Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR£0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five samples were included and were categorized according to treatment in three groups: control (A), 1,25(OH)2D3 0.5nM (B) and 1,25(OH)2D3 100nM (C).Total RNA was isolated using RNeasy kit and than was carried out according to microarray Affymetrix protocol. Data was then assessed with GeneSpring X software for background correction, normalization and summarization of raw data (CEL files) using the Robust Multi-Array Average (RMA). Five samples were included in a test and gene expression was analyzed by microarray (using SAM paired analysis). To establish a differential gene expression profile between vitamin D treated and untreated samples, SAM two class paired, provided on MEV (MultiExperiment Viewer – Boston, MA, USA) was used. Unsupervised hierarchical clustering based on Euclidean distance and average linkage was used to verify association patterns. The reliability of the clustering was assessed by the Bootstrap technique using MEV (MultiExperiment Viewer – Boston, MA, USA). Samples (16) were included in a validation group and was analyzed by qPCR.

Project description:Vitamin D may have anti-tumorigenic actions by influencing the gene expression profile of target tissues, which possess vitamin D receptors. We used a more physiological in vitro model, represented by short-term culture of breast cancer tissue slices, to study the transcriptional effects of a near physiological concentration of calcitriol. Methods. Breast cancer fragments were sliced and maintained in culture for 24 hours in the presence or absence (control) of calcitriol 0.5nM or 100nM (called physiological or supra-physiological concentrations). Five and 16 samples were included in a test or validation group and gene expression was analyzed by microarray (using SAM paired analysis) or qPCR, respectively. Results. Nine genes were regulated by calcitriol 0.5nM including CYP24A1, DPP4, EFTUD1 (FDR£0.01), which were up-modulated. However, using a less stringent FDR value (0.25) 61 genes were differentially expressed. Among the down-regulated transcripts were members of the MHC class II complex (HLA-DPA1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3) and IgG binding (FCGR2A, FCGR2B, FCGR2C). There was an enrichment of genes presenting transcription factor binding sites for vitamin D, among the up-regulated genes and for interferon regulatory factor IRF1, among the down-regulated genes. Analyzing calcitriol supra-physiological effects, a more impressive transcriptional modulation was identified and 136 and 60 genes (FDR 0.1) were more and less expressed in treated samples. Up-regulated genes were involved in vitamin metabolic process, regulation of leukocyte-mediated immunity and positive regulation of alpha-beta T cell activation. Many of the induced genes were already reported as vitamin D responsive genes, including CD14, which was also up-regulated in another set of 16 samples. Genes modulated by both calcitriol concentrations were CYP24A1, DPP4, CA2 (these three in both sets of samples), EFTUD1, TKTL1, KCNK3. Conclusion. Small increments in calcitriol concentration, within the physiological range, for a relatively short period of time may exert transcriptional effects in breast cancer samples. Further studies employing physiological concentrations of vitamin D for longer periods of time may help to elucidate the hormone effects in breast cancer treatment and prevention.

Project description:Background: Breast cancer patients present lower 1,25(OH)2D3 or 25(OH)D3 serum levels than unaffected women. Although 1,25(OH)2D3 pharmacological concentrations of 1,25(OH)2D3 may exert antiproliferative effects in breast cancer cell lines, much uncertainty remains about the effects of calcitriol supplementation in tumor specimens in vivo. We have evaluated tumor dimension (ultrassonography), proliferative index (Ki67 expression), 25(OH)D3 serum concentration and gene expression profile, before and after a short term calcitriol supplementation (dose to prevent osteoporosis) to post-menopausal patients. Results: Thirty three patients with operable disease had tumor samples evaluated. Most of them (87.5%) presented 25(OH)D3 insufficiency (<30 ng/mL). Median period of calcitriol supplementation was 30 days. Although tumor dimension did not vary, Ki67 immunoexpression decreased after supplementation. Transcriptional analysis of 15 matched pre/post-supplementation samples using U133 Plus 2.0 GeneChip (Affymetrix) revealed 18 genes over-expressed in post-supplementation tumors. As a technical validation procedure, expression of four genes was also determined by RT-qPCR and a direct correlation was observed between both methods (microarray vs PCR). To further explore the effects of near physiological concentrations of calcitriol on breast cancer samples, an ex vivo model of fresh tumor slices was utilized. Tumor samples from another 12 post-menopausal patients were sliced and treated in vitro with slightly high concentrations of calcitriol (0.5nM), that can be attained in vivo, for 24 hours In this model, expression of PBEF1, EGR1, ATF3, FOS and RGS1 was not induced after a short exposure to calcitriol. Conclusions: In our work, most post-menopausal breast cancer patients presented at least 25(OH)D3 insufficiency. In these patients, a short period of calcitriol supplementation may prevent tumor growth and reduce Ki67 expression, probably associated with discrete transcriptional changes. This observation deserves further investigation to better clarify calcitriol effects in tumor behavior under physiological conditions.

Project description:Background: Breast cancer patients present lower 1,25(OH)2D3 or 25(OH)D3 serum levels than unaffected women. Although 1,25(OH)2D3 pharmacological concentrations of 1,25(OH)2D3 may exert antiproliferative effects in breast cancer cell lines, much uncertainty remains about the effects of calcitriol supplementation in tumor specimens in vivo. We have evaluated tumor dimension (ultrassonography), proliferative index (Ki67 expression), 25(OH)D3 serum concentration and gene expression profile, before and after a short term calcitriol supplementation (dose to prevent osteoporosis) to post-menopausal patients. Results: Thirty three patients with operable disease had tumor samples evaluated. Most of them (87.5%) presented 25(OH)D3 insufficiency (<30 ng/mL). Median period of calcitriol supplementation was 30 days. Although tumor dimension did not vary, Ki67 immunoexpression decreased after supplementation. Transcriptional analysis of 15 matched pre/post-supplementation samples using U133 Plus 2.0 GeneChip (Affymetrix) revealed 18 genes over-expressed in post-supplementation tumors. As a technical validation procedure, expression of four genes was also determined by RT-qPCR and a direct correlation was observed between both methods (microarray vs PCR). To further explore the effects of near physiological concentrations of calcitriol on breast cancer samples, an ex vivo model of fresh tumor slices was utilized. Tumor samples from another 12 post-menopausal patients were sliced and treated in vitro with slightly high concentrations of calcitriol (0.5nM), that can be attained in vivo, for 24 hours In this model, expression of PBEF1, EGR1, ATF3, FOS and RGS1 was not induced after a short exposure to calcitriol. Conclusions: In our work, most post-menopausal breast cancer patients presented at least 25(OH)D3 insufficiency. In these patients, a short period of calcitriol supplementation may prevent tumor growth and reduce Ki67 expression, probably associated with discrete transcriptional changes. This observation deserves further investigation to better clarify calcitriol effects in tumor behavior under physiological conditions. Post-menopausal patients with early stage breast cancer, in the absence of distant metastasis, were invited to take part in the study. This protocol was approved by the Institutional Ethics Committee, and a written informed consent was signed by all participants. Patients had blood and tumor samples collected during biopsy, and were prescribed calcitriol supplementation, (Rocaltrol)TM 0.50 ÔÅ≠g/day PO, as recommended for osteoporosis prevention. Tumor specimens obtained during biopsy (pre-supplementation) or breast surgery (post-supplementation) were hand dissected and samples with at least 70% tumor cells were further processed. Breast surgery followed in about one month

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