Effects of the Macular Carotenoid Lutein in Human Retinal Pigment Epithelial Cells.

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Oxidative stress-induced damage to the RPE occurs as part of the pathogenesis of age-related macular degeneration and neovascular retinopathies (e.g., retinopathy of prematurity, diabetic retinopathy). The xanthophyll carotenoids, lutein and zeaxanthin, are selectively taken up by the RPE, preferentially accumulated in the human macula, and transferred to photoreceptors. These macular xanthophylls protect the macula (and the broader retina) via their antioxidant and photo-protective activities. This study was designed to investigate effects of various carotenoids (ß-carotene, lycopene, and lutein) on RPE cells subjected to either hypoxia or oxidative stress, in order to determine if there is effect specificity for macular pigment carotenoids. Using human RPE-derived ARPE-19 cells as an in vitro model, we exposed RPE cells to various concentrations of the specific carotenoids, followed by either graded hypoxia or oxidative stress using tert-butyl hydroperoxide (tBHP). The results indicate that lutein and lycopene, but not ß-carotene, inhibit cell growth in undifferentiated ARPE-19 cells. Moreover, cell viability was decreased under hypoxic conditions. Pre-incubation of ARPE-19 cells with lutein or lycopene protected against tBHP-induced cell loss and cell co-exposure of lutein or lycopene with tBHP essentially neutralized tBHP-dependent cell death at tBHP concentrations up to 500 µM. Our findings indicate that lutein and lycopene inhibit the growth of human RPE cells and protect the RPE against oxidative stress-induced cell loss. These findings contribute to the understanding of the protective mechanisms attributable to retinal xanthophylls in eye health and retinopathies.

Inhibition of pulmonary ß-carotene 15, 15'-oxygenase expression by glucocorticoid involves PPARα.

ß-carotene 15,15'-oxygenase (BCO1) catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. This enzyme is expressed in a variety of developing and adult tissues, suggesting that its activity may regulate local retinoid synthesis. Vitamin A and related compounds (retinoids) are critical regulators of lung epithelial development, integrity, and injury repair. A balance between the actions of retinoids and glucocorticoids (GCs) promotes normal lung development and, in particular, alveolarization. Alterations in this balance, including vitamin A deficiency and GC excess, contribute to the development of chronic lung disorders. Consequently, we investigated if GCs counteract retinoid effects in alveolar epithelial cells by mechanisms involving BCO1-dependent local vitamin A metabolism. We demonstrate that BCO1 is expressed in human fetal lung tissue and human alveolar epithelial-like A549 cells. Our results indicate A549 cells metabolize ß-carotene to retinal and retinoic acid (RA). GCs exposure using dexamethasone (DEX) decreases BCO1 mRNA and protein levels in A549 cells and reduces BCO1 promoter activity via inhibiting peroxisome proliferator-activated receptor γ (PPARγ) DNA binding. DEX also induces expression of PPARα, which in turn most likely causes a decrease in PPARγ/RXRα heterodimer binding to the bco1 gene promoter and consequent inhibition of bco1 gene expression. PPARα knockdown with siRNA abolishes DEX-induced suppression of BCO1 expression, confirming the requirement for PPARα in this DEX-mediated BCO1 mechanism. Taken together, these findings provide the first evidence that GCs regulate vitamin A (retinoid) signaling via inhibition of bco1 gene expression in a PPARα-dependent manner. These results explicate novel aspects of local GC:retinoid interactions that may contribute to alveolar tissue remodeling in chronic lung diseases that affect children and, possibly, adults.

Mitochondrial ß-Carotene 9',10' Oxygenase Modulates Prostate Cancer Growth via NF-κB Inhibition: A Lycopene-Independent Function.

Despite numerous inquiries into protective roles of lycopene in prostate cancer prevention or therapy, little is known about mechanisms by which lycopene or its metabolites inhibit prostate cancer. The enzyme ß-carotene 9',10'-oxygenase (BCO2), which catalyzes asymmetric cleavage of several carotenoids, is the principal regulator of lycopene metabolism, but the range of BCO2 biological functions is incompletely understood. This study investigated expression and functional roles of BCO2 in human prostate cancer. Expression of the bco2 gene is dramatically decreased in prostate cancer tissue and in a range of prostate cancer cell lines as compared with nonneoplastic prostate tissue and normal prostatic epithelial cells, respectively. Inhibition of DNA methyltransferase activity restored bco2 expression in prostate cancer cell lines tested. Treatment with lycopene or its metabolite, apo-10-lycopenal, also increased bco2 expression and reduced cell proliferation in androgen-sensitive cell lines, but lycopene neither altered bco2 expression nor cell growth in androgen-resistant cells. Notably, restoring bco2 expression in prostate cancer cells inhibited cell proliferation and colony formation, irrespective of lycopene exposure. Exogenous expression of either wild-type BCO2 or a mutant (enzymatically inactive) BCO2 in prostate cancer cells reduced NF-κB activity and decreased NF-κB nuclear translocation and DNA binding. Together, these results indicate epigenetic loss of BCO2 expression is associated with prostate cancer progression. Moreover, these findings describe previously unanticipated functions of BCO2 that are independent of its enzymatic role in lycopene metabolism. IMPLICATIONS: This study identifies BCO2 as a tumor suppressor in prostate cancer. BCO2-mediated inhibition of NF-κB signaling implies BCO2 status is important in prostate cancer progression. Mol Cancer Res; 14(10); 966-75. ©2016 AACR.

Basement membrane protein nidogen-1 is a target of meprin ß in cisplatin nephrotoxicity.

Meprins are oligomeric metalloproteinases that are abundantly expressed in the brush-border membranes of renal proximal tubules. During acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion, membrane-bound meprins are shed and their localization is altered from the apical membranes toward the basolateral surface of the proximal tubules. Meprins are capable of cleaving basement membrane proteins in vitro, however, it is not known whether meprins are able to degrade extracellular matrix proteins under pathophysiological conditions in vivo. The present study demonstrates that a basement membrane protein, nidogen-1, is cleaved and excreted in the urine of mice subjected to cisplatin-induced nephrotoxicity, a model of AKI. Cleaved nidogen-1 was not detected in the urine of untreated mice, but during the progression of cisplatin nephrotoxicity, the excretion of cleaved nidogen-1 increased in a time-dependent manner. The meprin inhibitor actinonin markedly prevented urinary excretion of the cleaved nidogen-1. In addition, meprin ß-deficient mice, but not meprin α-deficient mice, subjected to cisplatin nephrotoxicity significantly suppressed excretion of cleaved nidogen-1, further suggesting that meprin ß is involved in the cleavage of nidogen-1. These studies provide strong evidence for a pathophysiological link between meprin ß and urinary excretion of cleaved nidogen-1 during cisplatin-induced AKI.

ß-carotene regulates expression of ß-carotene 15,15'-monoxygenase in human alveolar epithelial cells.

ß-Carotene 15,15'-monooxygenase (CMO1, BCMO1) converts ß-carotene to retinaldehyde (retinal) and is a key enzyme in vitamin A metabolism. CMO1 activity is robust in the intestine and liver, where cmo1 gene transcription may be subject to negative feedback by accumulation of its metabolic products. Evidence from CMO1 null animals also indicates that non-gastrointestinal CMO1 may be required for tissue-specific conversion of ß-carotene into vitamin A. The aim of this study was to investigate the effects of the enzymatic substrate, ß-carotene, on regulation of CMO1 in a cell model of human alveolar pneumocytes. We demonstrate that CMO1 is expressed in human alveolar epithelial (A549) cells and converts ß-carotene into retinal and biologically active retinoic acids (RA). Exposure to ß-carotene suppresses CMO1 expression at both mRNA and protein levels. ß-Carotene, but not all-trans RA, decreases CMO1 promoter activity in a time- and dosage-dependent manner. This ß-carotene-mediated inhibition of CMO1 expression results from decreased binding of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα) in the CMO1 promoter. ß-Carotene treatment also antagonizes PPARγ activity in HEK293 cells that stably express CMO1 wild-type, but not in cells that express the CMO1 mutant or vector alone. These findings have implications for local vitamin A synthesis in the lung, especially during systemic vitamin A insufficiency and may also help to explain, in part, the mechanism underlying the increased lung cancer risk upon ß-carotene supplementation in smokers.

Single oral dose of micellar ß-carotene containing phospholipids improves ß-carotene metabolism and plasma lipids in vitamin A-deficient rats.

BACKGROUND: Vitamin A (VA) deficiency is still a major health problem in the developing world. It affects various cellular functions and causes hypolipidemic effects in the body. ß-Carotene (BC)-rich foods are promising sources of VA. Phospholipids are reported to improve BC bioefficacy in normal rats, but whether they show similar effects during VA deficiency is unknown. AIM: To compare the BC metabolism and plasma lipid responses in VA-sufficient (+VA) and VA-deficient (-VA) rats after a single oral dose of micellar BC containing phospholipids. METHODS: Groups of rats were fed with a VA-free diet and when they attained the weight-plateau stage of deficiency, both +VA and -VA rats were divided into 2 groups (phosphatidylcholine, PC and lysophosphatidylcholine, LPC). Each group was further divided into 4 subgroups (1, 2, 3, and 6 h; n = 5 rats/time point) and determined the BC metabolism and plasma lipid responses to a post-dose of micellar BC with phospholipids. RESULTS: Maximal plasma BC (pmol/mL) levels were observed at 2 h in PC (1330 ± 124) and at 1 h in LPC (1576 ± 144) groups of +VA rats, and at 3 h in the PC (1621 ± 158) and LPC (2248 ± 675) groups of -VA rats. Liver BC (pmol/g) was maximum at 1 h in the PC (218 ± 32) and LPC (249 ± 24) groups of +VA rats, and at 2 h in PC (228 ± 23) and at 3 h in LPC (277 ± 18) groups of -VA rats. Plasma and liver BC levels were significantly (P < 0.05) higher in -VA rats than +VA rats. Plasma retinyl palmitate (pmol/mL) was maximum at 3 h in PC (97 ± 18) and at 2 h in LPC (126 ± 14) groups of +VA rats, and at 2 h in the PC (92 ± 13) and LPC (134 ± 27) groups of -VA rats. The higher (P < 0.05) BC monoxygenase activity in -VA rats compared to +VA rats supports the BC bioefficacy. Plasma retinol level was improved in the PC and LPC groups, but the effect of LPC was higher (P < 0.05) than PC. Micellar phospholipids mitigate the VA deficiency-induced hypolipidemic effects. CONCLUSIONS: Micellar phospholipids improved BC metabolism and reinstated the hypolipidemic effects, perhaps by modifying the fat-metabolizing enzymes and repairing the altered intestinal membrane structure.

Bioefficacy of beta-carotene is improved in rats after solubilized as equimolar dose of beta-carotene and lutein in phospholipid-mixed micelles.

beta-Carotene (BC) is a potent dietary source of vitamin A for populations at risk of vitamin A deficiency, yet its bioavailability is influenced by several factors such as dietary fat, carotenoids type, and other components. We hypothesize that type of micellar phospholipids influence bioefficacy of carotenoids and activity of carotenoid metabolizing enzymes. This study determined the BC bioefficacy in rats (n = 5/time point) after an equimolar dose of BC and lutein (Lut) solubilized in micelles containing either phosphatidylcholine (PC) or lysophosphatidylcholine (LPC), or no phospholipid (NoPL). Results show that no BC and Lut was detected in the plasma of rats at 0 hour, but after gavage, the mean (SD) area under the curve (AUC; in picomoles per milliliter) of plasma BC for 6 hours in PC, LPC, and NoPL groups were 1145 (132), 965 (199), and 2136 (112), respectively. The AUC value of plasma Lut in LPC group (183 +/- 23 pmol mL(-1) h(-1)) was higher than the other 2 groups. Similarly, liver BC and Lut levels in the LPC group were significantly higher than the other groups. The activity of BC 15,15'-monooxygenase in the intestinal mucosa of LPC and PC groups was higher than NoPL group. Plasma retinyl palmitate level in LPC (AUC, 647 +/- 89 pmol mL(-1) h(-1)) group was 2-fold higher than that of PC and NoPL groups. Results indicate that phospholipids enhanced the BC and Lut absorption. beta-Carotene uptake was not affected by Lut when given with micellar phospholipids, but reduced plasma Lut level was observed, which may be due to the conversion of absorbed Lut into its metabolites.