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Originally published online as doi:10.1189/jlb.0705409 on June 12, 2006

Published online before print June 12, 2006
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(Journal of Leukocyte Biology. 2006;80:399-406.)
© 2006 by Society for Leukocyte Biology

Halofuginone inhibits NF-{kappa}B and p38 MAPK in activated T cells

M. Leiba*, L. Cahalon{dagger}, A. Shimoni*, O. Lider{dagger}, A. Zanin-Zhorov{dagger}, I. Hecht{dagger}, U. Sela{dagger}, I. Vlodavsky{ddagger} and A. Nagler*,1

* The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, and Sackler Medical School, Tel-Aviv University, Israel;
{dagger} Department of Immunology, the Weizmann Institute of Science, Rehovot, Israel; and
{ddagger} Cancer and Vascular Biology Research Center, Rappaport Faculty of Medicine, Technion, Haifa, Israel

1Correspondence: Division of Hematology, BMT and CBB, Chaim Sheba Medical Center, Tel-Hashomer, Israel 52621. E-mail: a.nagler{at}sheba.health.gov.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Halofuginone, a low molecular weight plant alkaloid, inhibits collagen {alpha}1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-ß (TGF-ß), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-{kappa}B activity, production of tumor necrosis factor {alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10–40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-{kappa}B activity (80% inhibition following incubation with 40 ng halofuginone, P=0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-{alpha}, IFN-{gamma}, interleukin (IL)-4, IL-13, and TGF-ß (P<0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P=0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-{kappa}B activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent.

Key Words: TGF-ß • TNF-{alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For centuries, the roots of Dichroa Febrifuga, a saxifragaceous plant, have been used in China in the treatment of malarial fever. Febrifugine and its stereoisomere, isofebrifugine, were identified as the active antimalarial components. Halofuginone [7-bromo-6-chloro-3-(3-hydroxy-2-piperidine)-2-oxopropyl-4(3H)-quinazoline] is one of the febrifugine analogoues used worldwide for almost 20 years in commercial poultry production to prevent coccidosis [1 ]. In previous studies, we have demonstrated that halofuginone inhibits collagen {alpha} I gene expression and collagen type I synthesis in avian, murine, and human fibroblasts [2 ]. Halofuginone is a potent inhibitor of fibrosis, decreasing skin collagen in scleroderma and in chronic graft-versus-host disease in animal models [3 ]. Halofuginone has also been shown to inhibit synthesis and deposition of extracellular matrix (ECM) by vascular smooth muscle [4 ] and mesangial [5 ] cells. Subsequently, halofuginone was found to inhibit expression of matrix metalloproteinase 2 (MMP2) and to suppress angiogenesis associated with bladder and brain carcinomas [6 , 7 ].

Several clinical trials have already been conducted with halofuginone [1 ]. Topical application of halofuginone in healthy volunteers showed no skin irritation or systemic absorption. In a phase II trial in patients with systemic sclerosis, daily topical application of halofuginone resulted in five out of 12 patients responding with a reduction in the mean total skin score after 3 months. A phase I trial of oral administration of halofuginone has shown that the compound is well-tolerated with some gastrointestinal adverse effects [1 ]. The exact mechanism of action of halofuginone is not well understood, although its action was recently found to be associated with inhibition of transforming growth factor-ß (TGF-ß) signaling [8 ]. TGF-ß is a pluripotent cytokine, regulating a variety of biological responses, including cell growth and differentiation, apoptosis, cell migration, immune cell function, and ECM deposition [9 ]. TGF-ß may also function as an anti-inflammatory or immunosuppressive agent, so that its blockage might exert an adverse effect by augmenting inflammation. In the present study, we have measured nuclear factor (NF)-{kappa}B activity, cytokine release, p38 mitogen-activated protein kinase (MAPK) phosphorylation, apoptosis, and chemotaxis in activated T lymphocytes before and after incubation with halofuginone to better delineate its pro- or anti-inflammatory properties.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and monoclonal antibodies (mAb)
The following reagents were used: stromal cell-derived factor-1{alpha} (SDF-1{alpha}) and fibronectin (FN; Chemicon, Temecula, CA); tumor necrosis factor {alpha} (TNF-{alpha}), interferon-{gamma} (IFN-{gamma}), interleukin (IL)-4, IL-13, and TGF-ß (PeproTech, Rocky Hill, NJ); bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO); HEPES buffer, antibiotics, fetal calf serum (FCS), and RPMI 1640 (Kibbutz Beit Ha-Emek, Israel); and Na51[Cr] (Amersham, High Wycombe, UK). The antibodies used were anti-human IFN-{gamma} and TNF-{alpha} mAb (BioSource International, Inc., Camarillo, CA) and anti-human IL-4, IL-13, and TGF-ß mAb (R&D Systems, Inc., Minneapolis, MN). Anti-CD3 mAb was purified from ascites fluid of mice injected with OKT3 hybridoma cell (American Type Culture Collection, Manassas, VA). Halofuginone was obtained from Collgard Ltd. (Tel-Aviv, Israel).

Purification of human T cell populations
T cells were purified from the peripheral blood of healthy human donors (Blood Bank, Tel-Hashomer, Israel). The whole blood was incubated (20 min, 22°C) with RosetteSepTM human T cell enrichment cocktail (CD3+ T cells) or with RosetteSepTM human CD4+ and CD8+ T cell enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada). The remaining unsedimented cells were then loaded onto a lymphocyte separation medium (ICN Biomedicals, Asse-Relegem, Belgium), isolated by density centrifugation, and washed with phosphate-buffered saline. The purified cells (>97% CD3+ T cells, >95% CD4+ T cells, >95% CD8+ T cells) so obtained were cultured in RPMI containing antibiotics and 10% heat-inactivated FCS. In a second round of purification, CD3+ T cells were labeled for negative selection with magnetically coupled mAb against CD45RA+ and CD45RO+ (Miltenyi Biotec, Auburn, CA). The purified cells obtained (usually >97% CD45RO+ or CD45RA+ T cells) were cultured in RPMI containing antibiotics and 10% heat-inactivated FCS. In a third round of purification, CD45RO+ T cells were labeled with anti-CC chemokine receptor 7 (CCR7)-fluorescein isothiocyanate (FITC), and the CD45RO+ CCR7+ and CD45RO+ CCR7– T cells were isolated by sorting using FACSVantage (Becton Dickinson, Mountain View, CA; >95% CD45RO+ CCR7+ or CD45RO+ CCR7– T cells). In four rounds of purification, CD4+ T cells and CD8+ T cells were labeled for negative and positive selection with magnetically coupled mAb against CD27 (Miltenyi Biotec). The purified cells obtained (usually >95% CD4+CD27+ or CD4+CD27– or CD8+CD27+ or CD8+CD27–) were cultured in RPMI containing antibiotics and 10% heat-inactivated FCS.

Cytokine secretion
T cells (2x106 cells/ml) were incubated with the indicated concentrations of halofuginone in serum-free RPMI containing 0.1% BSA and plated on 24-well plates (nontissue-culture grade), precoated with anti-CD3 mAb (1 µg/ml; overnight). Supernatants were collected, and their cytokine content (TNF-{alpha}, IFN-{gamma}, IL-4, IL-13, and TGF-ß) [10 ] was determined by enzyme-linked immunosorbent assay (ELISA) using the appropriate mAb, according to the manufacturer’s instructions. Each experiment was done in quadruplicate, and the results are expressed as the mean ± SD.

NF-{kappa}B activation
T cells (1.5x107) were activated as above, incubated with various concentrations of halofuginone, and plated into plates precoated with anti-CD3 mAb. NF-{kappa}B concentration was determined using a NF-{kappa}B/p65-active ELISA kit (Imgenex, San Diego, CA), according to the manufacturer’s instructions. NF-{kappa}B activation index is defined as NF-{kappa}B concentration in the nucleus of activated T cell divided by NF-{kappa}B concentration in the nucleus of nonactivated T cells. Nuclear extracts were prepared, and nuclear fraction was subjected to ELISA using specific anti-NF-{kappa}B antibodies. Each experiment was performed in quadruplicate, and the results are expressed as the mean ± SD.

p38 phosphorylation
T cells (1.5x107) were activated as above, incubated with various concentrations of halofuginone, and plated into plates precoated with anti-CD3 mAb. After 18 h, cells were collected and lysed. Phosphorylation of p38 was determined using the Human Phospho-p38{alpha} (T180/Y182) DuoSet IC ELISA kit (R&D Systems), according to the manufacturer’s instructions. Cytoplasmic extracts were prepared, and the latter were subjected to ELISA using specific anti-p38 and antiphosphorylated p38 antibodies. Each experiment was done in quadruplicate, and the results are expressed as the mean ± SD.

Chemotaxis assays
Cell migration was measured in Transwells (6.5 mm diameter, Corning, NY), fitted with polycarbonate filters (5 µm pore size). The filters separating the upper and lower chambers were preincubated (1 h, 37°C) with FN (25 µg/ml). Aliquots (100 µl) of 51[Cr]-labeled T cells (2x106 cells/ml RPMI containing 0.1% BSA, 0.1% L-glutamine, and antibiotics) were added to the upper chamber. The lower chamber contained 0.6 ml of the same medium, with or without human SDF-1{alpha} (250 ng/ml). After a 3-h incubation (37°C, 7.5% CO2-humidified atmosphere), T cell migration through the FN-coated filter was determined by collecting the transmigrated cells from the lower chamber. The cells were centrifuged and resuspended in 100 µl distilled water containing 1 M NaOH and 0.1% Triton X-100, and the radioactivity associated with the resulting supernatant was measured with a {gamma}-counter. Cell migration was calculated as the number of migrating cells [counts per minute (cpm) in the lower chamber] expressed as the percentage of the total cpm per 100 µl aliquot of the starting cell mixture (i.e., per 2x105 cells). Each experiment was done in quadruplicate, and the results are expressed as the mean ± SD.

T cell apoptosis
T cells (2x106 cells/ml) were preincubated with the indicated concentrations of halofuginone in RPMI medium containing 10% FCS and plated into 24-well plates (nontissue-culture grade), precoated with anti-CD3 mAb (1 µg/ml; overnight), and cultured for 72 h. Then, the percentage of cells undergoing apoptosis was determined using the annexin V detection assay. Briefly, cells were incubated (10 min in the dark) at room temperature in 200 µl buffer containing FITC-conjugated human annexin V (5 ml, Bender MedSystem, San Bruno, CA). Propidium iodide (PI; 10 µl) was then added to each sample, and the percentage of cells undergoing apoptosis was analyzed by FACScan at 525 nm using CellQuest software. Annexin V-positive, PI-negative cells corresponded to apoptotic cells. Each experiment was done in quadruplicate, and the results are expressed as the mean ± SD.

Delayed-type hypersensitivity (DTH) assay
Groups of five female, inbred BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were sensitized on the shaved abdominal skin with 2% oxazalone (100 µl), dissolved in acetone/olive oil [4:1 (vol/vol)], and applied topically. DTH was elicited 6 days later by challenging the mice with 0.5% oxazalone in acetone/olive oil (10 µl given topically to each side of the ear). A constant area of the ear was measured immediately before and 24 h after the challenge using a Mitutoyo engineer’s micrometer. The percent inhibition of DTH is calculated as follows: % inhibition = {1–[(treated–negative control)/(positive control–negative control)]} x 100.

The positive control is the DTH reaction to oxazalone elicited in sensitized mice without treatment. The negative control is the background swelling produced by the oxazalone antigen in naïve, nonsensitized mice [11 ]. Halofuginone was adminstered to the mice by two intraperitoneal (i.p.) injections (5, 10, and 20 µg per mouse), starting at 1 day and 2 h before oxazalone challenging.

Statistical analysis
Statistical analysis was performed using Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of halofuginone on NF-{kappa}B activation
NF-{kappa}B activity is dictated by its translocation to the nucleus, where it controls the transcription of genes responsible for the regulation of cell proliferation, cell survival, and inflammation. NF-{kappa}B activation index, in response to T cell activation by anti-CD3 mAb, was 8.2 ± 0.8. Treatment with halofuginone (6 h at 37°C, 10 ng/ml) decreased NF-{kappa}B activity by 36% (P=0.01; Fig. 1 ). This inhibition was dose-dependent. Halofuginone (40 ng/ml) decreased NF-{kappa}B activation index by 72% (P=0.002; Fig. 1 ).


Figure 1
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  		Figure 1. Halofuginone inhibits the activation of NF-{kappa}B in anti-CD3-activated T cells. Halofuginone inhibits the activation of NF-{kappa}B in anti-CD3-activated T cells. The inhibition is dose-dependent. The data are means (±SD) of one representative experiment out of three performed.

 
Effect of halofuginone on cytokine secretion
As NF-{kappa}B mediates transcription of TNF-{alpha} and IFN-{gamma}, we examined the effect of halofuginone on the regulation of cytokine secretion by anti-CD3-activated human T cells.

Figures 2 and 3 show that halofuginone markedly decreased the secretion of TNF-{alpha} and IFN-{gamma} by activated T cells. Halofuginone (40 ng/ml) decreased TNF-{alpha} and IFN-{gamma} levels by 56% (P<0.001). Thus, halofuginone appears to inhibit NF-{kappa}B and its related proinflammatory cytokines, IFN-{gamma} and TNF-{alpha}, in a dose-dependent manner. To assess the inhibitory effect of halofuginone relative to the commonly used immunosuppressive drugs, we compared the effect of increasing concentrations of halofuginone on cytokine secretion with that of Cyclosporine A. Halofuginone was found to inhibit secretion of IFN-{gamma} and TNF-{alpha} to the same extent as Cyclosporine A (not shown). Next, we have tested the effect of increasing concentrations of halofuginone on secretion of IL-4, IL-13, and TGF-ß by activated T cells, as these cytokines are known to modulate collagen gene expression [10 ]. Inhibition (20–30%) was obtained already at 5 ng/ml, reaching an almost complete inhibition at 20–40 ng/ml halofuginone (not shown).


Figure 2
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  		Figure 2. Effect of halofuginone on TNF-{alpha} secretion by human T cells. Halofuginone inhibits TNF-{alpha} secretion by anti-CD3-activated T cells. The inhibition is dose-dependent. The data are means (±SD) of one representative experiment out of the three performed.

 

Figure 3
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  		Figure 3. Effect of halofuginone on IFN-{gamma} secretion by human T cells. Halofuginone inhibits IFN-{gamma} secretion by anti-CD3-activated T cells. The inhibition is dose-dependent. The data are means (±SD) of one representative experiment out of the three performed.

 
In subsequent experiments, we tested the effect of halofuginone on various T cell subsets, including naïve, memory, and effector c,ells. For this purpose, CD3+ T cells were separated to the following subpopulations: CD45RA+, CD45RO+, CD45RO+CCR7+, and CD45RO–CCR7–, as well as CD4+, CD4+CD27+, CD4+CD27–, CD8+, CD8+CD27+, and CD8+CD27– T cells. The different, purified T cell subsets were activated and incubated with halofuginone, and the supernatants were assessed for IFN-{gamma} and TNF-{alpha} by ELISA. Halofuginone effectively inhibited the secretion of both cytokines by the various T cell subsets. It is notable that the inhibitory effect on CD4+ cells was higher (90±15%) than that on CD8+ cells (70±20%). This differential effect may explain the partial inhibition of cytokine secretion obtained upon addition of halofuginone to the unseparated T cell population.

Effect of halofuginone on p38 phosphorylation
p38, a member of the MAPK family, is a stress-activated protein kinase, which modulates nuclear NF-{kappa}B activity and TNF-{alpha} synthesis and secretion [12 ]. As demonstrated in Figure 4 , halofuginone inhibited the phosphorylation of p38 MAPK in activated T cells in a dose-dependent manner. The degree of p38 phosphorylation was reduced by 47% (P=0.005) and 63% (P=0.0001) in the presence of 10 ng/ml and 40 ng/ml halofuginone, respectively.


Figure 4
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  		Figure 4. Effect of halofuginone on p38 MAPK phosphorylation. Halofuginone inhibits p38 MAPK phosphorylation in anti-CD3-activated T cells. The inhibition is dose-dependent. The data are means (±SD) of one representative experiment out of two performed.

 
Effect of halofuginone on T cell apoptosis
Inflammation is often associated with programmed cell death (apoptosis). The percentage of T cell apoptosis after activation with anti-CD3 mAb was 56.6 ± 5.7%, as revealed by the annexin V detection assay. Treatment with halofuginone (20 ng/ml) yielded a 63% reduction in the number of apoptotic cells (P=0.005; Fig. 5 ). To assess whether the antiapoptotic effect of halofuginone is a result of its suppression of TNF secretion (Fig. 2) , CD3+ T cells were incubated with halofuginone (40 ng/ml) in the absence and presence of various concentrations (5–20 ng/ml) of TNF-{alpha}. After 72 h, the percentage of cells undergoing apoptosis was determined. As demonstrated in Figure 6 , adding TNF-{alpha} to T cells treated with halofuginone did not restore the inhibition of apoptosis induced by halofuginone.


Figure 5
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  		Figure 5. Effect of halofuginone on T cell apoptosis. Halofuginone inhibits apoptosis in anti-CD3-activated T cells. The data are means (±SD) of one representative experiment out of three performed.

 

Figure 6
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  		Figure 6. Effect of TNF-{alpha} on the inhibition of apoptosis by halofuginone. CD3+ T cells were incubated with halofuginone (40 ng/ml) and various concentrations of TNF-{alpha} and plated on anti-CD3-precoated plates. After 72 h, the percentage of cells undergoing apoptosis was determined by the annexin V detection assay. The data are means (±SD) of three independent experiments.

 
Effect of halofuginone on T cell chemotaxis
SDF-1-mediated cell migration is a key event in the inflammatory response [13 ]. We have therefore assessed whether this process is affected by halofuginone. As demonstrated in Figure 6 , T cell chemotaxis, measured by migration of Cr-51-labeled cells toward SDF-1, was not affected by halofuginone.

Effect of halofuginone on DTH response
To assess the immunosuppressive activity of halofuginone on T cells in vivo, we applied the DTH mouse model. For this purpose, mice were sensitized on the abdominal skin with oxazalone, and DTH was elicited 6 days later by challenging the mice with oxazalone, given topically to each side of the ear. Halofuginone was administered i.p., starting at 1 day and 2 h before challenging. As demonstrated in Figure 7 , treatment with halofuginone (20 µg/mouse, i.p. injections 24 h and 2 h before challenge) inhibited the DTH response to an extent similar to dexamethasone, indicating suppression of T cell-mediated inflammation in vivo.


Figure 7
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  		Figure 7. Effect of halofuginone on DTH response. Groups of BALB/c mice (five each) were sensitized to oxazolone by skin painting, and the degree of DTH reactivity was assessed 7 days later by applying oxazolone (on Day 6) to the ears and measuring the ear thickness 24 h later. The mice were treated by i.p. injections of halofuginone [20 µg (mcg), 10 µg, or 5 µg per mouse], given 1 day and 2 h before the second sensitization. Dexamethasone (DEXA), 40 µg, administered 1 day before the second sensitization, was used as a positive control. The percent inhibition was calculated in comparison with control-sensitized, untreated mice. The average percentage derived from three different experiments is shown. *, P value = 0.01; **, P value = 0.0004.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was undertaken to assess the effect of halofuginone on T cell NF-{kappa}B activation, proinflammatory cytokine secretion, p38 MAPK phosphorylation, cell migration, apoptosis, and DTH response.

Preincubation of activated human T cells with halofuginone resulted in inhibition of NF-{kappa}B nuclear translocation. NF-{kappa}B plays a critical role in the regulation of immunity and inflammation by stimulating the transcription of a wide range of cytokine-encoding genes, including TNF-{alpha} and IFN-{gamma} [14 ]. Indeed, pretreatment of T cells with halofuginone resulted in a marked, although partial, decrease in the levels of secreted TNF-{alpha} and IFN-{gamma}, as well as IL-4, IL-13, and TGF-ß. These cytokines are known to regulate collagen synthesis [10 ] and thus, are likely to be involved in the antifibrotic effect of halofuginone [1 2 3 4 5 ]. In fact, halofuginone inhibited secretion of IFN-{gamma} and TNF-{alpha} to the same extent as Cyclosporine A, emphasizing its potential immunosuppressive capabilities. The suppressive effect of halofuginone was equally demonstrated for naïve, memory, and effector T cells.

It is notable, however, that the inhibitory effect on CD4+ cells was higher than that on CD8+ cells, which may account for the partial inhibition of cytokine secretion obtained upon addition of halofuginone to the entire nonseparated T cell population.

By itself, TNF-{alpha}, as well as other cytokines, activates NF-{kappa}B and thus, might initiate a vicious, inflammatory cycle [15 ]. In addition to its ability to regulate cytokine production, NF-{kappa}B is involved in regulation of the acute-phase response of inflammation, which provides systemic defense and restores homeostasis after infection or injury [16 , 17 ]. Anti-inflammatory drugs such as salicylates and corticosteroids have been reported to inhibit NF-{kappa}B [17 18 19 20 ]. Thus, halofuginone appears to share similar anti-inflammatory properties.

In recent years, a strong linkage between NF-{kappa}B and tumorigenesis has been demonstrated [21 ]. Active NF-{kappa}B was identified in tumor tissues derived from patients with hematological malignancies, including multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, as well as in patients with prostate and breast cancers [22 ]. Suppression of NF-{kappa}B in these tumors inhibits cell proliferation, causes cell cycle arrest, and leads to apoptosis [21 ]. Bortezomib, a recently described proteosome inhibitor and a potent inhibitor of NF-{kappa}B, showed substantial activity against multiple myeloma and other cancers [23 24 25 26 ]. We have previously demonstrated that halofuginone inhibits angiogenesis and tumor growth in several experimental models in vitro and in vivo [27 , 28 ]. The antineoplastic effect of halofuginone was attributed in part to the inhibition of MMP2, known to be involved in metastatic spread of tumor cells, and to inhibit basic fibroblast growth factor-mediated tumor angiogenesis [27 ]. In addition, halofuginone was found to induce apoptosis in various types of cancer cells [29 ]. Based on our current findings, we propose an additional, putative mechanism by which halofuginone exerts its antitumor activity, namely, inhibition of NF-{kappa}B and thereby, several gene products involved in cancer metastasis and angiogenesis, including MMPs.

We found that halofuginone inhibits apoptosis of normal, activated T cells. It is conceivable that this inhibition is mediated primarily by the inhibition of TNF-{alpha} secretion, the most potent, physiologic inducer of apoptosis [30 ]. However, addition of TNF-{alpha} to activated T cells treated with halofuginone failed to reverse the protective, antiapoptotic effect of halofuginone. Apoptosis inhibition might also be mediated by inhibition of NF-{kappa}B, although the exact role of NF-{kappa}B in apoptosis is controversial [31 , 32 ]. Lin et al. [33 ] showed that NF-{kappa}B can be pro- or antiapoptotic, depending on the timing of modulating NF-{kappa}B activity relative to the death stimulus.

The observed inhibition of NF-{kappa}B by halofuginone may be direct or indirect. We have previously found that halofuginone inhibits phosphorylation of p38 MAPK. p38, a member of the MAPK family, is a stress-activated protein kinase and is a central component in transducing signals generated by growth factors and stress agents [34 ]. p38 MAPK was found recently to modulate NF-{kappa}B and thereby, TNF-{alpha} in human macrophages [12 ]. The observed inhibition of p38 MAPK by halofuginone might thus explain the indirect effect of halofuginone on NF-{kappa}B and cytokine production, reflected by its anti-inflammatory and antitumor properties.

Another mechanism by which halofuginone could influence T cells is via the inhibition of TGF-ß, and inhibition of TGF-ß secretion by halofuginone was, in fact, demonstrated in the present study. TGF-ß has been shown to regulate multiple fundamental cellular processes, including cell growth, migration, adhesion, ECM deposition, and apoptosis. TGF-ß also activates unique transmembrane serine/threonine kinase receptors (TGF-ß receptors type I and II), which activate Smad2 and Smad3 [35 36 37 ], and Smad3 is an essential mediator of the TGF-ß signaling pathway [8 ]. McGaha et al. [38 ] were able to show that in fibroblasts from a tight skin mouse (a model of scleroderma), halofuginone inhibited TGF-ß-induced activation of Smad3. Moreover, Xavier et al. [8 ] have recently demonstrated that halofuginone induced expression of the inhibitory Smad7 and decreased the levels of TGF-ß receptor II, altogether inhibiting the activation of Smad2 and Smad3. Moreover, Furukawa et al. [34 ] have shown that blockade of the p38 MAPK pathway resulted in a decrease of Smad 3 phosphorylation. TGF-ß can also induce activation of the p38 MAPK pathway through an upstream mediator—TGF-ß-activated kinase [39 , 40 ]. Our current finding that halofuginone inhibits the phosphorylation of p38 MAPK is in accordance with this observation, an effect that may be mediated via its inhibition of the TGF-ß signaling pathway.

Halofuginone failed to inhibit SDF-1-mediated chemotaxis of activated T cells (Fig. 8 ). We can therefore conclude that the effects of halofuginone on the regulation of inflammation are most probably a result of its long-term, anti-inflammatory activities (i.e., inhibition of proinflammatory cytokine secretion) rather than an immediate effect on chemotaxis by SDF-1 [41 ].


Figure 8
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  		Figure 8. Effect of halofuginone on T cell chemotaxis induced by SDF-1{alpha}. Halofuginone did not inhibit T cell chemotaxis induced by SDF-1. The results are means (±SD) of three different experiments.

 
Finally, to assess the anti-inflammatory activity of halofuginone in vivo, we applied the DTH mouse model. Treatment with halofuginone effectively inhibited the DTH response, indicating suppression of T cell-mediated inflammation in vivo. In conclusion, we showed that halofuginone is a potent inhibitor of NF-{kappa}B, proinflammatory cytokines, and p38 MAPK phosphorylation in activated T cells in vitro, as well as DTH inflammatory response in vivo. These findings may strengthen its therapeutic potential as an antitumoral and anti-inflammatory compound, in addition to its well-established antifibrotic effect.

Halofuginone can be administered orally, making halofuginone-based therapy clinically attractive. Although the characteristics of a putative halofuginone receptor and the exact downstream signaling pathways are still obscure, our findings provide additional information toward elucidating its mode of action and therapeutic potential.


   ACKNOWLEDGEMENTS
 
This study is dedicated to the memory of Professor Ofer Lider (the Weizmann Institute of Science), a leading scientist in the field of inflammation and autoimmunity, who died from a terminal disease.

Received July 23, 2005; revised February 18, 2006; accepted March 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pines, M., Snyder, D., Yarkoni, S., Nagler, A. (2003) Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma Biol. Blood Marrow Transplant. 9,417-425[CrossRef][Medline]
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