ABSTRACT
We studied effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], its analog seocalcitol (EB 1089), and 25-hydroxyvitamin D3 [25(OH)D3], on the growth of seven melanoma cell lines. While three cell lines (MeWo, SK-Mel-28, SM) responded to antiproliferative effects of active vitamin D analogs, the others (SK-Mel-5, SK-Mel-25, IGR, MeUuso) were resistant. A strong induction (7000-fold) of 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase, CYP24) mRNA was detected in responsive cell lines along with 1,25(OH)2D3-treatment, indicating functional integrity of vitamin D receptor (VDR)-mediated transcription. In contrast, induction of 24OHase was much lower in resistant melanoma cells (70-fold). VDR mRNA was induced up to 3-fold both in responsive and resistant cell lines along with 1,25(OH)2D3-treatment. RNA for vitamin D-activating enzymes vitamin D-25-hydroxylase (25OHase, CYP27A1) and 25-hydroxyvitamin D-lalpha-hydroxylase (lalphaOHase, CYP27B1) was detected in all melanoma cell lines analyzed, additionally we show splicing variants of lalphaOHase in SK-Mel-28 cells. Expression of 250Hase and laOHase was marginally modulated along with treatment. Proliferation of melanoma cells was not inhibited by treatment with 25(OH)D3, indicating no significant stimulation of endogeneous production of antiproliferative acting 1,25(OH)2D3. In conclusion, we characterize the vitamin D endocrine system in melanoma cells and demonstrate that the majority of melanoma cell lines analyzed is resistant to antiproliferative effects of 1,25(OH)2D3. It can be speculated that these alterations are of importance for pathogenesis and growth of metastasizing malignant melanoma. Additionally, our findings indicate that only a minority of cases with metastasizing melanoma may represent a promising target for palliative treatment with new vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of 1,25(OH)2D3-synthesis/metabolism.
Subject(s)
Antineoplastic Agents/pharmacology , Calcitriol/pharmacology , Melanoma/metabolism , Skin Neoplasms/metabolism , Vitamin D/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Alternative Splicing , Calcitriol/analogs & derivatives , Cell Line, Tumor , Cell Proliferation/drug effects , Cholestanetriol 26-Monooxygenase/genetics , Cholestanetriol 26-Monooxygenase/metabolism , Drug Resistance, Neoplasm , Endocrine System/drug effects , Endocrine System/metabolism , Humans , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Steroid Hydroxylases/genetics , Steroid Hydroxylases/metabolism , Vitamin D3 24-HydroxylaseABSTRACT
Increasing evidence points at an important function of Vitamin D metabolites for growth regulation in various tissues, including MM. Using array CGH, amplification of 24-OHase was recently detected as a likely target oncogene of the amplification unit 20q13.2 in breast cancer cell lines and tumors. Additionally, amplification of 1alpha-OHase has been reported in human malignant glioma. Using immunohistochemistry, we have now detected nuclear Vitamin D receptor (VDR) immunoreactivity in primary cutaneous malignant melanoma (MM), indicating that Vitamin D metabolites may be of importance for the growth regulation in these tumors. Using Southern analysis, we have analyzed MM and metastases for evidence of amplification of 1alpha-OHase or 24-OHase genes. Our results do not support the hypothesis that amplification of 1alpha-OHase or 24-OHase genes may be of importance for pathogenesis or progression of MM.
Subject(s)
Gene Amplification , Melanoma/genetics , Skin Neoplasms/genetics , Steroid Hydroxylases/genetics , Blotting, Southern , Chromosomes, Human, Pair 20 , Humans , Immunohistochemistry , Melanoma/enzymology , Receptors, Calcitriol/metabolism , Skin Neoplasms/enzymologyABSTRACT
1,25-DihydroxyVitamin D(3) and analogs have been shown to inhibit proliferation and to induce differentiation in different cell types, including human melanocytes. However, various tumor cell lines that fail to respond to the antiproliferative effects of Vitamin D analogs have also been reported. Using real-time PCR (LightCycler), we have compared mRNA expression of Vitamin D receptor (VDR), Vitamin D-25-hydroxylase (25-OHase), 25-hydroxyVitamin D-1alpha-hydroxylase (1alpha-OHase), and 1,25-dihydroxyVitamin D-24-hydroxylase (24-OHase) in a melanoma cell line that responds to antiproliferative effects of Vitamin D (MeWo) with a non-responsive melanoma cell line (SkMel5). Additionally, modulation of cell proliferation by calpain inhibitors, as well as regulation of mRNA expression of VDR, 1alpha-OHase, and 24-OHase genes by Vitamin D analogs were assessed in melanoma cell lines in vitro using a WST-1 based colorimetric assay and real-time PCR, respectively. RNA for VDR, 25-OHase, 1alpha-OHase, and 24-OHase was detected in melanoma cell lines. In contrast to SkMel5 cells, treatment of MeWo cells with calcitriol resulted in a dose-dependent increase in mRNA for VDR and 24-OHase as well as in a suppression of cell proliferation (up to approximately 50%). Our findings demonstrate that local synthesis or metabolism of Vitamin D metabolites may be of importance for growth regulation of MM and melanoma cell lines. Additionally, metastasizing MM represents a promising target for palliative treatment with new Vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.
Subject(s)
Calcitriol/pharmacology , Cell Differentiation/drug effects , Cell Division/drug effects , Melanoma/pathology , Base Sequence , Cell Line, Tumor , DNA Primers , Humans , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptors, Calcitriol/genetics , Steroid Hydroxylases/geneticsABSTRACT
BACKGROUND: Increasing evidence points at an important function of vitamin D metabolites for growth regulation in various tissues, and new vitamin D analogs are interesting candidates for the treatment of malignancies, including squamous cell carcinomas (SCC). METHODS: We have analyzed expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25-OHase), 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-OHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24-OHase) in SCC. RESULTS: Intensity of VDR immunoreactivity was increased in SCCs as compared to normal human skin. VDR staining did not correlate with histological type or grading, nor with markers for proliferation, differentiation, or apoptotic cells. Incubation of SCC cell lines (SCL-1, SCL-2) with calcitriol resulted in a dose-dependent suppression of cell proliferation (approximately up to 30%) in vitro, as measured by a tetrazolium salt (WST-1)-based colorimetric assay. RNA levels for VDR, 25-OHase, 1 alpha-OHase, and 24-OHase were significantly elevated in SCCs as compared to HS, as measured by real-time polymerase chain reaction. CONCLUSIONS: Our findings demonstrate that modulation of VDR expression and local synthesis or metabolism of vitamin D metabolites may be of importance for growth regulation of SCCs. Additionally, SCCs represent potential targets for therapy with new vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.
