1,25 dihydroxyvitamin D-mediated orchestration of anticancer, transcript-level effects in the immortalized, non-transformed prostate epithelial cell line, RWPE1

<p>Abstract</p> <p>Background</p> <p>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, 1α,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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Molecular regulation of prostate biology and prostate cancer by signaling through the vitamin D receptor

High vitamin D status (serum 25-hydroxyvitamin D) and UV light exposure are associated with reduced risk of prostate cancer. The biologically active form of vitamin D, 1 alpha, 25-dihydroxyvitamin D (1,25(OH)2D) regulates expression of its target genes via its ligand activated transcription factor, the vitamin D receptor (VDR). To clarify the role of VDR signaling in the prevention of prostate cancer, we conducted three experiments. In the first experiment we used androgen depletion/repletion model in mice to test whether prostate epithelial cell (PEC) VDR level was important for the control of PEC proliferation and apoptosis. Analysis of the data revealed that loss of VDR signaling in mouse PEC increased proliferation by 97% (p\u3c0.05) and reduced apoptosis by 44% (p\u3c0.05) after androgen depletion and repletion. PEC VDR deletion also had a growth inhibitory effect on stromal cells during phases of active growth indicating a possible role of cell-cell communication in the regulation of prostate biology. In the second experiment we used APT121 mouse model of prostate cancer to test whether PEC VDR level was important for the control of prostate cancer. The data revealed that PEC VDR deletion increased the incidence of advanced lesions (i.e. adenocarcinomas) and reduced the incidence of less-advanced preneoplastic lesions in anterior prostate lobe. The number of foci and the area affected by adenocarcinoma was also significantly greater in animals with VDR knockout. These data suggest that increased progression of carcinogenesis occurs in the absence of VDR signaling in PEC. In the final experiment we used microarray analysis to identify potential vitamin D target genes and pathways affected by 1,25(OH)2D. Immortalized but non-tumorigenic prostate epithelial cells, (RWPE1), were treated with 1,25(OH)2D or vehicle for 6, 24, or 48 h and the transcript profile was assessed. Many transcript level changes were observed at each time point at a 5% false detection rate. Over 260 transcripts were regulated by 1,25(OH) 2D at all time points (79% up) and are considered potential direct vitamin D target genes that may modulate prostate carcinogenesis. Several of these were identified as direct targets of 1,25(OH)2D by using ChIP assay. Functional analysis of the microarray data revealed modulation of several functionally distinct pathways by 1,25(OH)2D, i.e. suppression of pro-proliferative and WNT signaling, suppression of pro-angiogenic VEGF signaling, suppression of pro-inflammatory cytokine signaling pathways, up-regulation of transcripts involved in the protection of cells from oxidative stress or DNA damage. This data demonstrated that VDR signaling is critical for anticancer effects of vitamin D. 1,25(OH)2D can suppress growth of normal prostate epithelial cells via multiple mechanism and initiate mechanisms preventing carcinogenic changes

Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome

Changes of Slfn3, AKT and p38 signaling in defunctionalized intestine.

<p>Levels of Slfn3 (A), Glut2 (B), villin (C), SI (D), phosphorylated p38/total p38 (pp38/p38) (E), phosphorylated AKT/total AKT (pAKT/tAKT) (F) were lower in the intestinal mucosa of defunctionalized (Roux) bowel seven days after anastomosis compared to sham-operated controls or proximal or distal bowel in the same animals (p<0.05, n = 6). * - significantly different from area representing defunctionalized region of intestine in control (sham-operated) animals (p<0.05).</p

Slfn3 modulates expression of enterocyte differentiation markers in vivo.

<p>(A) 10¹¹ vector particles in 200 µL of Ad-GFP or Ad-GFP-Slfn3 were injected intraluminally into temporarily obstructed jejunal segments of anesthetized 8-12 weeks old male rats. 72 hours after exposure to the Ad-GFP-Slfn3 virus there was a significant increase of Slfn3 transcript (A), villin (B), Glut2 (C), and SI (D) expression compared to respective mRNA from control animals inoculated with intraluminal Ad-GFP by real-time qPCR of mRNA from jejunal mucosal scrapings (n = 6–7, *p<0.01). Induced gene expression was confirmed by grading of slides stained with appropriate antibody. Overexpression of Slfn3 significantly increase expression of Glut2 (C) on brush border membrane (BMM, arrows) and compared to Ad-GFP treated mucosa. (F) Intestinal mucosa villous length was significantly increased after Ad-GFP-Slfn3 infection (n = 3, *p<0.05, #p<0.1). Representative images of control (Ad-GFP) and Ad-GFP-Slfn3 virus injected intestines. Scale bar in (A–E, 400× magnification) is 20 µm and in (F, 100× magnification) 100 µm.</p