Acitretin and Retinoic Acid Derivatives Inhibit BK Polyomavirus Replication in Primary Human Proximal Renal Tubular Epithelial and Urothelial Cells.

Small-molecule drugs inhibiting BK polyomavirus (BKPyV) represent a significant unmet clinical need in view of polyomavirus-associated nephropathy or hemorrhagic cystitis, which complicate 5% to 25% of kidney and hematopoietic cell transplantations. We characterized the inhibitory activity of acitretin on BKPyV replication in primary human renal proximal tubular epithelial cells (RPTECs). Effective inhibitory concentrations of 50% (EC50) and 90% (EC90) were determined in dilution series measuring BKPyV loads, transcripts, and protein expression, using cell proliferation, metabolic activity, and viability to estimate cytotoxic concentrations and selectivity indices (SI). The acitretin EC50 and EC90 in RPTECs were 0.64 (SI50, 250) and 3.25 µM (SI90, 49.2), respectively. Acitretin effectively inhibited BKPyV replication until 72 h postinfection when added 24 h before infection until 12 h after infection, but decreased to <50% at later time points. Acitretin did not interfere with nuclear delivery of BKPyV genomes, but it decreased large T-antigen transcription and protein expression. Acitretin did not inhibit the initial round of BKPyV replication following transfection of full-length viral genomes, but it affected subsequent rounds of reinfection. Acitretin also inhibited BKPyV replication in human urothelial cells and in Vero cells, but not in COS-7 cells constitutively expressing Simian virus 40 (SV40) large T antigen. Retinoic acid agonists (all-trans retinoic acid, 9-cis retinoic acid [9-cis-RA], 13-cis-RA, bexarotene, and tamibarotene) and the RAR/RXR antagonist RO41-5253 also inhibited BKPyV replication, pointing to an as-yet-undefined mechanism. IMPORTANCE Acitretin selectively inhibits BKPyV replication in primary human cell culture models of nephropathy and hemorrhagic cystitis. Since acitretin is an approved drug in clinical use reaching BKPyV-inhibiting concentrations in systemically treated patients, further studies are warranted to provide data for clinical repurposing of retinoids for treatment and prevention of replicative BKPyV-diseases.

One-Step, Low-Cost, Operator-Friendly, and Scalable Procedure to Synthetize Highly Pure N-(4-ethoxyphenyl)-retinamide in Quantitative Yield without Purification Work-Up.

It is widely reported that N-(4-hydroxyphenyl)-retinamide or fenretinide (4-HPR), which is a synthetic amide of all-trans-retinoic acid (ATRA), inhibits in vitro several types of tumors, including cancer cell lines resistant to ATRA, at 1-10 µM concentrations. Additionally, studies in rats and mice have confirmed the potent anticancer effects of 4-HPR, without evidencing hemolytic toxicity, thus demonstrating its suitability for the development of a new chemo-preventive agent. To this end, the accurate determination of 4-HPR levels in tissues is essential for its pre-clinical training, and for the correct determination of 4-HPR and its metabolites by chromatography, N-(4-ethoxyphenyl)-retinamide (4-EPR) has been suggested as an indispensable internal standard. Unfortunately, only a consultable old patent reports the synthesis of 4-EPR, starting from dangerous and high-cost reagents and using long and tedious purification procedures. To the best of our knowledge, no article existed so far describing the specific synthesis of 4-EPR. Only two vendors worldwide supply 4-ERP, and its characterization was incomplete. Here, a scalable, operator-friendly, and one-step procedure to synthetize highly pure 4-EPR without purification work-up and in quantitative yield is reported. Additionally, a complete characterization of 4-EPR using all possible analytical techniques has been provided.

Pharmacoepigenomics circuits induced by a novel retinoid-polyamine conjugate in human immortalized keratinocytes.

Retinoids are widely used in diseases spanning from dermatological lesions to cancer, but exhibit severe adverse effects. A novel all-trans-Retinoic Acid (atRA)-spermine conjugate (termed RASP) has shown previously optimal in vitro and in vivo anti-inflammatory and anticancer efficacy, with undetectable teratogenic and toxic side-effects. To get insights, we treated HaCaT cells which resemble human epidermis with IC50 concentration of RASP and analyzed their miRNA expression profile. Gene ontology analysis of their predicted targets indicated dynamic networks involved in cell proliferation, signal transduction and apoptosis. Furthermore, DNA microarrays analysis verified that RASP affects the expression of the same categories of genes. A protein-protein interaction map produced using the most significant common genes, revealed hub genes of nodal functions. We conclude that RASP is a synthetic retinoid derivative with improved properties, which possess the beneficial effects of retinoids without exhibiting side-effects and with potential beneficial effects against skin diseases including skin cancer.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Acyclic retinoid and angiotensin-II receptor blocker exert a combined protective effect against diethylnitrosamine-induced hepatocarcinogenesis in diabetic OLETF rats.

BACKGROUND: Insulin resistance (IR) is closely associated with the progression of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR) targets retinoid X receptor α and reportedly prevents HCC recurrence in clinical practice. Angiotensin-II receptor blocker (ARB) can also inhibit experimental hepatocarcinogenesis and HCC development. These are reported to suppress IR-based hepatocarcinogenesis; however, limited data are available regarding the combined effects of both these agents. This study aimed to investigate the combined chemopreventive effect of ACR and ARB on liver tumorigenesis on rats with congenital diabetes. METHODS: Male diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats underwent 70% partial hepatectomy following a single intraperitoneal injection of diethylnitrosamine to induce hepatocarcinogenesis and the administration of ACR (peretinoin, 40 mg/kg/day), ARB (losartan, 30 mg/kg/day), and a combination of ACR and ARB. Six weeks thereafter, we assessed the size and number of the pre-neoplastic lesions (PNL) as well as the altered angiogenesis, oxidative stress, and chronic inflammation in the liver. Moreover, we assessed the effects exerted by ACR and ARB on in vitro cell growth in human HCC cell lines and human umbilical vascular endothelial cells (HUVECs). RESULTS: OLETF rats showed increase in the size and number of PNLs compared to LETO rats. ACR suppressed the augmentation in size and number of PNLs in the OLETF rats with suppression of cell growth, intrahepatic angiogenesis, lipid peroxidation, oxidative DNA damage, and proinflammatory cytokine production. Combining ACR with ARB enhanced the tumor-suppressive effect and ameliorated intrahepatic angiogenesis, lipid peroxidation, and proinflammatory status; however, cell growth and oxidative DNA damage remained unchanged. IR-mimetic condition accelerated in vitro proliferative activity in human HCC cells, while ACR inhibited this proliferation with G0/G1 arrest and apoptosis. Furthermore, ACR and ARB significantly attenuated the HUVECs proliferation and tubular formation under the IR-mimetic condition, and a combination of both agents demonstrated greater inhibitory effects on HUVEC growth than each single treatment. CONCLUSIONS: ACR and ARB exert a combined inhibitory effect against IR-based hepatocarcinogenesis by the inhibition of cell growth, intrahepatic angiogenesis, and oxidative stress. Thus, this combination therapy appears to hold potential as a chemopreventive treatment therapy against HCC.

9-cis-13,14-Dihydroretinoic Acid Is an Endogenous Retinoid Acting as RXR Ligand in Mice.

The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand.

Phenosafranin inhibits nuclear localization of transglutaminase 2 without affecting its transamidase activity.

Transglutaminase 2 (TG2) localizes to the nucleus and induces apoptosis through a crosslinking inactivation of Sp1 in JHH-7 cells treated with acyclic retinoid. We screened an inhibitor suppressing transamidase activity in the nucleus without affecting transamidase activity itself. Phenosafranin was found to inhibit nuclear localization of EGFP-tagged TG2 and dose-dependently reduce nuclear transamidase activity without affecting the activity in a tube. We concluded that phenosafranin was a novel TG2 inhibitor capable of suppressing its nuclear localization.

SRD5A3-CDG: Expanding the phenotype of a congenital disorder of glycosylation with emphasis on adult onset features.

Increasing numbers of congenital disorders of glycosylation (CDG) have been reported recently resulting in an expansion of the phenotypes associated with this group of disorders. SRD5A3 codes for polyprenol reductase which converts polyprenol to dolichol. This is a major pathway for dolichol biosynthesis for N-glycosylation, O-mannosylation, C-mannosylation, and GPI anchor synthesis. We present the features of five individuals (three children and two adults) with mutations in SRD5A3 focusing on the variable eye and skin involvement. We compare that to 13 affected individuals from the literature including five adults allowing us to delineate the features that may develop over time with this disorder including kyphosis, retinitis pigmentosa, and cataracts. © 2016 Wiley Periodicals, Inc.

Retinoic acid isomers facilitate apolipoprotein E production and lipidation in astrocytes through the retinoid X receptor/retinoic acid receptor pathway.

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-ß (Aß) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aß-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ~4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aß in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy.

Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease.