Elucidation of the anti-tumor effect of the synthetic retinoid, CD437, in malignant melanoma

Abstract

The synthetic retinoid 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437/AHPN) has been shown to have an antiproliferative effect, as well as being a potent inducer of apoptosis, in many cancer cell lines. The mechanisms behind CD437s activities have been reported as cell-line dependent. In this master thesis, the anti-tumor effect of CD437 was elucidated in the two human malignant melanoma cell lines, FEMX1 and WM239. - CD437 was shown to have an antiproliferative effect in both cell lines. A reduction in cells relative to the control was first visually observed, and later confirmed by a cell count. As revealed by MTS, the reduction of viable cells was duration- and dosage dependent. - Treatment with CD437 caused a cell-line dependent cycle arrest, yielding an S phase- and a G1 phase arrest in FEMX1 and WM239 respectively, as demonstrated by flow cytometry. Western blot analysis revealed that the arrests were accompanied by upregulations of p53, p21 and E2F-1 protein levels. A down-regulation of cyclin D1 was found in FEMX1 cells. - Apoptosis was observed in both cell lines following treatment with CD437; Morphological features associated with apoptosis were seen in both cell lines. A cell count using trypan blue revealed an increase of dead cells after exposure to CD437. A TUNEL analysis demonstrated DNA fragmentation in WM239, but not in FEMX1. In both cell lines, cleavage of the initiator caspases 8 and -9 were observed, in addition to PARP cleavage. Cleavage of caspase 3 was found in FEMX1 cells. - An increase of the orphan nuclear receptor Nur77 was found using the western blot analysis, in treated the cells. In addition, activation of c-jun was observed in both cell lines following exposure to CD437. - Treatment with CD437 resulted in an activation of the p38/MAPK signaling pathway in both cell lines, as assessed by western blotting. Additionally, in the exposed FEMX1 cells the PI3K signaling pathway was activated. - Preliminary results suggest that an up-regulation of p53 caused by CD437 may be regulated by Nur77. The increase of p21 did not appear to be controlled by Nur77, which may suggest a p53-independent expression of p21. Malignant melanomas are notoriously resistant to chemotherapy, making the prognosis for patients with metastasis very poor. CD437 represent a potential new treatment option, however many more studies are needed to evaluate its effect both in vitro and in vivo.