Analysis of the vitamin D system in basal cell carcinomas (BCCs).

Using real-time PCR (LightCycler) and immunohistochemistry, we have analyzed expression of key components of the vitamin D system in basal cell carcinomas (BCCs) and normal human skin (NS). Increased VDR-immunoreactivity was demonstrated in BCCs using a streptavidin-peroxidase technique. RNA expression of vitamin D receptor (VDR) and of main enzymes involved in synthesis and metabolism of calcitriol (vitamin D-25-hydroxylase [25-OHase], 25-hydroxyvitamin D-1alpha-hydroxylase [1alpha-OHase], 1,25-dihydroxyvitamin D-24-hydroxylase [24-OHase]) was detected in BCCs and NS. Expression levels were determined as ratios between target genes (VDR, 1alpha-OHase, 25-OHase, 24-OHase) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Median of mRNA ratios for VDR/GAPDH (BCCs: 16.54; NS: 0.00021), 1alpha-OHase/GAPDH (BCCs: 0.739; NS 0.000803) and 24-OHase/GAPDH (BCCs: 0.00585; NS 0.000000366) was significantly (Wilcoxon-Mann-Whitney U-test) elevated in BCCs. In contrast, median of mRNA ratio for 25-OHase/GAPDH (BCCs: 0.17; NS: 0.016) was not significantly altered in BCCs as compared to NS. Additionally, we report for the first time expression of 1alpha-OHase splice variants in BCCs and NS, that were detected using conventional RT-PCR. In conclusion, our findings provide supportive evidence for the concept that endogeneous synthesis and metabolism of vitamin D metabolites as well as VDR expression may regulate growth characteristics of BCCs. New vitamin D analogs that exert little calcemic side effects, their precursors, or inhibitors of 24-OHase may offer a new approach for the prevention or therapy of BCCs. The function of alternative transcripts of 1alpha-OHase that we describe here for the first time in BCCs and NS and their effect on activity level has to be investigated in future experiments.

Analysis of the vitamin D system in cutaneous squamous cell carcinomas.

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.

Analysis of the vitamin D system in cervical carcinomas, breast cancer and ovarian cancer.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the biologically active metabolite of vitamin D and has been shown to regulate the growth of various cell types. There are two principal enzymes involved in the formation of circulating 1,25(OH)2D3 from vitamin D, the vitamin D 25-hydroxylase (25-OHase) and the 1alpha-hydroxylase (1alpha-OHase). Recently, extrarenal activity of 1alpha-OHase has been reported in various cell types. The aim of this study was to analyze expression of VDR and the main enzymes involved in the synthesis and metabolism of calcitriol in gynecological malignancies and corresponding normal tissue. Expression of VDR, 25-OHase, 1alpha-OHase, and 24-OHase was analyzed in breast carcinomas (BC), ovarian cancer (OC), cervix carcinomas (CC) and normal corresponding tissues using real-time PCR and specific hybridization probes as well as using immunohistochemistry. RNA for VDR, 1alpha-OHase, 24-OHase and 25-OHase was up-regulated in breast cervical and ovarian carcinomas as compared to normal tissue. VDR immunoreactivity was increased in breast and ovarian cancer and in cervix carcinomas as compared to normal corresponding tissue. Our findings indicate that cervical carcinomas, breast cancer and ovarian cancer may be considered as potential targets for prevention or therapy with new vitamin D analogs that exert little or no calcemic side effects or by pharmacological modulation of 1,25(OH)2D3 synthesis and metabolism in these tumor cells.

Analysis of the vitamin D system in cutaneous malignancies.

1,25-dihydroxyvitamin D3, the biological active metabolite of vitamin D, has great impact on keratinocyte growth and differentiation, and consequently has already been successfully used in the therapy of hyperproliferative skin disorders. We have now characterized the key components of the vitamin D system (VDR, 1alpha-OHase, 24-OHase and 25-OHase) in cutaneous basal cell carcinomas (BCC) and squamous cell carcinomas (SCC), using immunohistochemical and quantitative real-time PCR techniques. Additionally, proliferative activity (Ki-67 expression), differentiation status (cytokeratin 10 and transglutaminase K expression), rate of apoptosis (TUNEL assay) and the abundance of the main heterodimerization partners of VDR (RXRs) was determined for these tumours and correlated with the components of the Vitamin D system. Our findings indicate that the Vitamin D system may be of high importance for the growth behaviour of BCCs and SCCs and that new vitamin D analogues that exert less calcaemic side effects may be effective in the prevention or treatment of these tumours.

Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin.

Calpain, also named CAPN (for calcium-activated neutral protease), is a ubiquitous intracellular cytoplasmic non-lysosomal cysteine endopeptidase that requires calcium ions to exert its activity. Two major isoenzymes are known- micro -calpain (CAPN1) and m-calpain (CAPN2)-requiring micromolar and millimolar calcium concentrations for activation, respectively. Many known substrates of the different calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumour suppressor protein p53, protein kinase C, pp60src, or the adhesion molecule integrin, have been implicated in the pathogenesis of various malignancies including squamous (SCC) and basal (BCC) cell carcinomas of human skin, suggesting an important role of the calpain isoenzymes in malignant diseases. We have analysed the expression of CAP1 and CAPN2 protein and mRNA expression in BCCs and SCCs of human skin. Interestingly, CAPN1 immunoreactivity (streptavidin-peroxidase technique) was markedly reduced in BCCs compared to normal human skin or SCCs, while in contrast CAPN1 mRNA levels (determined by real-time PCR) were markedly elevated in BCCs and SCCs compared to normal human skin. No differences were found analysing CAPN2 protein and mRNA expression in normal human skin, BCCs and SCCs. In conclusion, we have demonstrated for the first time alterations in calpain mRNA expression and protein content in malignant skin tumours that may be of importance for the tumorigenesis and growth characteristics of BCCs and SCCs. However, our results do not allow conclusions on the function of CAPN1 and CAPN2 in BCCs and SCCs. It is not known if the CAPN genes in BCCs or SCCs exhibit functionally inactivating mutations or whether decreased CAPN1 protein expression in BCCs and elevated CAPN1 mRNA in BCCs and SCCs reflect a feedback loop coupled with increased degradation or proteolysis of CAPN1 protein.