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[Cancer Research 63, 1325-1332, March 15, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Carnosic Acid Potentiates the Antioxidant and Prodifferentiation Effects of 1{alpha},25-Dihydroxyvitamin D3 in Leukemia Cells but Does Not Promote Elevation of Basal Levels of Intracellular Calcium1

Michael Danilenko, Qing Wang, Xuening Wang, Joseph Levy, Yoav Sharoni and George P. Studzinski2

Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel [M. D., J. L., Y. S.], and Department of Pathology and Laboratory Medicine, UMD–New Jersey Medical School, Newark, New Jersey 07103 [Q. W., X. W., G. P. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiation therapy of cancer remains an only partially attained goal. Agents currently under active investigation include derivatives of vitamin D, modeled on its physiological hormone form, 1{alpha},25-dihydroxyvitamin D3 (1,25D3), but the calcemic effects of these compounds preclude their use in the clinic. An approach that may obviate this problem is to combine 1,25D3 or its derivatives with other agents that increase the antineoplastic effects of low, nontoxic concentrations of vitamin D compounds. We have recently used the plant-derived polyphenolic antioxidant, carnosic acid (CA), to demonstrate an increase in the differentiating action of 1,25D3 on human leukemia cells under these conditions (M. Danilenko et al., JNCI, 93: 1224–1233, 2001). We now show that treatment of HL60-G cells with either CA or 1,25D3 alone resulted in a decrease in the intracellular levels of reactive oxygen species. Furthermore, the combination of 10 µM CA and a low concentration of 1,25D3 (1 nM) produced an enhanced antioxidant effect, which correlated with the potentiation of monocytic differentiation. Other plant antioxidants tested (curcumin, silibinin, and the organoselenium antioxidant ebselen) also potentiated differentiation induced by 1,25D3, although alone, they had only minor differentiating effects. Differentiation induced by CA/1,25D3 combinations was associated with increased intracellular glutathione content, whereas buthionine sulfoxime decreased both differentiation and the cellular glutathione content. This combination also enhanced the activation of the Raf-mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase mitogen-activated protein kinase module and increased the binding of the activator protein-1 (AP-1) transcription factor to its cognate DNA element in the promoter regions of vitamin D receptor gene, suggesting that the mechanism of potentiation is at least in part attributable to induction and activation of components of this mitogen-activated protein kinase pathway. Cell treatment with a high concentration of 1,25D3 (100 nM) resulted in a substantial elevation of basal intracellular calcium concentration. In contrast, importantly for an eventual clinical application of these studies, the potentiating action of CA on differentiation induced by a low concentration of 1,25D3 (1 nM) was not accompanied by an elevation of basal intracellular calcium concentration. These findings suggest that combinations of CA with derivatives of vitamin D should be evaluated for use in differentiation therapy of myeloid leukemias.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The goal of differentiation therapy of cancer is to arrest the growth of malignant cells by inducing normalization of cellular phenotypes without damage to normal tissues. A notable example of the success of this approach is provided by the use of vitamin A derivatives, which are in varying stages of development for treatment of malignant diseases, e.g., ATRA3 is particularly effective in the treatment of acute promyelocytic leukemia (reviewed in Refs. 1 and 2 ). Similarly, 1,25D3 and its derivatives are currently under investigation as differentiating agents in a variety of tumor types and seem especially suited for clinical applications: (a) 1,25D3 is a human hormone with a known physiological function in the control of calcium homeostasis; (b) there is substantial epidemiological evidence that levels of circulating 1,25D3 near the top of the physiological range play a part in the reduction of incidence of the common human cancers that affect the female breast, prostate, and colon (3) ; and (c) differentiation and apoptosis-inducing effects of 1,25D3 have been demonstrated in neoplastic cells established in culture from these and other tissues (reviewed in Ref. 4 ), showing that under appropriate conditions, 1,25D3 can indeed control the growth of these cells.

The well-known limitation to the therapeutic use of 1,25D3 is its hypercalcemic effect. When the hypercalcemia is sufficiently prolonged and severe, widespread calcifications take place in tissues. Current attempts to overcome this problem focus on the synthesis of analogues of 1,25D3, which retain the prodifferentiation activities but have lower calcemic effects (e.g., Ref. 5 ). However, although various Phase I/II trials have been conducted (e.g., Refs. 6, 7, 8, 9, 10 ), these vitamin D analogues have as yet not been successfully used for the treatment of cancer, including myeloid leukemias. An alternative approach is to combine nonhypercalcemic concentrations of 1,25D3 or its analogues with compounds that have different mechanisms of action, e.g., increased antitumor activity has been reported when dexamethasone or cytotoxic agents, such as paclitaxel, were combined with 1,25D3 (11, 12, 13) , whereas several plant-derived antioxidant compounds, such as polyphenols curcumin (14 , 15) and silibinin (16) , and carotenoids lycopene and ß-carotene (17) were found to potentiate the differentiating and antiproliferative actions of 1,25D3 on leukemic cell lines.

We have recently reported a marked potentiation of the 1,25D3-induced differentiation of HL60 human myeloid leukemia cells by CA, a polyphenol derived from the plant rosemary (Rosmarinus officinalis; Refs. 18 and 19 ). We also found that this potentiation relates to the ability of CA to enhance a program of gene expression consistent with monocytic differentiation (19) . In the present study, we investigated the mechanism of the synergy between 1,25D3 and CA and found that 1,25D3 and CA cooperatively decreased the levels of ROS in this system, increased total cellular glutathione content, activated the Raf/MEK/ERK MAPK module, and enhanced the binding of AP-1 to its DNA response element. Furthermore, we demonstrated that, in contrast to induction of differentiation with a high concentration of 1,25D3 (100 nM), which had a differentiating effect accompanying a considerable elevation of basal intracellular calcium levels in HL60 cells, the combination of a low (1 nM) 1,25D3 concentration with CA only moderately affected the basal [Ca2+]i.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies.
CA was obtained from Alexis Biochemicals (Läufenfingen, Switzerland). Curcumin, ebselen, silibinin, L-buthionine, BSO, fMLP, DCFH-DA, TPA, GSH, glutathione reductase from bakers yeast, 5,5'-dithiobis(2-nitrobenzoic acid), 5-sulfosalicylic acid, and NADPH were purchased from Sigma (St. Louis, MO). Complete protease inhibitor cocktail was from Roche Molecular Biochemicals (Mannheim, Germany). Poly [d(I-C)] was from Roche Diagnostics (Mannheim, Germany). NP40 was from Calbiochem-Novabiochem Corp. (San Diego, CA). 1,25D3 was a gift from Dr. Milan Uskokovic (BioXell, Nutley, NJ). [{gamma}-32P]ATP was purchased from NEN Life Science Products, Inc. (Boston, MA). The antibodies against Raf-1 (c-12, rabbit polyclonal), MEK-1 (c-18, rabbit polyclonal), ERK1/2 (k-23, rabbit polyclonal), and p90RSK (c-21, rabbit polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies used to detect phospho-Raf (Ser 259), phospho-MEK (Ser 217/221), phospho-ERK (Thr 202/Tyr 204), and phospho-p90RSK (Ser 381), all rabbit polyclonal antibodies, were purchased from Cell Signaling Technology (Beverly, MA). Anticalreticulin antibody was purchased from Affinity Bioreagents (Golden, CO). Horseradish peroxidase-conjugated antirabbit IgG and antimouse IgG were obtained from Santa Cruz Biotechnology. Anti-CD14 (MY4-RD-1) and anti-CD11b (MO1-FITC) antibodies were obtained from Coulter Corp. (Brea, CA). Stock solutions of CA, curcumin, silibinin, ebselen (10 mM each), and 1,25D3 (0.25 mM) were prepared in absolute ethanol.

Cell Culture and Proliferation Assay.
HL60-G cells (20) , a subclone of human promyeloblastic leukemia HL60 cells (21) , were routinely cultured at 37°C in RPMI 1640 (Mediatech, Washington, D.C., or Biological Industries, Beth Haemek, Israel), supplemented with 10% heat-inactivated, iron-enriched bovine calf serum (HyClone, Logan, UT). Cell culture was passaged two to three times weekly to maintain a log phase growth. Cells were seeded into fresh culture medium at 0.5–1 x 105 cells/ml in 25 cm2 tissue culture flasks and incubated with test agents for 24–96 h. To demonstrate the enhancement of differentiation and growth inhibition induced by 1,25D3, cells were treated with a low concentration of this inducer (1 nM) in the presence of other agents, whereas 100 nM 1,25D3 was used to illustrate the maximal effect. Cell growth was estimated by counting cells with a Coulter Counter after dilution in Isoton-II (Coulter Electronics, Hialeah, FL). Cell viability was determined using trypan blue (0.25%) exclusion. To determine the maximal subtoxic concentration of each antioxidant, the cells were cultured for 48 h in the presence of graded concentrations of the compound, and cell viability was measured as described above.

Determination of Markers of Differentiation.
Aliquots of 1 x 106 cells were harvested, washed twice with PBS, and suspended in 10 µl of PBS. The cell suspensions were incubated for 45 min at room temperature with 0.5 µl of MY4-RD-1 and 0.5 µl of MO1-FITC (1:20 dilution of the stock antibodies) to analyze the expression of surface cell markers CD14 and CD11b, respectively (19) . The cells were then washed three times with ice-cold PBS and resuspended in 1 ml of PBS. Two-parameter analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Isotypic mouse IgG1 was used to set threshold parameters.

Cell Extracts.
All procedures were carried out at 4°C. Cells (1–2 x 107) were harvested and washed twice with ice-cold PBS. Whole-cell extracts were prepared essentially as described previously (19) . Washed cell pellets were solubilized with a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Equal amounts of 3 x SDS sample buffer containing 150 mM Tris-HCl (pH 6.8), 30% glycerol, 3% SDS, 1.5 mg/ml bromphenol blue dye, and 100 mM DTT were then added to each sample. Nuclear extracts were prepared by the procedure described before (19 , 22) with minor modifications. Briefly, cell pellets were resuspended in 0.5 ml of ice-cold hypotonic buffer [10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and Complete protease inhibitor cocktail]. The cells were kept on ice for 10 min to allow them to swell, vortexed for 10 s, and centrifuged at 16,000 x g for 30 s. Supernatant was discarded, and the pellet was resuspended in 50 µl of nuclear extraction buffer [20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and Complete protease inhibitor cocktail], placed on ice for 20 min, and centrifuged at 16,000 x g for 15 min. The supernatant was saved as the nuclear extract and stored at -80°C.

Western Blotting.
Equal amounts of whole-cell extracts (40 µg of protein) were separated on 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were blocked with 5% milk in Tris-buffered saline/0.1% Tween 20 for 1 h, subsequently blotted with primary antibodies, and then the membranes were blotted with a horseradish-linked secondary antibody for 1 h. The protein bands were visualized with a chemiluminescence assay system (Amersham). The protein loading of the gel and efficiency of the transfer were controlled by stripping the membrane and reprobing for calreticulin, a constitutively expressed protein in HL60 cells. The absorbance of each band was quantitated using an image quantitator (Molecular Dynamics, Sunnyvale, CA).

Electrophoretic Mobility Shift Assay.
AP-1 binding to its cognate DNA element (TPA-response element) in a 1,25D3-responsive gene, hVDR, was evaluated as described previously (23) with the following modifications. Double-stranded oligonucleotides from promoter regions of hVDR containing the proximal (-77 to -97 relative to the transcription start site) binding site for AP-1 (5'-CTGGCAAGAGAGGACTGGACC-3' hVDR-AP-1#1), mutated AP-1 (5'-CTGGCAAGAGAGtgCTGGACC-3'), and the distal (-1023 to -1043) hVDR-AP-1#2 (5'GATTAGCTGAGTCATGTTGG-3') were synthesized by the Molecular Resource Facility of the New Jersey Medical School. The reference sequence accession number for hVDR in the GenBank of National Center for Biotechnology is AB002157. Nuclear extracts (10 µg of protein) were preincubated with 0.02 A260 units of poly [d(I-C)] for 15 min on ice in buffer containing 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM EDTA, 100 mM KCl, 2 mM DTT, 20% (volume for volume) glycerol, and 0.1% (volume for volume) NP40. The extracts were then incubated for an additional 30 min at room temperature with 50–60 pg (50,000–75,000 cpm) of 32P-labeled, double-stranded oligonucleotide. Specificity of the AP-1 binding was estimated by competition with a 10x molar excess of the unlabeled double-stranded "self" nucleotide (see above) or unrelated double-stranded nucleotide containing the Sp1 response element (5'-ATTCGATCGGGGCGGGGCGAGC-3') added to parallel samples during the preincubation period. To examine the effects of oxidation of nuclear proteins on their AP-1 DNA-binding activity, samples of nuclear extracts were preincubated with the oxidizing agent diamide (20 mM). The complexes were separated on 4% polyacrylamide gel under nondenaturing conditions with a constant current of 20–25 mA, for 3–4 h at 4°C. The gel was dried and exposed overnight to Kodak X-Omat LS film.

Measurement of Intracellular Peroxides by Flow Cytometry.
The intracellular peroxide levels were determined using the oxidation-sensitive fluorescent probe DCFH-DA (24 , 25) . Intracellular peroxides oxidize this probe to a highly fluorescent compound, DCF. Cells (1–2 x 106/ml) were harvested at the time points indicated and washed with HEPES-buffered HBSS. Cells were then loaded with 5 µM DCFH-DA for 15 min with horizontal agitation in a shaking water bath at 37°C. In some experiments, after loading with DCFH-DA, cells were washed with PBS and incubated for an additional 30 min with 0.01–1 mM H2O2 under the same conditions. The fluorescence intensity was measured with a FACSCalibur flow cytometer (Becton Dickinson). For each analysis, 10,000 events were recorded.

Superoxide Anion Measurement.
The production of superoxide (O2-) was measured in a 96-multiwell format by the superoxide dismutase-inhibitable reduction of cytochrome c as described previously (18) . Cells were washed in HBSS and suspended (2.5 x 105 cells/well) in 100 µl of HBSS 150 µM cytochrome c. Cells were stimulated by the addition of either TPA (100 nM) or fMLP (10 µM), and the reduction of cytochrome c was monitored at 550 nm (650-nm reference wavelength) for 30–40 min at 37°C in a VERSAmax microplate spectrophotometer (Molecular Devices, Menlo Park, CA). The maximal rates of superoxide generation were determined and expressed as nmol O2-/106 cells/min using extinction coefficient E550 = 21 mM-1/cm-1.

Assay for Glutathione.
Cells (2 x 106) were collected by centrifugation (1,000 x g for 5 min), washed with ice-cold PBS, and resuspended in 200 µl of 5% 5-sulfosalicylic acid. After 15 min on ice with intermittent vortexing, the suspension was centrifuged at 16,000 x g for 5 min to remove protein precipitates. Total glutathione was determined in the supernatants by the glutathione reductase recycling assay as described by Griffith (26) with minor modifications (27) .

Determination of [Ca2+]i.
Calcium assay was performed as described previously (28) with minor modifications. Briefly, after a 96-h incubation, cells were harvested, washed with PBS, and resuspended in HBSS containing 10 mM HEPES and 1 mg/ml BSA. Cells were incubated with 2 µM fura-2 a.m. (Molecular Probes, Inc., Eugene, OR) for 30 min at 25°C. Before [Ca2+]i measurements, 0.5–1 x 106 cells were aliquoted to microfuge tubes, centrifuged for 5 s, resuspended in 100 µl of HEPES and 1 mg/ml BSA, and injected into cuvettes containing 1.9 ml of the same solution. Data were collected at 2-s intervals at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Fluorescence was monitored at 37°C with constant stirring in a Perkin-Elmer Model 50-LS fluorescence spectrophotometer. Basal [Ca2+]i and the peak Ca2+i response of cells stimulated with 10 µM fMLP were monitored. Calibration of the signal was achieved by exposing cells to 0.1% Triton X-100 in the presence of saturating Ca2+ or HBSS containing 15 mM EGTA (pH 8.0). Autofluorescence of solutions, drugs, and fura-2-free-treated cells was subtracted from the fluorescence spectra before [Ca2+]i calculations (28 , 29) .

Statistical Analysis.
All experiments were repeated at least three times. The significance of the differences between the means of the various subgroups was assessed by two-tailed Student’s t test. The computations were performed with an IBM-compatible personal computer using Microsoft EXCEL and GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) programs.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CA and 1,25D3 Cooperate As Antioxidants in HL60-G Cells.
CA and related polyphenols have been known to act as both antioxidant and pro-oxidant in different biological systems (30, 31, 32) . Therefore, we set out to investigate whether the potentiating effect of this polyphenol on cell differentiation (18 , 19) is accompanied by any detectable changes in the levels of ROS in HL60-G cells. For these experiments, the cells were incubated with 0, 5, and 10 µM CA alone for 96 h, followed by measurements of the levels of intracellular peroxides by flow cytometry using the fluorescent oxidation-sensitive probe DCFH. As shown in Fig. 1ACitation , CA produced a concentration-dependent decrease in the intracellular levels of ROS, as compared with untreated control cells. Interestingly, 1,25D3 alone induced an even greater decrease in ROS levels (Fig. 1BCitation ; see also Table 1Citation ), and cell treatment with a combination of 10 µM CA with 1 nM 1,25D3 resulted in a cooperative effect in at least a large part of the cell population (Fig. 1C)Citation . The data summarized in Table 1Citation indicate that although the combined antioxidant effect of CA + 1,25D3 is bimodal (Fig. 1C)Citation , its overall magnitude is similar to that obtained with 100 nM 1,25D3 alone. Concurrently, a similar extent of cell differentiation was observed under these treatments (Fig. 7, C and D)Citation . The capacity of both CA and 1,25D3 to reduce ROS levels increased with time reaching a saturation by 96 h (data not shown).



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  		Fig. 1. CA and 1,25D3 cooperate in the reduction of intracellular ROS levels. HL60-G cells were treated for 96 h with increasing concentrations of CA (A) and 1,25D3 (B) or the combination of 10 µM CA and 1 nM 1,25D3 (C). In C, the effect of 100 nM 1,25D3 is depicted for comparison. The cells (1 x 106) were then incubated without (A, "negative cells") or with 5 µM DCFH-DA for 15 min at 37°C, as described in "Materials and Methods." Cell-associated DCF fluorescence was analyzed by flow cytometry. Representative flow cytometry data obtained in at least four similar experiments are shown.

 

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  		Table 1 Combination of carnosic acid with 1 nM 1,25D3 promotes a reducing intracellular environment similar to that produced by 100 nM 1,25D3

The percentage of "high ROS"-containing cells and the mean "ROS index" (mean fluorescence intensity) in cultures were estimated by flow cytometry after treatments that are shown in the table and loading with DCFH-DA. High ROS are defined as those cells in which the fluorescence exceeds an arbitrary threshold. The threshold was defined experimentally and set so that 96.3% of cells in untreated ("control") cultures were positive for high ROS. This value is presented in the table as 100%, and all other values were normalized for conversion of the control value to 100%. Mean fluorescence intensity was calculated using a CellQuest program. The means ± SD of three experiments are shown.

 


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  		Fig. 7. CA enhances the chemotactic peptide-stimulated [Ca2+]i increase in 1,245D3-treated cells but does not promote elevation of basal [Ca2+]i. Cells were treated with the indicated compounds for 96 h. Cells (1 x 106) were then loaded with 2 µM fura-2 and cytosolic-free calcium; concentration ([Ca2+]i) was monitored by spectrofluorometry, as described in "Materials and Methods." A, a representative experiment showing [Ca2+]i monitoring in HL60 cells: 1, control; 2, CA (10 µM); 3, 1,25D3 (1 nM); 4, CA (10 nM) + 1,25D3 (1 nM); 5, 1,25D3 (100 nM). B, average values of basal [Ca2+]i and fMLP-induced changes in [Ca2+]i. C and D present superoxide generation and percentage of CD11b/CD14 double-positive cells, respectively, determined in experiments illustrated in A and B. The means ± SE of at least three similar experiments are shown in B–D.

 
To further evaluate the antioxidant effects of CA and 1,25D3, after DCFH loading, control cells and cells treated with 10 µM CA, 1 nM 1,25D3, and the combination of 1 nM 1,25D3 and 10 µM CA or 100 nM 1,25D3 were challenged with increasing concentrations of H2O2 (0–1 mM). The results demonstrate that in the CA-treated cells, a 30-min incubation with H2O2 induced less pronounced increases in ROS levels, as compared with control cells (Fig. 2)Citation . Treatment with 1 nM 1,25D3 had a minor effect on the H2O2-induced elevation of ROS, whereas a high concentration of 1,25D3 (100 nM) substantially inhibited the oxidant action. Importantly, a similar inhibition of ROS production was obtained in cells incubated with 1 nM 1,25D3 together with CA. Taken together, these results indicate that the two agents are capable of protecting the cells against oxidative stress and that there is correlation between the differentiating effects of 1 nM 1,25D3 + CA and 100 nM 1,25D3, and the antioxidant effects of these corresponding treatments.



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  		Fig. 2. CA and 1,25D3 reduce intracellular ROS levels in H2O2-treated HL60-G cells. Control cells (1 x 106 cells; A) and those treated with 10 µM CA (B) or 100 nM 1,25D3 (C) were incubated with 5 µM DCFH-DA for 15 min at 37°C and washed once with HEPES-buffered HBSS. Cells were treated for an additional 30 min with the indicated concentrations of H2O2. Fluorescence intensity of the oxidized product (DCF) was monitored by flow cytometry. A representative of three similar experiments is shown. In D, quantitation is presented (mean fluorescence intensity ± SD, n = 4) of the experiments illustrated in A–C, as well as of experiments in which 1 nM 1,25D3, alone or together with 10 µM CA, was used in analogous assays.

 
Diverse Compounds with Antioxidant Action also Potentiate Differentiation Induced by 1,25D3.
If the antioxidant properties of CA have a role in potentiation of differentiation by 1,25D3, other antioxidants would be expected to increase the prodifferentiation effects of 1,25D3 to some extent. We therefore tested several such compounds at their maximal subtoxic concentrations (viability >= 90%) in HL60-G cells and found that although the synthetic antioxidants ebselen (Fig. 3)Citation , hydroxyurea, and diphenylene iodonium (data not shown) had slight to moderate potentiating effects on 1,25D3-induced differentiation, these effects were more marked when plant polyphenols curcumin and silibinin were used (Fig. 3)Citation . Conversely, the pro-oxidant BSO, which depletes cellular glutathione by inhibiting {gamma}-glutamylcysteine synthetase (33) , moderately decreased the differentiating effect of 1,25D3 alone and markedly inhibited the enhanced differentiation induced by its combination with CA (Fig. 4A)Citation . On the other hand, cell treatment with CA resulted in a substantial increase in the intracellular glutathione levels, which was further elevated by adding 1 nM 1,25D3, and both these increases were inhibited by BSO (Fig. 4B)Citation . These findings indicate that reducing conditions (e.g., elevation of cellular glutathione) favor monocytic differentiation induced by 1,25D3.



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  		Fig. 3. Diverse antioxidants potentiate cell differentiation induced by 1,25D3. Cells were incubated with the indicated compounds for 48 h followed by analysis of the expression of monocytic differentiation markers CD11b and CD14 by flow cytometry, as described in "Materials and Methods." The means ± SE of at least four experiments are shown. The experimental groups marked with an asterisk were significantly increased compared with cells treated with 1,25D3 alone (*P < 0.05).

 


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  		Fig. 4. The effects of glutathione-depleting agent, buthionine sulfoximine, on differentiation and glutathione levels in cells treated with 1,25D3 and its combination with CA. HL60-G cells were incubated with the indicated compounds for 96 h followed by analysis of CD11b and CD14 expression by flow cytometry and the measurement of glutathione by an enzymatic assay, as described in "Materials and Methods." In A, buthionine sulfoximine inhibits differentiation. Determination of CD14 and CD11b double positive cells was carried out by flow cytometry. The means ± SE of three experiments are shown. In B, buthionine sulfoximine blocks increases in intracellular glutathione levels. Glutathione was determined by an enzymatic assay, as described in "Materials and Methods." The means ± SE of four experiments are shown.

 
The Raf/MEK/ERK MAPK Module Is Activated by CA in 1,25D3-induced Differentiation.
It has been shown in several laboratories that ERK 1/2 MAPK pathway participates in 1,25D3-induced differentiation of HL60 cells (34, 35, 36) . To initiate studies of the mechanisms of the potentiating effect of CA on this form of differentiation, we examined the activation of several components of this pathway, as evidenced by the level of phosphorylation of these signaling proteins. Consistent with the suggested role of this pathway in differentiation, in cells treated with CA alone, which showed borderline differentiation, a minimal increase was noted in phosphorylation of Raf-1 and MEK-1 (Fig. 5)Citation . Interestingly, phosphorylation appeared to stabilize these proteins, because the total protein levels also increased after exposure to CA.



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  		Fig. 5. CA and 1,25D3 cooperatively activate the ERK MAPK pathways. HL60-G cells were treated with the indicated compound for 96 h, followed by preparation of whole-cell lysates. Lysates were subjected to 10% SDS-PAGE and Western blot analysis, as described in "Materials and Methods." Calreticulin protein levels are displayed to demonstrate similar protein loading and transfer to nitrocellulose membrane. In A, one representative of three similar experiments is shown. B, quantitation of these experiments.

 
More pronounced increases in activation of each member of the ERK 1/2 cascade, as well as of RSK-1, one of the known downstream targets of this pathway, were noted when CA was combined with 1,25D3, and these increases were of similar magnitude as those that resulted from exposure to a high (100 nM) concentration of 1,25D3, whereas calreticulin, a constitutively expressed cellular component, showed no changes in its level of expression (Fig. 5)Citation . Thus, the data suggest that enhanced activation of the ERK 1/2 pathway is associated with the potentiation by CA of monocytic differentiation of HL60 cells.

A Possible Role of the AP-1 Transcription Factor in the Enhancement of Differentiation by CA.
Activation of the ERK cascade has been associated with increased functional activity of AP-1 transcription factor (reviewed in Refs. 37 and 38 ), and recent studies have indicated the role of AP-1 activation in myeloid differentiation of leukemic cells (23 , 39, 40, 41) , and in the expression of the VDR gene (42) . Importantly, AP-1 is regulated by cellular redox status (43) . Therefore, we determined whether the enhancement of 1,25D3-induced monocytic differentiation of HL60 cells was accompanied by changes in the DNA-binding capacity of nuclear proteins to the AP-1 motifs present in the promoter of the human VDR gene (GenBank accession no. AB002157). Consistent with the data reported previously (23) , cell treatment with 1,25D3 resulted in increases in binding to both these AP-1 motifs. This effect was pronounced in cells treated with 100 nM 1,25D3, whereas at 1 nM, this inducer showed only a moderate effect (Fig. 6)Citation . However, combining 1 nM 1,25D3 with CA, which was also relatively ineffective in this assay, caused a substantial elevation of AP-1 binding activity. Elimination of binding because of competition with unlabeled AP-1 response element probe but not with a mutated AP-1 sequence or a "nonself" probe (Sp-1 response element) indicates the specificity of the AP-1 gel-shift assay in this system. These data suggest that the AP-1 transcription factor regulates genes that participate in the CA-enhanced program of monocytic differentiation.



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  		Fig. 6. CA potentiates 1,25D3-induced AP-1 binding to its cognate DNA elements. HL60-G cells were treated with the indicated compounds for 96 h, followed by preparation of nuclear extracts. In A, an oligonucleotide corresponding to the proximal AP-1 binding site in the promoter region of the human VDR gene (AP-1 hVDR#1) was synthesized and 5'-labeled with a 32P-ATP. The extracts were incubated with this labeled oligonucleotide, and binding of the AP-1 complex was analyzed by electrophoretic mobility shift assay, as described in "Materials and Methods." Unlabeled AP-1 hVDR #1, mutant hVDR, and Sp1 oligonucleotides were used to compete with the nuclear protein binding to indicate the specific nature of AP-1 hVDR interaction in this assay. In B, the binding of nuclear proteins to the distal AP-l element (AP-1 hVDR #2) was similarly studied. Inhibition of this binding when the nuclear extracts were incubated with 20 mM diamide, an oxidizing agent, is also demonstrated here. A representative gel analysis of three similar experiments is shown for each probe. Note that a longer exposure of the gel was used in the experiment shown in B, to display the decrease in AP-1 binding in the presence of the oxidizing agent.

 
CA Does Not Augment the 1,25D3-induced Rise of Basal Cytosolic Calcium Levels.
Several studies have shown that the 1,25D3-induced monocytic differentiation of myeloid leukemia cells is accompanied both by an increase in basal cytosolic calcium levels and the expression of fMLP receptors, which mediate the chemotactic peptide-induced transient [Ca2+]i elevations (28 , 29 , 44, 45, 46, 47) . Because disturbances of calcium homeostasis may have profound implications for any potential use of CA/1,25D3 in differentiation therapy of myeloid leukemia, we determined whether along with its enhancement of the differentiating and antiproliferating effects of 1,25D3, CA can also potentiate the ability of this inducer to affect cytosolic calcium levels. Changes in both basal [Ca2+]i and its elevations in response to fMLP were monitored in HL60-G cells treated for 96 h with 1,25D3, CA, and their combination using the fluorescent calcium probe fura-2. Cells treated with 100 nM 1,25D3 showed a substantial increase (P < 0.01) in the basal [Ca2+]i as compared with untreated control cells (Fig. 7)Citation , whereas neither 1 nM 1,25D3 nor 10 µM CA had a significant effect. The addition of 10 µM fMLP to control or to CA-treated cells did not affect their basal [Ca2+]i, whereas in cells incubated with 1,25D3, this peptide induced a transient elevation of cytosolic calcium. These data are consistent with the induction of chemotactic peptide receptors in 1,25D3-differentiated HL60 cells (18 , 48 , 49) . The peak values of the fMLP-stimulated [Ca2+]i rises depended on 1,25D3 concentration (Fig. 7)Citation . The addition of CA to 1 nM 1,25D3 during 96-h incubations resulted in the enhanced calcium response to fMLP, which was comparable with that observed in the 100 nM 1,25D3-exposed cells. Most interestingly, however, an increase in the basal [Ca2+]i was much lower in cells treated with the CA/1,25D3 combination than in those incubated with 100 nM 1,25D3 (Fig. 7, A and B)Citation , although the extent of differentiation was comparable (Fig. 7, C and D)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The dramatic enhancing effect of CA on the 1,25D3-induced monocytic differentiation of HL60-G cells has recently been reported (19) , and here we present studies of the mechanistic basis of this potentiation. Our results, taken together with previous studies (39 , 50) , show that modulation of the cellular redox state appears to enhance monocytic differentiation. The cumulative data indicate that many structurally distinct antioxidants, such as polyphenols, carotenoids, vitamin E, ascorbate, and lipoic acid, all potentiate leukemic cell differentiation induced by various agents, such as 1,25D3 and ATRA (14, 15, 16, 17 , 50 , 51) . Thus, the chemical structure of the antioxidant compounds that enhance differentiation does not appear to be important, and their only common property is that they alter the redox state.

The differentiation-enhancing agent used in this study, CA, a polyphenolic diterpene, displays strong antioxidant effects in foods and biological systems, compared with those of {alpha}-tocopherol and butylated hydroxytoluene (30 , 52 , 53) . CA effectively scavenges peroxyl and hydroxyl radicals, H2O2, and hypochlorous acid, a compound produced in sites of inflammation that may damage biological membranes (30) . CA (1–30 µM) has been shown to inhibit mitochondrial and microsomal lipid peroxidation (30 , 54 , 55) and protect red cells against oxidative hemolysis (54) . It also inhibits oxidation of low-density lipoprotein induced by human aortic endothelial cells (56) . However, in several other studies (30, 31, 32) , CA and other polyphenols were found to act as pro-oxidants probably because of different experimental conditions.

Here, we examined whether treatment of HL60 cells with CA, 1,25D3, and their combinations affects intracellular ROS levels. For this purpose, a fluorescent probe, DCFH, was used, which is a widely used indicator of ROS in leukemic and other cell types (24 , 25 , 57) . We found for the first time that incubation with nontoxic concentrations of CA alone produced a clear concentration-dependent reduction of ROS levels in a cellular model (HL60 cells) concomitant with a substantial increase in total glutathione levels. Furthermore, CA-treated cells were appreciably more resistant to the oxidative stress induced by H2O2 than the untreated control cells. These data indicate that CA has antioxidant action under experimental conditions that are used to induce differentiation of leukemia cells, although it is possible that additional mechanisms contribute to its potentiation of differentiation.

Interestingly, exposure of HL60 cells to 1,25D3 also led to reduction of ROS levels and protection against H2O2 stress with even greater capacity than CA. It appears that the effect of 1,25D3 on the cellular redox status is cell/tissue-dependent, because in breast cancer cells (MCF-7), 1,25D3 was reported to act as a pro-oxidant (58 , 59) , whereas in keratinocytes and in neurons, 1,25D3 protects against various stress stimuli, including H2O2 (60 , 61) . In our study, 1,25D3 is also an antioxidant and synergizes with CA to markedly reduce the intracellular ROS levels, which may at least in part be related to a synergistic increase in cellular glutathione abundance. Interestingly, although 1,25D3 alone did not change the basal glutathione content, it greatly potentiated the CA-induced elevation of this peptide concentration. The mechanism of this potentiation is unclear, although it may be related to synergy of the two compounds at the level of glutathione biosynthesis.

The relationship between the extent of differentiation and redox status has not been clearly elucidated, and the available data may seem inconsistent, although cell context and/or experimental conditions must also be taken into consideration, e.g., in HL60 cells induced to differentiate by either DMSO or ATRA, lower levels of ROS and DNA damage and higher levels of GSH were observed, as compared with undifferentiated cells (62) . Furthermore, many antioxidants have been shown to induce or enhance differentiation in leukemic cells (Refs. 14, 15, 16, 17, 18, 19 , 50 , and 51 and this study). However, similar differentiation effects were also demonstrated for some agents that promote intracellular ROS formation, e.g., topoisomerase inhibitor ß-lapachone (63) or the well-known differentiation inducer butyric acid (64) . Granulocytic differentiation of HL60 cells can be induced by the elevation of hydroxyl radicals generated by a Fenton reaction, involving an ADP-Fe2+ (or ATP-Fe2+) complex and H2O2 (65) . We demonstrate here that the extent of differentiation in HL60 cells is associated with reducing conditions. Both CA and 1,25D3 decrease the intracellular ROS levels; various antioxidants potentiate the differentiating effect of 1,25D3, whereas the pro-oxidant BSO decreases it. Importantly, BSO inhibits not only the differentiation induced by 1,25D3 but also its potentiation by CA. BSO, a specific inhibitor of {gamma}-glutamylcysteine synthetase that catalyzes the rate-limiting step in glutathione synthesis (33) , blocked the increases in glutathione levels by either CA or its combination with 1,25D3. Thus, the BSO reduction of 1,25D3-induced differentiation and, particularly, its enhancement by CA suggest that glutathione is involved in both effects, perhaps by protecting the cells from oxidative damage expected to be generated during the oxidative burst that characterizes the function of the maturing monocyte (66) . Furthermore, changes in GSH levels can lead to the redox-sensitive modulation of transcription regulators, such as AP-1, e.g., via the SH group of its component tripeptide lysine-cysteine-arginine (KCR) (67) , as depicted in Fig. 8Citation , or by glutathionylation of diverse proteins that can modify protein function (68) .



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  		Fig. 8. Schematic representation of the hypothesis for the cooperation between antioxidants and 1,25D3 in the facilitation of AP-1 function. Activation of the c-Jun-NH2-terminal kinase pathway is known to be associated with 1,25D3-induced differentiation (36) , and antioxidants may increase the activity of AP-1 up-regulated by 1,25D3. KCR is a tripetide in the DNA-binding region of both Jun and Fos proteins with reduced cysteine (SH), and the maintenance of the reduced state of these groups is required for DNA binding (modified from Ref. 67 ).

 
Activation of AP-1 by 1,25D3 has been reported previously (23 , 69) . In the present study, we show that CA/1,25D3 combinations increase the activation of AP-1 binding by 1,25D3, whereas CA alone has a minor effect (Fig. 6)Citation . Although the mechanism of this activation is still unknown, one possibility is that an up-regulation of the MAPK pathways by the CA/1,25D3 combination demonstrated here (Fig. 5)Citation also contributes to increased AP-1 activity (Fig. 8)Citation . This is supported by previous reports that 1,25D3 activates ERK in various cells, (34 , 35 , 70) , e.g., Wang and Studzinski (35) have shown that ERK phosphorylation in HL60 cells is much greater during short-term (<=24 h) than long-term (48–96 h) incubations and suggested that the MEK/ERK pathway maintains cell proliferation during the early phase of 1,25D3-induced monocytic differentiation of HL60 cells but that the ERK1/2 activity becomes suppressed during the later stages of differentiation. However, in the current study, the longer (96 h) 1,25D3/CA treatment resulted in somewhat elevated levels of P-MEK and P-ERK and inhibition of cell proliferation, as reported previously (19) . This shows that although the RAF-ERK pathway is important for monocytic differentiation of HL60 cells, because its inhibition reduces the extent of 1,25D3-induced differentiation (34 , 35) , the activity of this pathway is not sufficient to ensure continuation of proliferation in differentiating cells. A possible explanation is that the up-regulation of p35/Cdk5 complex, which occurs in CA/1,25D3-treated cells (see Fig. 4Citation in Ref. 19 ), overrides the pro-proliferation actions of the Raf-ERK pathway, as reported recently (71, 72, 73) .

The well-known limitation to the therapeutic use of 1,25D3 is its hypercalcemic affect. Current attempts to overcome this problem focus on the synthesis of analogues of 1,25D3, which retain the prodifferentiation activities but have lower calcemic effects (74) . However, in contrast to its effectiveness in the treatment of osteoporosis (75) and psoriasis (76) , and the efficient induction of differentiation in cultured human leukemia cells (reviewed in Ref. 77 ), 1,25D3 and its analogues have not as yet been successfully applied to the treatment of myeloid leukemias (78) . In an attempt to determine whether the CA/1,25D3 combination offers a potential therapeutic advantage over analogues of 1,25D3, we also studied the effects of CA/1,25D3 combinations on cellular calcium homeostasis. Previously, it was shown that CA enhances 1,25D3 induction of fMLP receptors (18) . Here, we show that although the fMLP-induced Ca transients are augmented by CA, the effect on basal Ca level was only moderate. Although this result cannot be extrapolated to reflect the possible systemic action of CA on the calcemic effects of 1,25D3, the result is promising and indicates that the CA/1,25D3 combinations, although inducing a similar extent of differentiation as high doses of 1,25D3, may produce less disturbance of calcium homeostasis.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Uskokovic (BioXell) for his generous gift of 1,25D3.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grant RO-1 CA44722 from the National Cancer Institute and Grant 2001041 from the US-Israel Binational Science Foundation, Jerusalem, Israel. Back

2 To whom requests for reprints should be addressed, at UMD–New Jersey Medical School, Department of Pathology and Laboratory Medicine, 185 South Orange Avenue, C543, Newark, NJ 07103. Phone: (973) 972-5869; Fax: (973) 972-7293; E-mail: studzins{at}umdnj.edu Back

3 The abbreviations used are: ATRA, all-trans retinoic acid; 1,25D3, 1{alpha},25-dihydroxyvitamin D3; TPA, 12-O-tetradecanoylphorbol 13-acetate; CA, carnosic acid; ROS, reactive oxygen species; [Ca2+]i, intracellular calcium concentration; DCFH-DA, 5,6-carboxyl-2',7'-dichlorofluorescein-diacetate; fMLP, N-formyl-methionyl leucyl-phenylalanine; AP-1, activator protein; hVDR, human vitamin D receptor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; GSH, reduced glutathione; BSO, buthionine sulfoxime; MAPK, mitogen-activated protein kinase; KCP, tripeptide lysine-cysteine-arginine. Back

Received 8/ 8/02. Accepted 1/15/03.


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 DISCUSSION
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