Endocrine Abstracts

Introduction: In our previous studies we found that the combination of 13-cis retinoic acid (13cRA) and lovastatin significantly reduced tumour growth in a mouse phaeochromocytoma allograft model, with the lowest microvessel density in the combination-treated tumours. We have now investigated the effect of 13cRA plus lovastatin on neuroendocrine (BON1, H727) and non-endocrine tumour (HepG2, Huh7) cell viability and signalling pathways (EGFR, AKT, ERK, p70S6K) to elucidate the underlying mechanism of action.

Methods: Cell viability was assessed with the MTS assay, the effect on signalling pathways by Western blotting. For cell viability data, the multiple-comparison Kruskal-Wallis-Test was used, followed by pairwise comparisons with the Mann-Whitney-Test. Interaction effects were analysed with Linear-Mixed-Effects-Models. Statistical significance was defined at P≤0.05.

Results: 3.75 μM or 7.5 μM 13cRA significantly reduced endocrine and non-endocrine tumour cell viability (P≤0.001). The combination of 10 μM lovastatin plus 7.5 μM 13cRA showed synergistic inhibition of BON1 cell viability (P≤0.05). We found similar results in the non-endocrine tumour cells (P≤0.01). In H727 cells, 10 μM lovastatin plus 3.75 μM or 7.5 μM 13cRA significantly more strongly reduced cell viability than each drug separately (P≤0.05), but this effect was not synergistic. In BON1, HepG2 and Huh7 cells, combination-treatment decreased pEGFR by more than 50% relative to each drug separately, and by more than 80% relative to the untreated control. This effect on pEGFR was less pronounced in H727 cells. In all cell lines investigated, combination treatment reduced pAKT by more than 50% compared to each drug separately, and strongly up-regulated pERK relative to the control.

Conclusion: We found a synergistic effect of 13cRA plus lovastatin in BON1 cells at clinically relevant doses, associated with enhanced EGFR inhibition. This is compatible with our recent in vivo data in a phaeochromocytoma allograft model showing slowest tumour growth and lowest microvessel density after 13cRA/lovastatin-treatment suggesting a novel potentially highly effective therapy.