calcitriol and Cell-Transformation--Neoplastic

Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Topics: Adenocarcinoma; Aniline Compounds; Animals; Caco-2 Cells; Calcium; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Dietary Supplements; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Male; Mice; Mice, Transgenic; Mutation; Phenethylamines; Propylamines; Receptors, Calcium-Sensing; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Transfection; Vitamin D

Prostate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1alpha,25 dihydroxyvitamin D3 (1,25(OH)2D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown.. We examined the effect of 1,25(OH)2D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D (Affymetrix U133 Plus 2.0, n = 4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h.. Our data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.

Topics: Antineoplastic Agents; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Gene Expression Profiling; Gene Expression Regulation; Humans; Male; Prostate; Prostatic Neoplasms; Response Elements; Signal Transduction; Transcription, Genetic; Vitamin D

Bone is the preferred site for prostate cancer (PCa) metastases. Once the tumor has established itself within the bone there is virtually no cure. To better understand the interactions between the PCa cells and bone environment in the metastatic process new model systems are needed. We have established a two-compartment in vitro co-culturing model that can be used to follow the trans-activation of bone and/or tumor cells. The model was validated using two PCa tumor cell lines (PC-3; lytic and LNCaP; mixed/osteoblastic) and one osteolytic inducing factor, 1,25-dihydroxyvitamin D(3) (D3). Results were in accordance with the expected bone phenotypes; PC-3 cells and D3 gave osteolytic gene expression profiles in calvariae, with up-regulation of genes needed for osteoclast differentiation, activation and function; Rankl, CathK, Trap and MMP-9, and down-regulation of genes associated with osteoblast differentiation and bone mineralization; Alp, Ocl and Dkk-1. LNCaP cells activated genes in the calvarial bones associated with osteoblast differentiation and mineralization, with marginal effects on osteolytic genes. The results were strengthened by similar changes in protein expression for a selection of the analyzed genes. Furthermore, the osteolytic gene expression profiles in calvarial bones co-cultured with PC-3 cells or with D3 were correlated with the actual ongoing resorptive process, as assessed by the release of collagen fragments from the calvariae. Our results show that the model can be used to follow tumor-induced bone remodeling, and by measuring changes in gene expression in the tumor cells we can also study how they respond to the bone microenvironment.

Topics: Bone and Bones; Bone Neoplasms; Bone Remodeling; Cell Communication; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Coculture Techniques; Gene Expression; Gene Expression Profiling; Humans; Male; Models, Biological; Osteolysis; Prostatic Neoplasms; Up-Regulation; Vitamin D

In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1alpha,25-dihydroxyvitamin D3. BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1alpha,25-dihydroxyvitamin D3. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

Topics: Amino Acid Sequence; Cell Differentiation; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation; Humans; Keratinocytes; Molecular Sequence Data; Osteocalcin; Promoter Regions, Genetic; Protein Isoforms; Protein Precursors; Receptors, Calcitriol; Transcription Factors; Transcriptional Activation; Vitamin D