The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

BACKGROUND: Bexarotene (Targretin®) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. OBJECTIVES: To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. METHODS: With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. RESULTS: Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC50) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. CONCLUSIONS: UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs.

Expression of p27Kip1, a cell cycle repressor protein, is inversely associated with potential carcinogenic risk in the genetic rodent models of obesity and long-lived Ames dwarf mice.

INTRODUCTION: The association of genetic rodent models of obesity and cancer still remains a controversial issue. Although this controversy has largely been resolved in recent years for homozygous leptin receptor-deficient obese Zucker rats and homozygous long-lived Ames dwarf mice, it is still unresolved for homozygous leptin-deficient obese ob/ob mice. OBJECTIVE: The objective of the present study described below was to investigate whether the expression of the cell cycle repressor protein p27(Kip1) is (a) down-regulated in the tumor-free homozygous leptin receptor-deficient obese Zucker rats as well as tumor-free homozygous leptin-deficient obese ob/ob mice and (b) up-regulated in the tumor-free homozygous long-lived Ames dwarf mice. METHODS: To achieve this objective, we first performed western immunoblot analysis of the hepatic expression of p27. We then performed western immunoblot analysis and proteomic analysis of the hepatic expression of the proteins involved in the upstream molecular signaling pathways for the expression of p27. Lastly, we analyzed the serum levels of glucose, insulin, and branched-chain amino acids, all of which have been shown to regulate, causally and inversely, the expression of p27. RESULTS/CONCLUSIONS: The results indicated that the hepatic expression of p27 was down-regulated in the homozygous leptin receptor-deficient obese Zucker rats and up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the hepatic expression of p27 was down-regulated in the homozygous leptin-deficient obese ob/ob mice. This last observation was not completely consistent with all of the results of the published studies where homozygous leptin-deficient obese ob/ob mice were used.

Upstream molecular signaling pathways of p27(Kip1) expression in human breast cancer cells in vitro: differential effects of 4-hydroxytamoxifen and deficiency of either D-(+)-glucose or L-leucine.

BACKGROUND: The objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D-(+)-glucose could down-regulate and, conversely, (b) the deficiency of D-(+)-glucose or certain L-amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1. RESULTS: Using human MDA-MB-231 breast cancer cells in vitro, these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D-(+)-glucose or L-leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3. CONCLUSIONS: (a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D-(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D-(+)-glucose or L-leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.

Upstream molecular signaling pathways of p27(Kip1) expression: effects of 4-hydroxytamoxifen, dexamethasone, and retinoic acids.

BACKGROUND: p27(Kip1) is a cyclin-dependent kinase inhibitor that inhibits G1-to-S phase transition of the cell cycle. It is known that a relatively large number of nutritional and chemopreventive anti-cancer agents specifically up-regulate expression of p27 without directly affecting the expression of other G1-to-S phase cell cycle regulatory proteins including p21(Cip1Waf1). However, the upstream molecular signaling pathways of how these agents up-regulate the expression of p27 have not been well characterized. The objective of this study was to identify such pathways in human breast cancer cells in vitro using 4-hydroxytamoxifen, dexamethasone, and various retinoic acids as examples of such anti-cancer agents. RESULTS: Experimental evidence presented in the first half of this report was obtained by transfecting human breast cancer cells in vitro with proximal upstream region of p27 gene-luciferase reporter plasmids. 1) The evidence indicated that 4-hydroxytamoxifen, dexamethasone, and various retinoic acids up-regulated expression of p27 in both estrogen receptor-positive and negative human breast cancer cells in vitro. 2) The degree of up-regulation of p27 expression by these anti-cancer agents in human breast cancer cells in vitro linearly correlated with the degree of inhibition of methylnitrosourea (MNU)-induced rat mammary adenocarcinoma in vivo. 3) Lastly, up-regulation of the expression of p27 was likely due to the activation of translation initiation rather than transcription of p27 gene. The experimental evidence presented in the second half of this report was obtained by a combination of Western immunoblot analysis and transfection analysis. It indicated that 4-hydroxytamoxifen and dexamethasone up-regulated expression of p27 by down-regulating phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) at Ser65 and this phosphorylation was likely to be mediated by upstream receptor tyrosine kinases/phosphoinositide-3-kinase/Akt/5'-AMP-activated protein kinase/mammalian target of rapamycin (RTKs/PI3K/Akt/AMPK/mTOR) protein kinase signaling pathways. Retinoic acids up-regulated expression of p27 without using either 4E-BP1 or RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways. CONCLUSIONS: 4-Hydroxytamoxifen and dexamethasone up-regulated translation initiation of p27 by down-regulating 4E-BP1 phosphorylated at Ser65 and this down-regulation seemed to be mediated by upstream RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways. Retinoic acids also up-regulated translation initiation of p27, but without using any of these pathways.

"Nutritional and chemopreventive anti-cancer agents up-regulate expression of p27Kip1, a cyclin-dependent kinase inhibitor, in mouse JB6 epidermal and human MCF7, MDA-MB-321 and AU565 breast cancer cells".

BACKGROUND: p27(Kip1) is a cyclin-dependent kinase inhibitor. When up-regulated, p27 inhibits G1-to-S phase transition of the cell cycle. This report addresses the question of whether various nutritional and chemopreventive anti-cancer agents up-regulate the expression of p27 in preneoplastic and neoplastic cells. RESULTS: Experimental evidence presented in the first half of this report shows that these agents fairly faithfully up-regulate expression of p27 in mouse epidermal (JB6) and human breast cancer (MCF7, MDA-MB-321, and AU565) cells. Up-regulation appears to be specific to p27 because expression of cyclin D1, E, and A, and p21Cip1/Waf1 was not modulated by these agents. Up-regulation of the expression of p27 is likely due to the activation of translation rather than transcription of p27 because (a) up-regulation is mediated by the 5'-untranslated region (-575) of the p27 gene and (b) the antibiotic actinomycin D, an inhibitor of transcription, did not attenuate the up-regulation of p27. This latter finding is likely to preclude the existence of cryptic transcription factor binding site(s) in the 5'-untranslated region of p27 gene. The experimental evidence, presented in the second half of this report, was obtained using the 5'-untranslated region (-575) of p27 gene. The evidence suggests that cancer preventive agents up-regulate expression of p27 by at least four different molecular signaling pathways: (a) Caloric restriction is likely to up-regulate p27 expression via 5'-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR). Amino acid deficiencies also up-regulate the expression of p27 using some components of this pathway. (b) 4-Hydroxytamoxifen (but not tamoxifen), genistein (but not genistin), daidzein, and probably other nutritional and chemopreventive anti-cancer agents could up-regulate expression of p27 via receptor protein tyrosine kinases (RPTKs), phosphoinositide 3-kinase (PI3K), phosphoinosite-dependent kinase (PDK), Akt/PKB and mTOR. (c) Expression of p27 could also be up-regulated via RPTKs followed by MAPKs--MEK, ERK and p38MAPK--and probably MNK. Finally, (d) global hypomethylation of 5'-m7G cap of mRNAs could also up-regulate expression of p27. CONCLUSION: Based on these findings, we conclude that various nutritional and chemopreventive anti-cancer agents up-regulate expression of p27 in (pre)neoplastic cells.

Efficacy of new retinoids in the prevention of mammary cancers and correlations with short-term biomarkers.

A number of retinoid X receptor (RXR) agonists have proven to be highly effective in preventing methylnitrosourea (MNU) induced mammary cancers. However, these agonists have side effects; particularly causing an increase in serum triglyceride levels. A series of ligands for RXR were designed based on computer modeling to the ligand binding domain (LBD) of the RXR receptors and on structure-activity relationships. The chemopreventive effects of these retinoids were evaluated in the relatively long-term MNU model. As a short-term assay to predict their efficacy, the ability of the retinoids to modulate cell proliferation and apoptosis was also determined in mammary cancers after only 7 days of treatment. The five UAB retinoids evaluated included two Class I UAB retinoids (UAB20, UAB112) and three Class II UAB retinoids (UAB30, 4-methyl-UAB30 and the benzosuberone-analog of UAB30). The previously evaluated RXR agonist targretin and the pan-agonist 9-cis-retinoic acid (9-cis-RA), which interacts with both RAR and RXR receptors, were included as positive agonists known to prevent cancer in the MNU model. In the prevention studies, in which the agents were administered beginning 5 days after MNU until the end of the study, targretin (150 mg/kg diet) and 4-methyl-UAB30 (200 mg/kg diet) were highly effective in decreasing cancer numbers by 75-85%. UAB30 (200 mg/kg diet) and 9-cis-RA (60 mg/kg diet) gave intermediate inhibitions of 60 and 45%, respectively. Targretin (15 mg/kg diet), UAB20 (200 mg/kg diet) and the benzosuberone analog of UAB30 (200 mg/kg diet) showed limited activity by decreasing cancer multiplicity 25-30%, while UAB112 had no effect on mammary cancer multiplicity. A direct correlation was observed between the long-term chemopreventive efficacy of these agents and their ability to decrease cell proliferation in mammary cancers after short-term treatment. Furthermore, the highly effective agents (4-methyl-UAB30 and targretin at 150 mg/kg diet) increased apoptosis 3-5 times, while agents with moderate or limited preventive efficacy failed to significantly increase apoptosis. Although the more effective retinoid treatments increased serum triglycerides 2.5- to 4.0-fold, one moderately effective agent (UAB30) had no significant effect on lipid levels. In summary, a short-term in vivo method has been identified for screening newly synthesized retinoids both for chemopreventive efficacy and for their adverse effect on serum triglycerides.

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis.

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.

Efficacy of 9-cis-retinoic acid and N-(4-hydroxyphenyl) retinamide alone and in combination in mammary cancer prevention.

As demonstrated in several in vitro and in vivo cancer models, retinoids have chemopreventive activity. The present studies were performed to evaluate the efficacy of 9-cis-retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl) retinamide (4-HPR), alone and combined, in preventing mammary cancers. Female Sprague-Dawley rats received N-methyl-N-nitrosourea (MNU), 50 mg/kg BW, either at 50 days of age (experiment I, young rats) or at 100 days of age (experiment II, older rats). In experiment I, 9-cis-RA (60 mg/kg of diet), 4-HPR (586 mg/kg of diet), or the combination were evaluated; in experiment II, 9-cis-RA (30 mg/kg of diet), 4-HPR (196 mg/kg of diet), or the combination were tested. There were no signs of toxicity in either study. In the young rats, there were only slight reductions (15-20%) in the number of mammary cancers when the agents were given alone. In the older rats, lower doses of 9-cis-RA or 4-HPR alone were highly effective; with 61% and 46% reductions in the number of mammary cancers, respectively. The combination of retinoids in the young rats caused a 49% reduction in mammary cancers, while in the older rats the combination resulted in a 96% reduction. Thus, lower doses of the retinoids caused more striking inhibition of mammary cancers in older rats than the higher doses given to younger animals. In both experiments, the two retinoids in combination produced an additive effect, suggesting that they may inhibit mammary cancers by different mechanisms.

9cUAB30, an RXR specific retinoid, and/or tamoxifen in the prevention of methylnitrosourea-induced mammary cancers.

Studies were performed in female Sprague-Dawley rats to determine the efficacy of a new RXR specific retinoid (9cUAB30) when combined with tamoxifen in the prevention of mammary cancers and to determine various pharmacokinetic parameters of the retinoid. When administered by gavage, 9cUAB30 was rapidly absorbed and had a serum t(1/2) of 13.5 h. Since the retinoid was administered in the diet for the chemoprevention study, a 28-day study in which 9cUAB30 was given at dose levels of 200, 400, and 600 mg/kg diet revealed fairly constant serum levels regardless of dose or length of treatment; possibly accounting for the observed low toxicity of this compound. When suboptimal doses of 9cUAB30 were given in the methylnitrosourea (MNU)-induced mammary cancer model, the following average number of mammary cancers were observed: 9cUAB30 (150 mg/kg diet), 4.3; tamoxifen (0.4 mg/kg diet), 4.6; 9cUAB30 (150 mg/kg diet)+tamoxifen (0.4 mg/kg diet), 2.6; and controls, 6.0. Thus, the combination of the agents resulted in an increased effect in preventing mammary cancers; suggesting that cancer cell proliferation was inhibited by the compounds blocking different pathways.

G1 cell cycle regulatory proteins in chemically-induced rat mammary adenocarcinomas in vivo and tumor promotion-sensitive, -resistant and transformed mouse epidermal cells in vitro.

Tumor promotion is characterized by selective proliferation of initiated cells resulting in their clonal expansion. Cyclin Dl is frequently upregulated in this process, but its expression does not necessarily correlate positively with cyclin A. In the present article, expression of G1 cell cycle regulatory proteins was systematically analyzed using two models of carcinogenesis: (a) N-methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas and normal rat mammary epithelial cells in vivo and (b) promotion-sensitive, -resistant, and transformed JB6 mouse epidermal cells in vitro. The results of this analysis revealed that p27Kipl negatively correlated with cyclin Dl. In addition, there were two types of correlations between p27Kipl and cyclin A. First, p27Kipl negatively correlated with cyclin A (type-l correlation). This scenario was observed in normal rat mammary epithelial cells in vivo and promotion-sensitive (P+) JB6 mouse epidermal cells, stimulated with phorbol ester (TPA) in vitro. Second, p27Kipl positively correlated with cyclin A (type-ll correlation). This correlation was observed in MNU-induced rat mammary adenocarcinomas in vivo and TPA-stimulated (P+) JB6 cells, treated with retinoic acid in vitro.

Effects of body weight gain reduction resulting from chemopreventive agent treatment on mammary gland morphology.

Moderate reductions (< or = 15%) in body weight gain, similar to those observed after administration of some chemopreventive agents in chemically induced mammary cancer models, will result in decreased mammary cancers (up to 55%). The objective of this study was to determine whether changes in mammary gland differentiation, proliferation, apoptosis, and estradiol and progesterone levels are affected by moderate reductions in body weight induced after chemopreventive agent treatment and dietary restriction. The body weights of female Sprague-Dawley rats were reduced by dietary restrictions to match those of rats receiving 4-hydroxyphenylretinamide (4-HPR) at a dose known to inhibit methylnitrosourea (MNU)-induced mammary cancers. 4-HPR supplementation or dietary restrictions began 1 wk before MNU administration at 50 days of age. Mammary gland differentiation, proliferation, apoptosis, and serum levels of estradiol and progesterone were measured at 50, 57, and 71 days of age. Casein expression, proliferating cellular nuclear antigen expression, and apoptosis were not significantly different from controls in the dietary-restricted group. Proliferating cellular nuclear antigen expression was significantly lower in 4-HPR-treated animals than in controls at 57 days of age. The diameter of the mammary gland ducts was smaller at 71 days of age in the treatment groups. A decrease in estradiol levels for each group was observed at 50 days of age, but not at later time points. Progesterone levels were reduced in the 4-HPR group, but not in the dietary-restricted group, during each time period. It would appear that the observed decrease in mammary cancers observed with moderate reductions in body weight gain might be due to multiple related factors different from those related to 4-HPR treatment.

A randomized, 4-month mango and fat supplementation trial improved vitamin A status among young Gambian children.

Supplementation with carotene-rich fruits may be an effective and sustainable approach to prevent vitamin A deficiency. To test the effectiveness of mango supplementation, 176 Gambian children, aged 2 to 7 y, were randomly assigned to one of four treatments: 75 g of dried mango containing approximately 150 micro g retinol activity equivalents with (MF) or without (M) 5 g of fat, 5 d/wk for 4 mo or 60,000 micro g of vitamin A (A) or placebo (P) capsule at baseline. After 4 mo, plasma beta-carotene was greater in both the M (P < 0.05) and MF (P = 0.07) groups compared with the P group. After controlling for baseline plasma retinol, elevated acute phase proteins and age, plasma retinol concentrations in the A and MF, but not M, groups were higher than in the P group at the end of the study (P < 0.01). Increases in retinol concentrations, however, were small in both groups. These results support the use of dietary supplementation with dried mangoes and a source of fat as one of several concurrent strategies that can be used to help maintain vitamin A status of children in developing countries where there is a severe seasonal shortage of carotenoid-rich foods.

Prevention of methylnitrosourea-induced mammary cancers by 9-cis-retinoic acid and/or vitamin D3.

Two cancer chemopreventive agents, vitamin D3 and 9-cis-retinoic acid (9-cis-RA), were evaluated alone and in combination in the methylnitrosourea (MNU)-induced mammary cancer model. In this study, female Sprague-Dawley rats received MNU (50 mg/kg BW) at 50 days of age. Vitamin D3 and 9-cis-RA were administered in the diet beginning three days later. The groups were: Group 1, vehicle only; Group 2, 9-cis-RA (60 mg/kg diet); Group 3, vitamin D3 (10 microg/kg diet); Group 4, vitamin D3 (3.3 microg/kg diet); Group 5, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (10 microg/kg diet); and Group 6, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (3.3 microg/kg diet). Animals were observed daily for signs of toxicity and were palpated 2x/week for mammary tumors. The study was terminated 150 days after treatment with MNU. The average number of mammary cancers was 6.7 in the animals receiving only the carcinogen. 9-cis-RA alone caused a 23% decrease in mammary cancer multiplicity, while vitamin D3 alone actually caused slight increases of 17 and 16% at 10 and 3.3 microg/kg diet dose levels, respectively. When the agents were given in combination, however, the 9-cis-RA plus the high dose of vitamin D caused a statistically significant decrease (44%) in mammary cancer number, while the 9-cis-RA plus the low dose resulted in a 37% decrease. Thus, low doses of these agents that were not effective in preventing mammary cancer when given alone appeared to be active when given in combinations. Possible interactions between the retinoic acid receptors and vitamin D receptor may be responsible for the observed inhibition of mammary carcinogenesis.