POSITIVE AND NEGATIVE INTERACTION OF 1,25-DIHYDROXYVITAMIN D 3 AND THE RETINOID CD437 IN THE INDUCTION OF HUMAN MELANOMA CELL APOPTOSIS Carina DANIELSSON 1,2 , Hans TO ¨ RMA ¨ 1 , Anders VAHLQUIST 1 and Carsten CARLBERG 2 * 1 Department of Medical Sciences, Section of Dermatology, Uppsala University, Uppsala, Sweden 2 Institut fu ¨ r Physiologische Chemie I, Heinrich-Heine-Universita ¨ t, Du ¨ sseldorf, Germany The natural ligands of the nuclear receptors vitamin D receptor (VDR) and retinoic acid receptor (RAR), i.e., 1,25- dihydroxyvitamin D 3 (VD) and all-trans retinoic acid, have important effects on the proliferation, differentiation and apoptosis of a variety of malignant cells, including melanoma. The therapeutic potential of the 2 nuclear hormones can be enhanced by the use of synthetic analogues. In this study, the 2 human melanoma cell lines W M1341 and MeW o were compared for the combined effect of VD and synthetic retinoids. Both cell lines expressed reasonable amounts of VDR, RAR and retinoid X receptor  and differed only in the relative expression of RAR and . From 9 functional variants of retinoids, only the RAR-selective retinoid CD437 showed, in both cell lines, a significant anti-proliferative effect. In MeW o cells, but not in W M1341 cells, VD induced growth arrest but showed no synergistic interaction with the effects of CD437. In contrast, VD induced apoptosis in W M1341, but not in MeW o, cells. CD437 was a strong inducer of apoptosis in both melanoma cell lines. Parallel treatment with CD437 and VD resulted in synergistic enhancement of apoptosis in W M1341 cells, whereas a clear decrease in induction of apoptosis in MeW o cells occurred. O ur results indicate that a combined treatment of melanoma with VD and selected retinoids is promising but should be adapted to individual typesof tumor. Int. J. Cancer 81:467–470, 1999.  1999 Wiley-Liss, Inc. The seco-steroid 1,25-dihydroxyvitamin D 3 (VD), the biologi- cally active form of vitamin D 3 , and retinoids, natural and synthetic derivatives of vitamin A, regulate a broad range of biological processes, including growth, differentiation and apoptosis in nor- mal and neoplastic cells (Walters, 1992; Chambon, 1994). VD and the retinoids are nuclear hormones; i.e., their effects are mediated by nuclear receptors, which are the vitamin D receptor (VDR) for VD (Carlberg and Polly, 1998) and the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) for retinoids (Gigue `re, 1994). Each RAR and RXR is encoded by 3 distinct genes (,  and ), whereas there is only 1 gene known for VDR. Although VD and retinoids are very different in structure, their nuclear receptors show reasonable homology and all are members of the nuclear receptor superfamily (Mangelsdorf et al., 1995). VDR and the RARs modulate the expression of their target genes in response to their respective ligands by binding as heterodimeric complexes with RXR to specific DNA sequences in promoter sequences, referred to as response elements. These DNA elements are formed by 2 hexameric core-binding motifs in a directly repeated or inverted palindromic orientation that, if spaced by 5 or 2 nucleo- tides in a directly repeated orientation (DR5 and DR2), are specific RA response elements (RAREs) or, if spaced by 3 nucleotides in a directly repeated orientation (DR3) or by 9 nucleotides in an inverted palindromic orientation (IP9), are specific VD response elements (VDREs) (Carlberg, 1995; Carlberg and Saurat, 1996). VDR and RAR ligands have the potential to be used in the treatment of various skin diseases, including psoriasis and acne, and in the treatment or chemoprevention of cancers, including melanoma. Generally, human melanoma cells are notoriously resistant to chemotherapeutic drugs in vitro and in vivo. VD has been known for nearly 2 decades to inhibit cell growth and to induce differentiation in several normal and malignant cell types. Within the last few years, VD has also been shown to induce apoptosis in human breast cancer and leukemic cell lines (James et al., 1995; Elstner et al., 1996; Danielsson et al., 1997). One of the first types of cancer in which an anti-proliferative effect of VD could be shown was the human melanoma cell line Hs695T (Colston et al., 1981). Subsequently, the induction of human melanoma cell differentiation also was demonstrated (Mason et al., 1988). In vivo studies suggested that expression of the VDR is central for suppressing growth of tumor xenografts (Eisman et al., 1987), and this view was supported by an in vitro study with human melanoma cell lines (Evans et al., 1996). Moreover, the RAR- selective retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtha- lene carboxylic acid (AHPN or CD437) (Bernard et al., 1992) has been shown to inhibit the proliferation of different melanoma cell lines in vitro (Schadendorf et al., 1994) and to be a strong inducer of apoptosis in MeWo cells (late stage of melanoma) (Schadendorf et al., 1996b). VD and its analogues induce apoptosis in WM1341 cells (early stage of melanoma) but not in MeWo cells (Danielsson et al., 1998). In this study, the cell lines MeWo and WM1341 were compared for their response to a combined treatment with VD and retinoids. Only CD437 exhibited, in both cell lines, a significant anti- proliferative effect, whereas VD was effective only in MeWo cells and showed no synergistic interaction with the effects of CD437. However, parallel treatment with CD437 and VD resulted in synergistic enhancement of apoptosis in WM1341 cells but in a clearly decreased induction of apoptosis in MeWo cells. These results indicate that each type of melanoma has an individual response to a combined treatment with VD and selected retinoids. MATERIAL AND METHODS Compounds VD was kindly provided by Ms. C. Mørk Hansen (LEO, Ballerup, Denmark). It was dissolved in 2-propanol at a stock concentration of 4 mM and diluted with ethanol. The synthetic retinoids CD336, CD367, CD437, CD666, CD2314, CD2366, CD2409 and CD2425 were kindly provided by Dr. U. Reichert (Galderma, Sophia Antipolis, France) and dissolved in DMSO at a stock concentration of 2 mM. They were diluted with DMSO. Semi-quantitative RT-PCR The human melanoma cell lines WM1341 and MeWo were kindly provided by Dr. D. Schadendorf (Herlyn, 1990; Schaden- dorf et al., 1996a). They were chosen as representatives of early Grant sponsor: Swedish Medical Research Council; Grant number: 13X-07133; Grant sponsor: Sonderforschungsbereich 503; Grant number: A6; Grant sponsor: Medical Faculty of the Heinrich-Heine-University. *Correspondence to: Institut fu ¨r Physiologische Chemie I, Heinrich-Heine- Universita ¨t Du ¨sseldorf, Postfach 10 10 07, D-40001 Du ¨sseldorf, Germany. Fax: (49)211–208399. E-mail: carlberg@uni-duesseldorf.de Received 11 September 1998; Revised 20 November 1998 Int. J. Cancer: 81, 467–470 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer