Carnosic acid and promotion of monocytic differentiation of HL60-G cells initiated by other agents.

BACKGROUND: Carnosic acid is a plant-derived polyphenol food preservative with chemoprotective effects against carcinogens when tested in animals. Recently, we showed that carnosic acid potentiates the effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and of all-trans-retinoic acid (ATRA) on differentiation of human leukemia cells. We now examine the mechanisms associated with carnosic acid-induced enhancement of cell differentiation (in subline HL60-G) initiated by 1alpha,25(OH)2D3, ATRA, or 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: We evaluated monocytic differentiation markers (CD11b, CD14, and monocytic serine esterase), cell cycle parameters, and cell proliferation rates after treatment of cells with different agents with or without carnosic acid. We also assessed the abundance of the vitamin D receptor (VDR), retinoid X receptor (RXR)-alpha, retinoic acid receptor (RAR)-alpha, and cell cycle-associated proteins by immunoblot analysis (p27, early growth response gene [EGR]-1, and p35Nck5a), the expression of corresponding genes by reverse transcription-polymerase chain reaction (RT-PCR), and the activity of VDR by electrophoretic mobility shift analysis. The two-sided nonparametric Kruskal-Wallis one-way analysis-of-variance test with Dunn's adjustment was used for statistical analyses. RESULTS: Monocytic differentiation induced by low (1 nM) concentrations of 1alpha,25(OH)2D3, ATRA, or TPA was enhanced by carnosic acid (10 microM), as shown by the increased expression of monocytic serine esterase (P<.001, P<.001, and P =.043, respectively) and of CD11b (P =.008, P =.046, and P =.041, respectively). Increased expression of CD14 was seen only for 1alpha,25(OH)2D3 and ATRA (P =.009 and P =.048, respectively) and also for several cell cycle-associated proteins. Carnosic acid in combination with 1alpha,25(OH)2D3 and ATRA resulted in decreased cell proliferation and blocked the cell cycle transition from G1 to S phase (P<.05). Carnosic acid alone increased the expression of VDR and RXR-alpha, but the expression was greatly enhanced in the presence of 1alpha,25(OH)2D3 and ATRA. In combination with TPA, carnosic acid potentiated the expression of VDR and RAR-alpha. CONCLUSION: Carnosic acid enhances a program of gene expression consistent with 1alpha,25(OH)2D3-, ATRA-, or TPA-induced monocytic differentiation of HL60-G cells.

Suppression of mammary tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069.

Retinoids have been used in the clinic for the prevention and treatment of human cancers. They regulate several cellular processes including growth, differentiation, and apoptosis. Previously, we demonstrated that a pan-agonist retinoid 9-cis retinoic acid was able to suppress mammary tumorigenesis in the C3(1)-SV40 T-antigen (Tag) transgenic mouse model. However, significant toxicity was seen with this naturally occurring retinoid. We hypothesized that the cancer preventive effects of retinoids could be dissected from the toxic effects by using receptor-selective retinoids. In this study, we used TTNPB, an retinoic acid receptor-selective retinoid, and LGD1069, an retinoid X receptor-selective retinoid, to preferentially activate retinoic acid receptors and retinoid X receptors. In vitro, both compounds were able to inhibit the growth of T47D breast cancer cells. We then determined whether these retinoids prevented mammary tumorigenesis. C3(1)-SV40 Tag mice were treated daily by gastric gavage with vehicle, two different doses of TTNPB (0.3 or 3.0 microg/kg), or two different doses of LGD1069 (10 or 100 mg/kg). Mice were treated from approximately 6-8 weeks to 7-8 months of age. Tumor size and number were measured twice each week, and toxicities were recorded daily. Our data show that LGD1069 suppresses mammary tumorigenesis in C3(1)-SV40 Tag transgenic mice with no observable toxicity, whereas TTNPB had a modest chemopreventive effect, yet was very toxic. Median time to tumor development was 129 days in vehicle-treated mice versus 156 days in mice treated with 100 mg/kg LGD1069 (P = 0.05). In addition, tumor multiplicity was reduced by approximately 50% in mice treated with LGD1069 (2.9 for vehicle, 2.4 for 10 mg/kg LGD1069, and 1.4 for 100 mg/kg, P < or = 0.03). TTNPB-treated mice showed a delayed median time to tumor development (131 days for vehicle versus 154 days for 3.0 microg/kg TTNPB; P < or = 0.05), but no changes were seen in tumor multiplicity. However, toxicity (skin erythema, hair loss) was seen in all of the mice treated with TTNPB. These data demonstrate that receptor-selective retinoids suppress mammary tumorigenesis in transgenic mice and that preventive effects of retinoids can be separated from their toxicity, demonstrating that receptor-selective retinoids are promising agents for the prevention of breast cancer.

Inhibition of ectopic bone formation by a selective retinoic acid receptor alpha-agonist: a new therapy for heterotopic ossification?

Heterotopic ossification (HO) consists of formation of ectopic cartilage followed by endochondral bone and is triggered by major surgeries, large wounds, and other conditions. Current therapies, including low-dose irradiation, are not always effective and do not target the skeletogenic process directly. Because chondrogenesis requires a decrease of nuclear retinoic acid receptor alpha (RARalpha) action, we reasoned that pharmacologic activation of this receptor pathway should inhibit HO. Thus, we selected the synthetic retinoid NRX195183, a potent and highly selective RARalpha-agonist, and found that it did inhibit chondrogenesis in mouse limb micromass cultures. We established a mouse HO model consisting of subcutaneous implantation of Matrigel mixed with rhBMP-2. Control mice receiving daily oral doses of vehicle (peanut oil) or retinol (a natural nonactive retinoid precursor) developed large HO-like masses by days 9-12 that displayed abundant cartilage, endochondral bone, vessels, and marrow. In contrast, formation of HO-like masses was markedly reduced in companion mice receiving daily oral doses of alpha-agonist. These ectopic masses contained sharply reduced amounts of cartilage and bone, blood vessels, and TRAP-positive osteoclasts, and expressed markedly lower levels of master chondrogenic genes including Sox9, cartilage genes such as collagen XI and X, and osteogenic genes including Runx2. The data provide proof-of-principle evidence that a pharmacological strategy involving a selective RARalpha-agonist can indeed counteract an ectopic skeletal-formation process effectively and efficiently, and could thus represent a novel preventive treatment for HO.