Carnosic Acid and Promotion of Monocytic Differentiation of HL60-G Cells Initiated by Other Agents

Michael Danilenko, Xuening Wang, George P. Studzinski

Affiliation of authors: Department of Pathology and Laboratory Medicine, UMD—New Jersey Medical School, Newark.

Correspondence to: George P. Studzinski, M.D., Ph.D., Department of Pathology and Laboratory Medicine, UMD—New Jersey Medical School, 185 S. Orange Ave., C543, Newark, NJ 07103 (e-mail: studzins{at}umdnj.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 1{alpha},25-dihydroxyvitamin D3 (1{alpha},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 1{alpha},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 1{alpha},25(OH)2D3, ATRA, or TPA was enhanced by carnosic acid (10 µM), 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 1{alpha},25(OH)2D3 and ATRA (P = .009 and P = .048, respectively) and also for several cell cycle-associated proteins. Carnosic acid in combination with 1{alpha},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 1{alpha},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 1{alpha},25(OH)2D3-, ATRA-, or TPA-induced monocytic differentiation of HL60-G cells.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myeloid leukemias represent a largely unsolved challenge for chemotherapy of malignant disease. An important advance, however, was the demonstration that patients with a subset of this disease, acute promyelocytic leukemia (APL), can go into remission after treatment with all-trans-retinoic acid (ATRA) (13). In this situation, the immature hematopoietic cells are induced to differentiate toward nonproliferating, more mature granulocytes. The genetic defect in APL cells, i.e., a chromosomal translocation that results in a rearrangement of the gene for retinoic acid receptor (RAR)-{alpha}, can be compensated by increasing the concentration of ATRA, the physiologic ligand for RAR-{alpha} [reviewed in (4)]. Unfortunately, the ATRA-induced remissions of APL are usually followed by a relapse, and other forms of myeloid leukemias also carry a grim prognosis. Different approaches of differentiation therapy need to be evaluated.

Several compounds other than ATRA can induce differentiation of human leukemia cells in vitro, but the translation of these findings to the clinic has so far not been successful. For instance, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces monocyte–macrophage differentiation of HL60 human leukemia cells (5) as well as megakaryocytic differentiation of K562 leukemia cells (6,7); however, since this phorbol ester promotes carcinogenesis [e.g., (8,9)], clinical use of TPA cannot be contemplated. Similarly, dimethyl sulfoxide (DMSO) has diverse differentiation-inducing effects on leukemia cells, including morphologic granulocytic differentiation of HL60 cells (10), but it is a well-known chemical solvent unsuitable for internal human use. More promising is the potential use of the natural body metabolite, the vitamin–hormone 1{alpha},25-dihydroxyvitamin D3 (1{alpha},25[OH]2D3), and its chemically modified analogues, which induce monocyte-like differentiation of several human myeloid and myelomonocytic leukemia cell lines but which elicit no general cytotoxicity (11,12). However, even in this case, their clinical use is limited by systemic hypercalcemia resulting from the increased intestinal absorption of calcium and the calcium-mobilizing properties of 1{alpha},25(OH)2D3.

One approach to overcoming the problem of vitamin D-induced hypercalcemia, as well as the possible immunosuppressive effects of its analogues, is to combine relatively low doses of 1{alpha},25(OH)2D3 with the administration of another agent that augments the differentiation-inducing action of 1{alpha},25(OH)2D3 but that does not enhance the levels of circulating calcium and is not immunosuppressive. An example of such an approach is the combination of ketoconazole with 1-desoxy analogues of vitamin D3, which strongly potentiated the differentiation of HL60 cells in vitro but produced only minor changes in intracellular calcium homeostasis (13). While proof of principle was demonstrated, ketoconazole has substantial toxicity on its own and, so far, has not been introduced as a component of therapy for leukemia.

More recently, some plant-derived dietary antioxidants have been found not only to reduce the risk of cardiovascular disease and cancer in general (14) but also to enhance the differentiation of HL60 cells. Lycopene, a carotenoid present in fruits and vegetables but primarily present in tomatoes, can induce differentiation and can reduce the rate of growth of HL60 cells; however, more remarkably, lycopene cooperates with 1{alpha},25(OH)2D3 to produce these effects (15). Also, carnosic acid, an antioxidant polyphenol derived from the plant rosemary (Rosmarinus officinalis), augments the inhibition of growth and the induction of differentiation of HL60 and U937 leukemia cells by 1{alpha},25(OH)2D3 and ATRA (16). Since carnosic acid is a major component of rosemary extracts that are used widely as additives for the preservation of certain foods [e.g., (17)] and, therefore, appears to be safe for human administration, we investigated the nature and mechanisms of induction of differentiation of HL60 cells by using a combination of carnosic acid and low concentrations of several inducers of monocytic or granulocytic differentiation.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemicals and antibodies.

Carnosic acid was purchased from Alexis Biochemicals (Läufenfingen, Switzerland). 1{alpha},25(OH)2D3 was a gift from Dr. Milan Uskokovic (Hoffmann-La Roche Inc., Nutley, NJ). ATRA, TPA, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of carnosic acid (10 mM), 1{alpha},25[OH]2D3 (0.25 mM), ATRA (1 mM), and TPA (2 mM) were prepared in absolute ethanol. The antibodies against vitamin D receptor (VDR) (i.e., C-20), retinoid X receptor (RXR)-{alpha} (i.e., D-20), cyclin D1 (i.e., R-124), early growth response gene (EGR)-1 (i.e., 558), Cdk5 (i.e., DC17), and p35Nck5a (i.e., C-19) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-cyclin E (Ab-1) and anti-p27Kip1 antibodies were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA) and BD Biosciences—PharMingen (San Diego, CA), respectively. Phycoerythrin-conjugated anti-CD14 (MY4-RD-1) and fluorescein isothiocyanate-conjugated anti-CD11b (MO1-FITC) antibodies were obtained from Coulter Corp. (Miami, FL). Anti-calreticulin antibody (PA3-900) was purchased from Affinity BioReagents Inc. (Golden, CO). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were obtained from Santa Cruz Biotechnology Inc.

Cell culture and proliferation assay.

HL60-G cells (18), a subclone of human promyeloblastic leukemia HL60 cells (19), were cultured routinely at 37 °C in RPMI-1640 medium (Mediatech, Herndon, VA) supplemented with 1% glutamine and 10% heat-inactivated, iron-enriched bovine calf serum (HyClone Laboratories Inc., Logan, UT). The cultures were passaged two or three times weekly to maintain a log-phase growth. If not indicated otherwise, the cells were seeded at 1.0 x 105 cells/mL in 25-cm2 tissue culture flasks and incubated for 96 hours with carnosic acid, 1{alpha},25(OH)2D3, ATRA, TPA, DMSO, or combinations of carnosic acid with the above differentiation inducers. To demonstrate the enhancement of differentiation induced by 1{alpha},25(OH)2D3 or ATRA, we used low (1 nM) concentrations of these inducers, whereas we used 100 nM 1{alpha},25(OH)2D3 or 1 µM ATRA to illustrate the maximal effects of these inducers. Cell growth was estimated by counting the cells with a Coulter counter after dilution in Isoton-II (Coulter Electronics, Hialeah, FL). Cell viability was determined with the use of trypan blue dye (0.25%) exclusion.

Determination of markers of differentiation.

Aliquots of 1 x 106 cells were harvested, washed twice with phosphate-buffered saline (PBS), and suspended in 10 µL 1x PBS. The cell suspensions were incubated for 45 minutes at room temperature with 0.5 µL MY4-RD-1 and 0.5 µL MO1-FITC (1 : 20 dilution of the stock antibodies) to analyze the expression of the cell surface markers CD14 and CD11b, respectively. The cells were then washed three times with ice-cold 1x PBS and resuspended in 1 mL of PBS. Two-parameter analysis was performed with the use of an FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Isotypic mouse IgG1 was used to set threshold parameters. Monocytic differentiation was also monitored by cytochemical determination of the activity of monocytic serine esterase (MSE), also known as nonspecific esterase (NSE), as described previously (13).

Cell cycle distribution.

The cells (1 x 106) were washed twice with ice-cold 1x PBS and were fixed in 75% ethanol at –20 °C overnight. They were then washed twice with 1x PBS and incubated in 1 mL of a solution containing 1 µg (100 U/mL) ribonuclease (RNase) (BMB, Indianapolis, IN) and 10 µg/mL propidium iodide (Sigma Chemical Co.) for 1 hour at 4 °C in the dark. The cell DNA content was determined with the use of an FACSCalibur flow cytometer, and the cell cycle distribution was analyzed by a ModFit LT computer program (Verity Software House; AMPL Software, Turramurra, Australia), as described previously (20).

Cell extracts and western blotting.

The cells (approximately 1 x 107) were washed twice with ice-cold 1x PBS, snap-frozen in liquid N2, and stored at –80 °C. Whole-cell extracts were made by mixing the thawed cell pellets with an extraction buffer, i.e., 20 mM Tris–HCl, 0.25 M sucrose, 10 mM ethylene glycol-O,O`-bis-[2-amino-ethyl]-N,N,N`,N`-tetraacetic acid, 2 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.5), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% leupeptin, and 10 µg/mL aprotinin, followed by vigorous vortexing for 10 seconds. The extracts were mixed with an equal amount of 3x sodium dodecyl sulfate (SDS) sample buffer (i.e., 150 mM Tris, 30% glycerol, 3% SDS, 1.5 mg/mL bromophenol blue dye, and 100 mM dithiothreitol [DTT]). Equal amounts of extracts (40 µg of protein) were separated with the use of SDS–polyacrylamide gel electrophoresis (PAGE) (12% polyacrylamide gel containing SDS) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 for 1 hour and blotted for 1 hour with the specified primary antibody and then for 1 hour with a horseradish peroxidase-conjugated secondary antibody. The protein bands were detected with the use of a chemiluminescence assay system (Amersham Pharmacia Biotech Inc.) and visualized on Kodak X-Omat LS film. The optical density (OD) of each band was quantified with the use of an image quantitator (Molecular Dynamics, Sunnyvale, CA). The blots were stripped according to the manufacturer's protocol (Amersham Pharmacia Biotech Inc.) and successively reprobed with different antibodies and finally for calreticulin that is present constitutively.

Preparation of nuclear extracts.

The nuclear extracts used for gel mobility shift assays were prepared by the procedure described previously (21). All steps were performed at 4 °C. Briefly, 2 x 107 cells were harvested, washed twice with PBS, and resuspended in 0.2 mL of cell extraction buffer (i.e., 10 mM HEPES–KOH [pH 7.9], 1.5 mM MgCl2, 10 mM KC1, 0.5 mM DTT, 0.2 mM PMSF, and 10 µg/mL aprotinin). The cells were kept on ice for 10 minutes, vortexed for 10 seconds, and centrifuged at 4 °C at 16 000g for 30 seconds. The pellet was resuspended in 20–40 µL of nuclear extraction buffer (i.e., 20 mM HEPES–KOH [pH 7.9], 25% glycerol, 420 mM NaCl, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, and 10 µg/mL aprotinin), placed on ice for 20 minutes, and centrifuged at 4 °C at 16 000g for 2 minutes. The supernatant was saved as the nuclear extract and stored at –80 °C.

Gel mobility shift assay.

The vitamin D response element (VDRE) sequence—oligonucleotide 5'-CTAGTGCTCGGGTAGAGGTCAAGGAGGTCACTCGAC-3'—and its complement were synthesized and annealed, and the double-stranded DR3—the response elements are typically composed of two hexameric half-sites organized as directed repeats (DR), and the binding specificity is provided by the spacing between each half-site; following this rule, the consensus VDRE, which has a half-site spacing of three nucleotides (DR3), is called VDRE-DR3—was 5'-end phosphorylated by T4 polynucleotide kinase (Life Technologies Inc. [GIBCO BRL], Rockville, MD) in the presence of [{gamma}-32P]adenosine triphosphate (Amersham Pharmacia Biotech Inc.). The mutant VDRE-DR3 oligo, 5'-AGCTTCAGaaCAAGGAGaaCAGAGAGC-3' (residues changed in the DR3 sequence in the mutant oligonucleotides are indicated in lower case type; the bold letters are the consensus sequences), was used as a nonspecific competitor in the competition assay. The gel mobility shift assay was carried out with the use of the Gel Shift Assay Kit (Stratagene, La Jolla, CA). Nuclear extract proteins (10 µg) and the labeled VDRE probe (0.8 ng) were incubated for 30 minutes at room temperature; then the samples were separated on a 4% polyacrylamide gel with a constant current of 22 mA for 3 hours. The gel was dried and exposed to Kodak X-Omat LS film. Supershift assays were performed by adding 1 µg of anti-VDR (C-20, rabbit polyclonal antibody at dilution [1 : 25] of stock antibody [1 µg/µL] was used) and 1 µg of anti-RXR-{alpha} (D-20, rabbit polyclonal antibody at dilution [1 : 25] of stock antibody [1 µg/µL] was used) antibodies to the nuclear protein sample and incubating the mixture for 20 minutes at room temperature. The reaction was then continued for 30 minutes. This assay detects supershifted bands when VDR and RXR-{alpha} form a heterodimeric complex.

Reverse transcription–polymerase chain reaction (RT–PCR).

Total RNA was extracted from 5 x 106 cells with the use of the RNeasy Kit (Quiagen, Valencia, CA) according to the manufacturer's recommended procedure. The RNA concentration was determined spectrophotometrically. For RT–PCR, a 20-µL master mix of RT was prepared as follows: 5 mM MgCl2, PCR Buffer II, 1 mM deoxyguanosine triphosphate, 1 mM deoxyadenosine triphosphate, 1 mM deoxycytidine triphosphate, 1 mM deoxythymidine triphosphate, 1 U/µL RNase inhibitor, 2.5 µM random hexamers, a 1-µg sample of RNA, and diethyl pyrocarbonate-treated distilled water. The master mix was incubated in the Perkin-Elmer GeneAmp PCR system 9600 (Roche, Branchburg, NJ) at 42 °C for 15 minutes and then at 99 °C for 5 minutes and at 5 °C for 5 minutes. After RT, 78 µL of PCR master mix containing 2 mM MgCl2, PCR Buffer II, 2.5 U/µL AmpliTaq DNA polymerase, and the following primers was added at a 0.15 µM concentration: EGR-1, upstream primer (5'-AGATGATGCTGCTGAGCAAC-3'), downstream primer (5'-AGTAAATGGGACTGCTGTCG-3'); RXR-{alpha}, upstream primer (5'-TCACCTATGAACCCCGTCAG-3'), downstream primer (5'-TCGACTCCACCTCATTCTCG-3'); VDR, upstream primer (5'-AGGCCTTGAAGGACAGTCTG-3'), downstream primer (5'-GGTCTCTGAATCCTGGTATC-3'); 24-hydroxylase, upstream primer (5'-CCTTGACAAACCAACGGTT-3'), downstream primer (5'-TCCACAGGTTCATTGTCTGT-3'); human osteocalcin (hOC), upstream primer (5'-GAGCCCTCACACTCCTCGCCCTATT-3'), downstream primer (5'-GTAGAAGCGCCGATAGGCCTCCTGA-3'); and {beta}-actin, upstream primer (5'-TGACGGGGTCACCCACACTGTGCCCAGCTA-3'), downstream primer (5'-CTAGAAGCATTTGCCGGTGGACGATGGAGGG 3'). The complementary DNAs (cDNAs) in samples were amplified in the GeneAmpPCR System 9600 as follows: 105 seconds at 95 °C as an initial step, followed by 35 cycles of 15 seconds each at 95 °C and 30 seconds each at 60 °C and finally 7 minutes at 72 °C. The RT–PCR products were separated on 2% agarose gel. The intensity of the bands corresponding to VDR, RXR-{alpha}, and EGR-1 was measured with the use of the Image QuaNT Program (Molecular Dynamics, Sunnyvale, CA).

Statistical methods.

All experiments were repeated at least three times (n = 3–8). Measurement of cell number and viability as well as counting of cells stained for MSE (Table 1Go) in each experiment was carried out in triplicate. The statistical analysis was performed with the GraphPad Prism 3.0 Program (GraphPad Software, San Diego, CA). Data are reported as means ± 95% confidence intervals. Statistically significant differences among the multiple groups (see Figs. 2, 3, 6, and 7GoGoGoGo and Table 1Go) were tested with the use of the nonparametric Kruskal–Wallis one-way analysis-of-variance test, followed by Dunn's adjustment for individual groups versus control. Two compounds (A and B) were considered to show enhancement in the particular experiment if the effect of their combination (AB) was larger than the sum of their individual effects (AB>A + B), the data being compared after subtraction of the respective control values from A, B, and AB. Statistically significant differences between AB and A + B were estimated with the use of the nonparametric Wilcoxon matched pairs test. A P value less than .05 was considered to be statistically significant.


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Table 1. Effects of a combination of carnosic acid and other differentiation agents on monocytic differentiation (expression of monocytic serine esterase), viability, and proliferation of HL60-G cells*
 


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Fig. 2. A) Effect of 10 µM carnosic acid (CA) on expression of CD11b and CD14 differentiation markers in HL60-G cells exposed to various differentiating agents. Cells were treated with indicated compounds, as described in Fig. 1Go, and combinations of agents were used at the same concentrations as were used individually. B) Proportion of cells doubly positive for CD11b and CD14. The means ± 95% confidence intervals of eight separate experiments involving 1 nM 1{alpha},25-dihydroxyvitamin D3 (D3) and 1 nM all-trans-retinoic acid (ATRA) and four experiments involving 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.125% dimethyl sulfoxide (DMSO) are presented. Statistically significant differences from the sum of the individual effects of inducer and CA (see the "Materials and Methods" section): *P<.05 and **P<.001. Control = untreated HL60 cells at 96 hours.

 


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Fig. 3. Effects of 10 µM carnosic acid (CA) on cycle and proliferation of cells exposed to 1 nM 1,25-dihydroxyvitamin D3 (D3) and other differentiating agents. Panel A: cell cycle distribution. Cells were treated with chemical inducers of differentiation in the presence or absence of CA, as described in Figs. 1 and 2GoGo. The means ± 95% confidence intervals of eight separate experiments involving 1 nM D3 and 1 nM all-trans-retinoic acid (ATRA) and three experiments involving 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.125% dimethyl sulfoxide (DMSO) are presented. Combinations of agents were used at the same concentrations as were used individually. Statistically significant differences were observed between treated cells and untreated controls for cell fractions representing G0/G1 and S phases: *, P <.05; **, P <.01; ***, P <.001. Panels B and C: time-dependent inhibition of cell growth (B) and G1–S cell (the cells [%] in G0 and G1 phases combined and called G1 phase) cycle transition (C) by CA–D3 combinations. The means ± 95% confidence intervals of three experiments are shown. There was statistically significant enhancement of the individual effects of inducer and CA by drug combination (see legend to Fig. 2Go): *P<.05; ***P<.001.

 


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Fig. 6. Incrreased expression at transcriptional level of two vitamin D receptor (VDR) target genes by carnosic acid (CA). HL60-G cells were exposed for 96 hours to 10 µM CA and/or differentiating agents, i.e., 1 nM 1{alpha},25-dihydroxyvitamin D3 (D3), 1 nM all-trans-retinoic acid (ATRA), 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), and 0.125% dimethyl sulfoxide (DMSO), at the indicated concentrations; combinations of agents were used at the same concentrations as were used individually; and reverse transcription–polymerase chain reaction was performed as described in the "Materials and Methods" section. The levels of {beta}-actin transcripts were determined in the sample as internal controls. The upper three panels display an experiment representative of four similar determinations, which have been quantitated as shown in the lower two panels (means ± 95% confidence intervals; n = 4). There were statistically significant increases in human osteocalcin (hOC) in cells exposed to CA and/or the differentiating agents as compared with control (untreated HL60 cells at 96 hours); however, although a similar trend can be observed for 24-hydroxylase, the increases were not sufficiently marked to be statistically significant. *P <.05; **P<.01; ***P <.001. OD = optical density.

 


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Fig. 7. Effect of carnosic acid (CA) on transcription of the genes for vitamin D receptor (VDR), retinoid X receptor (RXR)-{alpha}, and early growth response gene (EGR)-1. The four upper panels display a representative reverse transcription–polymerase chain reaction experiment, and quantification (means ± 95% confidence intervals; n = 4) is shown in the lower three panels. There was a statistically significant increase in VDR, RXR-{alpha}, and EGR-1 transcripts in all groups exposed to 10 µM CA alone or together with differentiating agents, i.e., 1 nM 1{alpha},25-dihydroxyvitamin D3 (D3), 1 nM all-trans-retinoic acid (ATRA), 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), and 0.125% dimethyl sulfoxide (DMSO), as compared with control (untreated HL60 cells at 96 hours). *P<.05; **P<.01; ***P<.001. OD = optical density.

 

    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enhancement of HL60-G Cell Differentiation

It has already been reported that the plant antioxidant carnosic acid enhances the effects of 1{alpha},25(OH)2D3 to induce in wild-type HL60 cells the expression of the CD14 marker of monocytic differentiation as well as the oxidative burst activity and expression of chemotactic peptide receptors (16). In this study, we used a subline of HL60 cells, HL60-G cells established in our laboratory (18). Figs. 1 and 2GoGo illustrate that dual labeling of HL60-G cells with antibodies against CD14 and a general myeloid differentiation marker CD11b can demonstrate increased expression of the CD11b following a 96-hour treatment of these cells with either 1{alpha},25(OH)2D3 or ATRA at low concentrations (both at 1 nM), as compared with the control cells (P<.001). The expression of CD14 was increased appreciably in the 1{alpha},25(OH)2D3-treated cells (P<.001), but it was minimal in cells treated with ATRA, a finding consistent with the granulocytic lineage of differentiation induced by ATRA (10). TPA at 1 nM caused only a marginal but statistically not significant increase in the expression of CD11b (P = .074) and did not affect the CD14 expression (Fig. 2Go).



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Fig. 1. Effect of carnosic acid (CA) on differentiation of HL60-G cells by 1,25-dihydroxyvitamin D3 (D3) and all-trans-retinoic acid (ATRA). Cells were incubated for 96 hours with 10 µM CA, 1 nM D3, or 1 nM ATRA alone or with the indicated agent combinations at the same concentrations as were used individually. Control cells were treated with vehicle (0.1% ethanol). Surface markers of differentiation, i.e., CD14 (vertical scale) and CD11b (horizontal scale), were determined by flow cytometry as described in the "Materials and Methods" section. The total percentage of positive cells (CD14—two upper quadrants; CD11b—two right-side quadrants) are indicated within each panel. Data representative of four similar experiments are shown. Control = untreated HL60-G cells at 96 hours. Inset on the right shows the structural formula of CA. PE = phycoerythrin; FITC = fluorescein isothiocyanate.

 
The addition of 10 µM carnosic acid (see inset in Fig. 1Go for structure) to this differentiation system did not produce any substantial effect on the expression of the differentiation markers (i.e., CD14, CD11b, and MSE). However, this polyphenol (carnosic acid) augmented the expression of CD11b when the differentiating agent was either 1{alpha},25(OH)2D3 (P = .008), ATRA (P = .046), or TPA (P = .041) at a concentration of 1 nM (Figs. 1 and 2GoGo). Also, it greatly augmented the effects of 1{alpha},25(OH)2D3 on the induction of CD14 (P = .009), while it had no effect on the induction of this marker in the presence of TPA (P = .125). Surprisingly, carnosic acid caused a small but statistically significant (P = .048) enhancement of CD14 expression induced by ATRA (Figs. 1 and 2GoGo). A low concentration of DMSO (0.125%) had no effect on the expression of either CD11b or CD14 (Fig. 2Go). When carnosic acid was added along with DMSO, a slight but statistically not significant increase in the expression of CD11b was observed (P = .375), while the expression of CD14 was not affected.

Preferential Increase of Monocyte-Specific Markers by Carnosic Acid in Differentiating HL60-G Cells

The monocytic phenotype is characterized by the high expression of the CD14 marker (22), as exemplified by cells exposed to 1{alpha},25(OH)2D3 (Figs. 1 and 2GoGo) and by the expression of the cytoplasmic enzyme MSE (23). In view of the well-established consensus that ATRA induces granulocytic lineage of differentiation in HL60 cells (10), the unexpected synergistic increase in CD14 expression by the carnosic acid–ATRA combination (Figs. 1 and 2GoGo) prompted us to further analyze this system. The results showed that there was a marked increase in cells doubly positive for CD14 and CD11b (Fig. 2Go, B) when carnosic acid was added along with either 1{alpha},25(OH)2D3 (P<.001) or ATRA (P = .012) and in cells positive for MSE (P<.001 for both 1{alpha},25(OH)2D3 and ATRA, Table 1Go column A). A smaller increase in the CD14/CD11b- positive (P = .039; Fig. 2Go, B) and the MSE-positive (P = .043; Table 1Go column A) cells by the carnosic acid–TPA combination was noted, whereas no augmentation by the polyphenol was seen in the DMSO-treated cells (Fig. 2Go, B; Table 1Go column A). These data indicate that carnosic acid promotes the monocytic phenotype when it is combined with some, although not all, inducers of monocytic differentiation.

Effects of Carnosic Acid on Survival, Proliferation, and Cell Cycle Traverse of Differentiating Cells

Carnosic acid at a concentration of 10 µM, either alone or in combination with other differentiating agents, had no detectable cytotoxicity, as determined by microscopic examination of the nuclear morphology of the stained cells (data not shown) or exclusion of the trypan blue dye (Table 1Go column B). When added to HL60-G cells for 96 hours, carnosic acid alone only slightly affected cell proliferation (Table 1Go column C) compared with untreated control cells, but it statistically significantly reduced the rate of cell growth when it was combined with 1{alpha},25(OH)2D3 (P<.001), ATRA (P<.001), or TPA (P = .008). However, the enhancing effect of carnosic acid was statistically significant only when it was added together with 1 nM 1{alpha},25(OH)2D3 (P = .015) or 1 nM ATRA (P = .025) (Table 1Go column C). In view of the reduced rate of proliferation resulting from the exposure to carnosic acid together with differentiation agents, we examined the parameters of the cell cycle under these conditions. Small but statistically significant blocks of the G1- to S-phase transition manifested by the increase in the G0/G1 and a decrease in the S-phase cell populations were produced by both 1{alpha},25(OH)2D3 (P = .021 and P = .017 for G0/G1 and S, respectively) and ATRA (P = .019 and P = .026 for G0/G1 and S, respectively), whereas TPA and DMSO were without statistically significant effects at the concentrations used here (Fig. 3Go, A). It is interesting that, while it had no effect on the cell cycle progression when added alone, carnosic acid enhanced this increase in the G0/G1 phase (P = .004) and a decrease in the S phase (P = .027) induced by 1{alpha},25(OH)2D3 (Fig. 3Go, A). In contrast, carnosic acid did not statistically significantly alter the effects of ATRA (P = .074 and P = .136 for G0/G1 and S phases, respectively) or TPA (P = .215 and P = .236 for G0/G1 and S phases, respectively) and had no enhancing effect when it was combined with DMSO (P = .455 and P = .332 for G0/G1 and S phases, respectively) in these experiments (Fig. 3Go, A).

Further examination revealed that growth inhibition induced by the combination of carnosic acid and 1{alpha},25(OH)2D3 increased considerably with time, leading to a complete growth arrest after 168 hours of treatment (P<.001), without a statistically significant decrease in cell viability (mean [95% confidence intervals] for treated = 96.0% [93.4% to 98.6%] versus control = 98.1% [96.8% to 99.4%] at 168 hours; n = 3) (Fig. 3Go, B). This effect was accompanied by an increasingly higher G1/S ratio (Fig. 3Go, C), signifying a marked G1- to S-phase block (P<.001 at 168 hours). (For Fig. 3Go, B, = .032 and P = .018 at 96 and 120 hours, respectively; for Fig. 3Go, C, P = .012 and P<.001 at 96 and 120 hours, respectively.).

Consistent with the G1 block, the cyclin-dependent kinase inhibitor p27/Kip1, which controls the G1- to S-phase transition in 1{alpha},25(OH)2D3-treated HL60 cells (24,25), showed markedly elevated levels in cells treated with the carnosic acid–1{alpha},25(OH)2D3 combination. However, in cells treated with the carnosic acid–ATRA, the carnosic acid–TPA, or the carnosic acid–DMSO combinations, the changes in the p27/Kip1 levels were less marked or were not detectable, as shown by representative immunoblots (Fig. 4Go, A).



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Fig. 4. Immunoblot analysis of the effects of differentiating agents on HL60-G cells in the absence or presence of carnosic acid (CA), as described in Fig. 2Go. Combinations of agents were used at the same concentrations as were used individually. Calreticulin protein levels are displayed to demonstrate similar loading and transfer of proteins to the membrane. A) Levels in total cell extracts of three regulators of the cell traverse are enhanced by CA when the cells are treated with 1 nM 1{alpha},25-dihydroxyvitamin D3 (D3) or 1 nM all-trans-retinoic acid (ATRA) but not when treated with 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.125% dimethyl sulfoxide (DMSO). B) Levels of the receptors vitamin D receptor (VDR) and retinoid X receptor (RXR)-{alpha} in the total cell extract are increased by CA and enhanced by CA with D3 or ATRA. This enhancement is not evident with DMSO and is marginal with TPA. CA does not detectably increase the levels of retinoic acid receptor (RAR)-{alpha} but does enhance the effect of D3, ATRA, or TPA to increase the levels of this nuclear receptor. C) Total cell extract content of products of genes associated with monocytic lineage of differentiation. CA induces increases in the levels of early growth response gene (EGR)-1, p35Ncka5 (p35), and cyclin-dependent kinase (Cdk)-5 and enhances the effects of D3, of ATRA, and to a lesser extent of TPA. Enhancement by CA of the effect of DMSO is marginal for p35Nck5a, Cdk5, and EGR-1. Data representative of three similar experiments are shown. Control = untreated HL60 cells at 96 hours.

 
Further analysis of the G1 block was performed by the determination of the steady-state levels of the G1 cyclins, cyclin D1 and cyclin E. Previous study of this system showed that, in HL60 cells, as in other systems (26), cyclin D1 is maximally expressed in mid-G1, while cyclin E is maximally expressed at the G1/S boundary (Chen F, Studzinski GP: unpublished data). In the present experiments, the levels of cyclin D1 and cyclin E in cells treated with 1{alpha},25(OH)2D3 paralleled those of p27/Kip1 and the degree of G1 block (Fig. 4Go, A). These data suggest that the cell cycle block induced by the combination of 1{alpha},25(OH)2D3 and carnosic acid occurs in mid- to late-G1 phase rather than in G0 phase. Furthermore, increases in the G1 cyclins in cells treated with the carnosic acid–ATRA combination suggest that there is a partial G1 block in these cells, even though it was not statistically significant when cell cycle parameters were determined by flow cytometry. TPA alone also increased the expression of G1 cyclins, but no augmentation by carnosic acid in these or DMSO-treated cells was observed, as shown by representative immunoblots (Fig. 4Go, A).

Carnosic Acid-Induced Increases in Levels of Functional Nuclear Receptors VDR and RXR-{alpha} and of Monocyte-Specific Proteins

The nuclear receptors VDR and RXR-{alpha} heterodimerize to form a transcription factor that binds to the promoters of 1{alpha},25(OH)2D3-regulated genes, while RARs heterodimerize with RXRs to control ATRA-responsive genes (12). The heterodimers have low or absent transcription-activating activities that are increased by their ligands 1{alpha},25(OH)2D3 or ATRA, respectively. We therefore determined the protein abundance of VDR, RXR-{alpha}, and RAR-{alpha} in HL60-G cells. We found that carnosic acid increased the levels of VDR and RXR-{alpha} and increased the levels of VDR, RXR-{alpha}, and RAR-{alpha} further in the presence of 1{alpha},25(OH)2D3 and ATRA (Fig. 4Go, B). When added with carnosic acid, TPA, but not DMSO, also markedly increased the levels of VDR and RAR-{alpha}. RXR-{alpha} levels were increased by TPA and DMSO; however, in those cases, no enhancement was detectable in the presence of carnosic acid when added alone (Fig. 4Go, B).

Evaluation of the presence of active receptor complexes in nuclear extracts showed that carnosic acid treatment of HL60-G cells slightly increased the binding of active complexes to VDRE and potentiated the effects of 1{alpha},25(OH)2D3 and, to a lesser extent, the effects of ATRA when it was used (Fig. 5Go). This observation suggested that the increased levels of the VDRE-binding proteins VDR and RXR-{alpha} (Fig. 4Go, B) had functional significance and were examined at the level of expression of VDRE-activated genes, the 25-dihydroxyvitamin D3 24-hydroxylase (27), and the hOC gene (28). As shown in Fig. 6Go, the modest stimulation of 24-hydroxylase expression by carnosic acid was not statistically significant (P = .155), but carnosic acid did elevate the expression of hOC (P<.001). These disparate results on the activation of two VDRE-activated genes may be due to the lower sensitivity of the RT–PCR procedure used here to detect changes in 24-hydroxylase messenger RNA (mRNA) levels. Alternatively, it is possible that carnosic acid alters the composition or configuration of transcription factors or cofactors on the VDRE in a manner that depends on the context of surrounding sequences, resulting in gene-specific activation.



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Fig. 5. Electrophoretic mobility shift analysis of the effect of carnosic acid (CA) on the binding of the nuclear receptor vitamin D receptor (VDR) to its cognate DNA element vitamin D response element (VDRE). A) The intensity of the specific (VDR–retinoic X receptor (RXR)-{alpha}) binding to VDRE is evident at the 10 µM concentration of CA (lane 4) or at the 1 nM (lane 5) or 100 nM (lane 7) concentration of 1{alpha},25-dihydroxyvitamin D3 (D3) and shows enhancement by CA (10 µM) of the effects of D3 (1 nM, lane 6) or of all-trans-retinoic acid (ATRA) (1 nM, lane 9). Lane 10 (D3, 100 nM) shows that the unlabeled probe competes the binding of the complex, while the unlabeled mutant (as described in the "Materials and Methods" section) does not (100 nM D3, lane 11). B) In a parallel experiment, the nuclear extract was preincubated with antibodies to VDR and RXR-{alpha} and then subjected to electrophoretic mobility shift analysis, indicating the specific nature of the upper band in panel A. Data representative of three similar experiments are shown. Control = untreated HL60 cells at 96 hours. NS or ns = nonspecific band; Abs = antibodies.

 
In view of the marked enhancement of monocytic surface markers by carnosic acid in 1{alpha},25(OH)2D3- or ATRA-treated cells, we investigated if products of genes reported to be selectively activated in monocytic differentiation could also be detected. Fig. 4Go, C, indicates that EGR-1, the expression of which is reported to redirect the development of hematopoietic progenitor cells along the monocyte–macrophage lineage (29,30), and the p35 neuronal Cdk5 activator (p35Nck5a), also specifically associated with the monocytic lineage in hematopoietic cells (31,32), showed increased expression after exposure to carnosic acid (as well as to TPA, as expected in macrophage lineage differentiation) and that carnosic acid enhanced the effects of 1{alpha},25(OH)2D3 or ATRA.

Transcriptional Regulation of VDR, RXR-{alpha}, and EGR-1 Abundance by Carnosic Acid

Determination of specific mRNA levels in cell extracts showed that treatment with carnosic acid alone resulted in a statistically significant increase in the expression of VDR (P = .007), RXR-{alpha} (P = .011), and EGR-1 (P = .009) genes (Fig. 7Go), indicating that the increased expression of these genes by carnosic acid occurs at least in part at the transcriptional level. DMSO and 1{alpha},25(OH)2D3 also statistically significantly increased the transcription of VDR (P = .021) and RXR-{alpha} (P = .032), respectively, whereas all of the differentiation-inducing agents induced increased expression of the EGR-1 gene (P = .011 to P = .018) (Fig. 7Go), as was reported previously for the increased expression of EGR-1 by TPA (29). Statistical analysis revealed an enhancement by carnosic acid of the 1{alpha},25(OH)2D3-induced increase in the expression of VDR (P = .034). In addition, carnosic acid produced additive effects with DMSO on VDR expression (P = .097) and with 1{alpha},25(OH)2D3 on EGR-1 expression (P = .076), which were not statistically significant (Fig. 7Go). However, in general, the cooperation of carnosic acid with differentiation inducers at the mRNA level was less marked than at the protein level, suggesting both transcriptional and post-transcriptional controls.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our results provide potential mechanisms for the enhancement by carnosic acid of 1{alpha},25(OH)2D3- or ATRA-induced monocytic differentiation of HL60-G cells. First, carnosic acid increases the expression of VDR and RXR-{alpha}. The heterodimers of VDR–RXR-{alpha} thus formed can respond with increased sensitivity to 1{alpha},25(OH)2D3, while RAR-{alpha}–RXR-{alpha} heterodimers can activate ATRA-responsive genes when ATRA is added instead of 1{alpha},25(OH)2D3. The rationale for this suggestion is based on the previous findings that the increased expression of VDR in various cell types, as for instance by transfection of a VDR cDNA expression vector, increases the sensitivity of the cells to 1{alpha},25(OH)2D3 (33). Second, the increased expression of the EGR-1 gene product may, as reported (29,30), direct the differentiation of ATRA-treated HL60 cells toward the monocytic–macrophage lineage and further enhance the 1{alpha},25(OH)2D3-induced monocytic differentiation. Third, the activation of Cdk5 by p35Nck5a, the protein levels of which are also increased by carnosic acid (Fig. 4Go), also favors the development of the monocytic phenotype, since it has been shown previously that the inhibition of the Cdk5 activity in HL60 cells induced to monocytic differentiation by 1{alpha},25(OH)2D3 diverts the phenotype toward the immature granulocyte (29). Considered together, these results suggest that carnosic acid induces a program of gene expression that is characteristic of the monocytic phenotype. Furthermore, this monocytic phenotype appears to be dominant over the granulocytic phenotype in the presence of carnosic acid.

It was reported some time ago that HL60/MR1 cells, a subline of HL60 cells, derived from a transplantable HL60 tumor established in athymic nude mice, differentiate to monocytic rather than to granulocytic cells when they are cultured with ATRA in vitro (34). It is interesting that the cells from this xenotransplant-derived HL60 subline also showed other properties observed by us in carnosic acid-treated HL60 cells; the cells had markedly enhanced sensitivity to ATRA, and the differentiation pathways induced by TPA and DMSO were the same as those in the parental HL60 cells. More recently, M2 leukemic blast cells were found to undergo monocytic differentiation when they were treated with ATRA, which induces functionally active VDR in these cells (35). Thus, it appears that ATRA-induced granulocytic differentiation involves signaling circuits similar to those that are active in monocytic differentiation; however, in ATRA-treated HL60 cells, there is a lack of an additional input required for the monocytic form of differentiation. This input may be EGR-1, Cdk5 activated by p35Nck5a, or both of these, mediated by VDR–RXR-{alpha}-induced transcription. Taken together, our data support the notion that granulocytic phenotype results from a default pathway in myeloid lineage differentiation.

The effects of carnosic acid on differentiation induced by low concentrations of TPA or DMSO were less striking. An increase in differentiation resulting from the combination of TPA with carnosic acid was noted by the determination of MSE and CD11b markers, and this increase was statistically significant but was not apparent when CD14 was used as the marker. Conversely, a combination of carnosic acid and DMSO at several concentrations was less than additive (Table 1Go, A; Fig. 2Go; and data not shown). Consistent with this finding, there was no detectable enhancement by carnosic acid of VDR or RXR-{alpha} protein levels induced by DMSO. The enhancement by carnosic acid of a TPA-induced increase in RAR-{alpha} expression was marked, but at present its implications are unclear. In view of the low potential of TPA or DMSO for human use, this situation was not analyzed further; however, it seems that, while differentiation induced by the physiologically occurring compounds 1{alpha},25(OH)2D3 and ATRA is markedly promoted by carnosic acid, differentiation induced by exogenous xenobiotics is at best marginal.

The data also suggest that carnosic acid enhances 1{alpha},25(OH)2D3-induced and probably also ATRA-induced mid- to late-G1-phase cell cycle arrest. The support for this hypothesis is derived from the observation that cyclin D1 levels and cyclin E levels parallel the levels of p27Kip1 (Fig. 4Go, A), which have been shown previously to be regulating the G1-phase arrest in this system (24,25), and from the knowledge that the maximal expression of cyclin D1 is in mid-G1, while the maximal expression of cyclin E is at the G1/S-phase boundary (26). The increased levels of cyclin D1 in cells that are arresting in G1 phase (Fig. 3Go) can be explained by a progressive accumulation of cells at the point of G1, probably just prior to or at the R point (36), where they have maximal expression of cyclin D1. It appears that carnosic acid induces such an arrest in this window of the G1 phase and does not interfere with the normal expression of cyclin D1 or cyclin E. It is also possible that some or all of the other increases in expression in proteins studied here become elevated as a result of the same mechanism, i.e., arrest in mid- to late-G1 phase, already shown to exhibit maximal sensitivity to the differentiation-inducing activity of 1{alpha},25(OH)2D3 (37).

The fulfillment of the promise for the use of 1{alpha},25(OH)2D3 as an agent for differentiation therapy for leukemia is blocked currently by the hypercalcemia attendant on its use in vivo (11). The approaches employed to overcome this limitation have included the use of analogues of 1{alpha},25(OH)2D3 (3840), also referred to as "deltanoids" [e.g., (41)], or combinations of these deltanoid analogues with chemicals or cytokines to enhance differentiation [e.g., (13)]. For instance, the inhibitors of p38MAP kinase SB203580 and SB202190 were found to markedly potentiate differentiation of HL60 induced by low concentrations of 1{alpha},25(OH)2D3 (42). However, the potential of carnosic acid as a component of differentiation therapy regimen appears to be greater than that of other chemicals because of its anticipated safety and its great potency in enhancing the pro-differentiation of effects of low concentrations of the naturally occurring vitamin/hormone 1{alpha},25(OH)2D3 and the regulator of embryonic development ATRA.

In this study, we have focused on the mechanistic aspects of the action of carnosic acid that can identify the effectors of the expression of the monocytic phenotype induced by carnosic acid in combination with natural hormones/morphogens. Whether antioxidant properties of carnosic acid have any relevance to the increased expression of 1{alpha},25(OH)2D3-responsive genes and the enhancement of the monocytic phenotype is an open question, and the upstream regulators of the carnosic acid-induced increased abundance of the nuclear receptors VDR and RXR-{alpha}, the transcription factor EGR-1, or the Cdk5/p35 complex remain to be identified in future studies. However, the identification of these downstream regulators of monocytic differentiation, with its attendant G1 block, should provide new approaches to combined chemoprevention or differentiation therapy for myeloid leukemia.


    NOTES
 
At the time of this study, M. Danilenko was on leave from the Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel, which is now his present address.

Supported by Public Health Service grant CA44722 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

We thank Dr. Yan Ji (New Jersey Medical School, Newark) for valuable help with reverse transcription–polymerase chain reaction experiments. We also thank Dr. J. Levy and Dr. Y. Shavoni (Ben-Gurion University) for their support.


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 Materials and Methods
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 Discussion
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Manuscript received December 27, 2000; revised June 12, 2001; accepted June 22, 2001.


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