The antagonist for RAR-beta/gamma CD2665 as well as the selective agonists for RAR-alpha CD336 and RAR-beta CD2019 were less effective in growth inhibition than ATRA. In contrast, the synthetic RAR-gamma selective agonists CD437 and CD2325 induced a concentration- and time-dependent antiproliferative effect, which was similar or even more pronounced than ATRA. In contrast to ATRA, the adition of CD437 and CD2325 did not induce morphological changes typical of NB cell maturation but resulted in morphological features consistent with the occurrence of programmed cell death. Flow-cytometric analysis showed that in contrast to ATRA the addition of CD 437 and CD 2325 results in progressive time-dependent increase of apoptotic cells (25.9% and 57.7% after 72 hours). In conclusion, our study demonstrates RAR-gamma selectively binding retinoids dramatically suppress NB cell growth, primarily by inducing programmed cell death rather than by cell differentiation. Since advanced or disseminated NB tumors endogenously express low levels of RAR-gamma and lack of apoptosis is involved in tumor progression, RAR-gamma selectively binding retinoids may be more appropriate retinoids for clinical trials in NB.
Insights
Retinoid compounds targeting RAR-gamma receptors significantly inhibit neuroblastoma cell growth by inducing programmed cell death. These RAR-gamma selective retinoids show promise for clinical trials in neuroblastoma treatment.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Retinoids are known to inhibit tumor cell growth by interacting with retinoic acid receptors (RARs).
- Reduced expression of RAR-gamma in human neuroblastoma (NB) is linked to increased proliferation and malignancy.
- Understanding RAR subtype selectivity is crucial for developing effective retinoid therapies.
Purpose of the Study:
- To investigate the in vitro antiproliferative and pro-apoptotic effects of synthetic retinoids with varying RAR subtype selectivity on human neuroblastoma cells.
- To compare the efficacy of RAR-gamma selective agonists with all-trans-retinoic acid (ATRA) and other RAR subtype-selective compounds.
Main Methods:
- Utilized the human LAN-5 neuroblastoma cell line.
- Tested six synthetic retinoids selective for RAR-alpha, RAR-beta, and RAR-gamma, alongside ATRA.
- Assessed apoptosis via flow cytometry using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay.
- Evaluated cell morphology for signs of differentiation or apoptosis.
Main Results:
- RAR-gamma selective agonists (CD437, CD2325) demonstrated potent, dose- and time-dependent antiproliferative effects, comparable or superior to ATRA.
- Unlike ATRA, CD437 and CD2325 induced morphological changes indicative of programmed cell death, not cell differentiation.
- Flow cytometry confirmed a significant, time-dependent increase in apoptotic cells with CD437 and CD2325 treatment (up to 57.7% at 72 hours).
- RAR-alpha and RAR-beta/gamma selective compounds showed less efficacy in growth inhibition.
Conclusions:
- RAR-gamma selective retinoids potently suppress neuroblastoma cell growth, primarily through the induction of apoptosis.
- These findings suggest RAR-gamma selective retinoids are promising candidates for neuroblastoma clinical trials, especially given low RAR-gamma expression in advanced tumors.
- Targeting RAR-gamma may offer a more effective therapeutic strategy than ATRA for neuroblastoma.