Trp-P-1, a carcinogenic heterocyclic amine, inhibits lipopolysaccharide-induced maturation and activation of human dendritic cells

Cancer Letters 301 (2011) 63–74

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Trp-P-1, a carcinogenic heterocyclic amine, inhibits lipopolysaccharide-induced maturation and activation of human dendritic cells Jun Ho Jeon b,1, Sun Kyung Kim a,1, Jintaek Im a, Ki Bum Ahn a, Jung Eun Baik a, Ok-Jin Park a, Cheol-Heui Yun b, Seung Hyun Han a,⇑ a Department of Oral Microbiology & Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea b Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea

a r t i c l e

i n f o

Article history: Received 20 May 2010 Received in revised form 20 October 2010 Accepted 22 October 2010

Keywords: Trp-P-1 Heterocyclic amines Carcinogen Immunosuppression Dendritic cells Innate immunity

a b s t r a c t Carcinogens frequently provoke immunosuppressive effects thereby allowing cancer cells to persist in the host. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a carcinogenic heterocyclic amine that is abundantly produced by overcooking meat and fish. Here, we investigated the effect of Trp-P-1 on dendritic cells (DCs), which play a central role in the appropriate activation of the host immune system. When human monocyte-derived DCs were stimulated with lipopolysaccharide (LPS), the DCs became mature with an increase in the expression of co-stimulatory receptors such as CD80, CD86, and MHC molecules and a decrease in phagocytic capacity. Trp-P-1 inhibited all of these phenomena under the same conditions. In addition, Trp-P-1 inhibited production of the cytokines TNF-a and IL-12 in LPS-stimulated DCs. Furthermore, DCs that were pre-exposed to Trp-P-1 were less efficient in inducing activation and proliferation of autologous T cells than control DCs. Trp-P-1 also attenuated the ability of DCs to directly kill T-cell lymphoma Jurkat cells. Mechanism studies showed that Trp-P-1 did not inhibit LPS-binding to Toll-like receptor 4 but interfered with the signaling pathways mediated through p38 kinase. In conclusion, our results suggest that Trp-P-1 is immunosuppressive by inhibiting the functionality of DCs that play an essential role in the appropriate induction of anti-cancer immune responses. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Immune responses consist of innate and adaptive branches. The innate immunity serves as the first-line of defense, providing immediate responses against transformed cells or non-self antigens. In contrast, adaptive

⇑ Corresponding author. Address: Department of Oral Microbiology & Immunology, Dental Research Institute, and BK21 Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. Tel.: +82 2 740 8641; fax: +82 2 743 0311. E-mail address: [email protected] (S.H. Han). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.10.023

immunity provides a highly specific response to antigens for effective host defense. Dendritic cells (DCs) are one of the most potent antigen-presenting cells that play a pivotal role in the induction of immune responses against various antigens, including cancers, by orchestrating the innate and adaptive immunities [1]. DCs not only directly kill transformed cells but also induce the activation of cancer-specific adaptive immunity by phagocytosis, digestion, processing, and presentation of tumor antigens to CD4+ or CD8+ T-lymphocytes, eventually leading to effective elimination of the target cancer cells [2]. DCs can be found in almost all tissues throughout the body and perform immunosurveillance in an immature state in which they have high phagocytic capacity but low

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capacity for antigen presentation [3]. Once they encounter tumor antigens, pathogen-associated molecular patterns such as lipopolysaccharide (LPS), or damage-associated molecular patterns, DCs undergo maturation and activation [3]. Mature DCs have lowered phagocytic activity with increased antigen-presentation capacity [3]. Maturation is phenotypically characterized by augmented expression of co-stimulatory molecules such as CD80 and CD86, MHC molecules for antigen presentation, and cytokines such as TNF-a and IL-12, all of which influence the type, intensity, and duration of the subsequent adaptive immunity [4]. Heterocyclic amines (HCAs), also called heterocyclic aromatic amines, are food-borne dietary toxicants that are easily formed by overcooking meat and fish [5,6]. HCAs, including 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), manifest their carcinogenicity in various organs including the liver, prostate, small and large intestines, skin, mammary gland and lymphoid tissues [7–10]. Furthermore, epidemiological studies demonstrated that HCAs are highly associated with colon cancers in humans [11,12]. The carcinogenic effect appears to be mediated by reactive metabolites that are able to form DNA adducts [13,14]. These metabolites are generated through biotransformation by cytochrome P450 and peroxidases and are more potent in their carcinogenicity and mutagenicity than the native structures [15,16]. Carcinogens frequently provoke immunosuppression, in which transformed cells may not be easily removed from the host. Indeed, HCAs such as Trp-P-1, PhIP, and IQ have been reported to have immunosuppressive effects. Trp-P1 inhibits the proliferation of T- and B-lymphocytes in human [17] and animal models [18]. PhIP and IQ also exert an inhibitory effect on the proliferation of T-lymphocytes [19,20]. Aside from their suppressive effect on adaptive immunity, HCAs also show inhibitory effects on innate immunity based on recent findings that Trp-P-1 attenuates LPS-induced IL-8 expression in human promonocytic cell [21] and PhIP inhibits lipoteichoic acid-induced TNF-a expression in a murine macrophage line [22]. Although the immunosuppressive effects of HCAs have been reported, little is known about the effect of HCAs on the function of DCs that are necessary for appropriate activation of the immune system. In the present study, we investigated the effect of HCAs on the maturation and activation of human DCs and their ability to further induce autologous T-cell activation and proliferation.

2. Materials and methods 2.1. Reagents and chemicals Trp-P-1 and ultrapure LPS from Escherichia coli O111:B4 were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Invivogen (San Diego, CA, USA), respectively. Ficoll-Paque Plus was obtained from GE Healthcare Bioscience AB (Uppsala, Sweden). RPMI-1640 medium, fetal bovine serum (FBS), and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) were purchased from HyClone

(Logan, UT, USA). GM-CSF and IL-4 were purchased from Invitrogen (Grand Island, NY, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Phycoerythrin (PE)labeled anti-human CD80 (clone L307.4), allophycocyanin (APC)-labeled anti-human CD86 [clone 2331(FUN-1)], PECy5-labeled anti-HLA-A, B, C (clone G46-2.6) for MHC class I, fluorescein isothiocyanate (FITC)-labeled anti-HLA-DR, DP, DQ (clone Tu39) for MHC class II, PE-labeled antiHLA-DR (clone L243) for MHC class II, Alexa FluorÒ 647-labeled anti-human CD205 (clone MMRI-7), FITC-labeled anti-human CD206 (clone 19.2), PE-labeled anti-human CD69 (clone FN50), FITC-labeled anti-human CD25 (clone BC96), and APC-labeled anti-human CD3 (clone HIT3a) antibodies for flow cytometry were obtained from BD Biosciences (San Diego, CA, USA) or Biolegend (San Diego, CA, USA). Dextran-FITC and 5-(and-6)-carboxyfluoroscein diacetate succinimidyl ester (CFSE) were purchased from Molecular Probes (Eugene, OR, USA). Antibodies specific to p38, phospho-p38 (p-p38), ERK1/2, phospho-ERK1/2 (p-ERK1/2), JNK, phospho-JNK (p-JNK) and anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Berverly, Ma, USA). Monoclonal antibody to b-actin and anti-mouse IgG-HRP were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), respectively. SB203580 and SB202190 were purchased from Calbiochem (Darmstadt, Germany). All other reagents were obtained from Sigma– Aldrich unless otherwise stated. 2.2. Preparation of human monocyte-derived DC Heparinized human blood was obtained from the Red Cross Korea and subjected to density gradient centrifugation using Ficoll-Paque Plus for the preparation of peripheral blood mononuclear cells (PBMC). CD14+ monocytes were isolated from the PBMC using a magnetic bead-based isolation kit, I Mag™ anti-human CD14 particles-DM (BD Biosciences). The CD14+ monocytes (1  106 cells/ml) were then differentiated into immature DCs with 500 U/ml IL-4 and 800 U/ml GM-CSF in RPMI-1640 containing 10% FBS and antibiotics for 6 days. The culture medium containing IL-4 and GM-CSF was replaced every 3 days. All experiments using human blood were conducted under the approval of the Institutional Review Board of the Seoul National University (IRB No.: S-D20060001). 2.3. Analysis of cytotoxicity The immature DCs were treated with various concentration of Trp-P-1 (0, 0.1, 0.3, 1, 3, or 10 lg/ml) for 48 h and stained with annexin V-FITC and propidium iodide (PI) using the annexin V-FITC apoptosis detection kit I (BD Biosciences) according to the manufacturer’s instructions. All flow cytometric data were acquired by FACSCalibur with CellQuest software (BD Biosciences) and analyzed using Flow Jo software (Tree Star, San Carlos, CA, USA). 2.4. Phenotypic analyses of DCs Immature DCs (1  106 cells/ml) were treated with Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation

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with LPS (0.1 lg/ml) for 48 h. After washing with PBS, the cells were stained on ice for 30 min with PE-labeled anti-CD80 antibody; APC-labeled anti-CD86 antibody; PECy5-labeled anti-HLA-A, B, C antibody for MHC class I; FITC-labeled anti-HLA-DR, DP, DQ antibody for MHC class II; Alexa FluorÒ 647-labeled anti-human CD205 antibody; or FITC-labeled anti-human CD206 antibody. The cells were washed three times with PBS containing 2% FBS, then 1  104 cells were subjected to flow cytometry to analyze expression of the phenotypic markers as described above.

re-suspended in RPMI-1640, and plated on a U-bottom 96-well plate at 1  104 cells/well. Immature DCs or LPS-stimulated DCs in the presence or absence of Trp-P-1 were co-cultured with the target cell, Jurkat, at 5:1 or 10:1 ratios. After 18 h, the cells were harvested on Unifilter-96 GF/B (PerkinElmer, CT, USA) and radioactivity was measured by scintillation counting. The cytotoxicity of DC towards Jurkat cells was calculated by the following formula: [1 (cpm of Jurkat with DCs)/(cpm of Jurkat alone)]  100.

2.5. Analysis of phagocytic capacity of DCs

2.9. Measurement of LPS-binding to Toll-like receptor 4 (TLR4)

Immature DCs (1  106 cells/ml) were treated with TrpP-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for 48 h. The cells were washed with PBS, re-suspended in RPMI-1640 complete media, and exposed to 1 mg/ml dextran-FITC for an additional 1 h at 4 °C to measure nonspecific uptake and at 37 °C to measure specific uptake. The cells were then washed four times with ice-cold PBS containing 2% FBS followed by flow cytometric analysis to determine the fluorescence intensity of the cells as described above.

FITC-conjugated LPS was prepared as previously described [24]. The concentration of FITC-conjugated LPS was determined using Limulus amebocyte lysate test kit (QCL-1000; Cambrex Bioscience, Walkersville, MD, USA). Immature DCs (1  106 cells/ml) were treated with DMSO (0.1%) or with Trp-P-1 (1 lg/ml) for 1 h at 37 °C followed by incubation with FITC-labeled LPS at various concentrations (0, 0.1, 0.3, 1, and 3 lg/ml) for additional 1 h at 37 °C. Then, the cells were washed with PBS three times and subjected to flow cytometry as described above.

2.6. Determination of cytokine expression

2.10. Western blot analysis

Immature DCs (1  106 cells/ml) were treated with TrpP-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for 48 h. The culture supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) for the determination of TNF-a and IL-12p70 levels using commercial ELISA kits (DuoSet, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

Immature DCs were pre-treated with DMSO (0.1%) or Trp-P-1(1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for an additional 15 min. The cells were washed with ice-cold PBS and lysed with Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeoggi-Do, Korea) on ice for 30 min. The cell lysates were centrifuged at 13,000  g for 20 min at 4 °C and the protein concentration was measured by BCA protein assay kit (Pierce, Rockford, IL, USA) according to manufacturer’s instruction. Twenty-five micrograms of total proteins were separated by 10% SDS–PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in TBS (50 mM Tris–HCl, 150 mM NaCl, pH 7.6) at room temperature for 1 h and then incubated with primary antibodies in the 5% BSA at 4 °C for overnight.

DCs stimulated with LPS in the presence or absence of Trp-P-1 were prepared as described above. For the T-cell activation assay, the DCs were washed three times with PBS and co-cultured with autologous PBMC at a ratio of 1:10 for 4 days. The cells were then stained with APC-labeled anti-human CD3, PE-labeled anti-human CD69, and FITC-labeled anti-human CD25 antibodies followed by flow cytometric analysis as described above. For the T-cell proliferation assay, PBMC (1  107 cells/ml) were labeled with CFSE by incubation in complete RPMI-1640 containing 10 lM CFSE for 30 min at 37 °C followed by three washes with PBS. The CFSE-labeled PBMC were incubated with autologous DCs for 4 days, and then stained with APClabeled anti-human CD3 antibody for identification of the T-cell population. The cells were subjected to the flow cytometry as described above. 2.8. Measurement of cancer cell-killing activity of DCs The cancer cell-killing activity of DCs was examined by the Just Another Method (JAM) [23]. Briefly, Jurkat cells (a T-cell lymphoma line; 1  105 cells/ml), were pulsed with 1 lCi/ml [3H]-thymidine for 12 h at 37 °C. The radio-labeled cells were washed three times with PBS,

No staining

dide Propidium iod

2.7. Analysis of the capacity of DCs to stimulate T-cell activation and proliferation

0.9

0.1

No treatment 5.8

4.6

DMSO 4.4

8.6

Trp-P-1 Trp P1 (0.1 μg/ml) 3.6

5.9

0.0

11.5

18.9

11.9

Trp-P-1 p (0.3 μg/ml)

Trp-P-1 p (1 μg/ml)

Trp-P-1 p (3 μg/ml)

Trp-P-1 p (10 μg/ml)

3.9

5.5

8.5

6.5

4.3

6.8

9.8

5.9

7.3

5.8

55.2

27.6

Annexin V Fig. 1. Effect of Trp-P-1 on the viability of DCs. Immature DCs were treated with 0.1% DMSO as a vehicle control or various concentrations of Trp-P-1 (0, 0.1, 0.3, 1, 3, or 10 lg/ml) for 48 h. The cells were stained with annexin V-FITC and PI followed by flow cytometric analysis.

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After washing three times with TBST (50 mM Tris–HCl, 150 mM NaCl, 0.05% tween 20, pH 7.6) for 10 min each, the membrane was incubated with an HRP-conjugated

A

No treatment

DMSO

secondary antibody in the blocking buffer at room temperature for 1 h. The membrane was washed three times with TBST for 10 min each and the immuno-reactive bands were

Trp-P-1 (0.1 μg/ml)

Trp-P-1 (0.3 μg/ml)

Trp-P-1 (1 μg/ml)

53

52

57

51

59

374

379

317

269

187

_

LPS

CD80

B

18

20

19

14

15

440

459

326

221

111

_

LPS

CD86

C

28

29

27

27

27

88

81

79

72

56

_

LPS

MHC I

D

20

21

20

17

20

81

74

76

69

53

_

LPS

MHC II Fig. 2. Trp-P-1 attenuates the expression of co-stimulatory molecules and MHC proteins on DCs stimulated with LPS. Immature DCs were pre-treated with 0.1% DMSO as the vehicle control or with various concentrations of Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with 0.1 lg/ml LPS for an additional 48 h. The cells were stained with PE-labeled anti-CD80 antibody (A), APC-labeled anti-CD86 antibody (B), PE-Cy5-labeled anti-HLA-A, B, C antibody (clone G46-2.6) for MHC class I (C), and PE-labeled anti-HLA-DR antibody (clone L243) for MHC class II (D). Expression of each molecule was analyzed by flow cytometry. The dotted line and gray field area indicate the histograms of the isotype control and non-treatment group, respectively, and the black bold line indicates the DMSO or Trp-P-1 treatment groups. The results shown are representative of three separate experiments yielding similar results.

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detected with ECL western blotting reagents (Neuronex Co., Pohang, Korea). Band intensities for the phosphorylated and nonphosphorylated forms of MAP kinases were quantified using a densitometer (Bio-ID, Vilber-Lourmat, Marne La Vallee, France).

4

A

/

2.11. Statistical analysis All experiments were performed at least three times. Data were expressed as mean values ± standard deviation (SD) of triplicate samples in each experimental group.

37

No ttreatment t t

DMSO

Trp-P-1 (0 1 μg/ml) / l) (0.1

Trp-P-1 (0 3 μg/ml) / l) (0.3

Trp-P-1 (1 μg/ml) / l)

6 / 76

6 / 79

5 / 55

8 / 92

7 / 90

3/4

3/5

3/7

3/8

4 / 14

_

LPS

FITC-Dextran

B _

No treatment

DMSO

Trp-P-1 (0.1 μg/ml)

Trp-P-1 (0.3 μg/ml)

Trp-P-1 (1 μg/ml)

33

32

31

28

30

94

98

84

72

57

LPS

CD205

C _

No treatment

DMSO

Trp-P-1 p (0.1 μg/ml)

Trp-P-1 p (0.3 μg/ml)

Trp-P-1 p (1 μg/ml)

107

98

96

99

82

46

42

59

60

63

LPS

CD206 Fig. 3. Trp-P-1 retards the attenuation of phagocytic capacity of DCs at maturation induced by LPS. Immature DCs were pre-treated with 0.1% DMSO as the vehicle control or with various concentrations of Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with 0.1 lg/ml LPS for an additional 48 h. For analysis of phagocytic activity, the cells were incubated with dextran-FITC (1 mg/ml) for 1 h at 4 °C (gray field area) or 37 °C (black bold line) followed by flow cytometric analysis. MFI is shown in the upper right of each histogram (MFI at 4 °C/MFI at 37 °C). The results shown are representative of three separate experiments yielding similar results (A). For analysis of phagocytosis-related receptors, the cells were stained with Alexa FluorÒ 647-labeled anti-human CD205 antibody (B) or FITC-labeled anti-human CD206 antibody (C). Expression of each molecule was analyzed by flow cytometry. The dotted line and gray field area indicate the histograms of the isotype control and non-treatment group, respectively, and the black bold line indicates the DMSO or Trp-P-1 treatment groups. One of three similar results is shown.

J.H. Jeon et al. / Cancer Letters 301 (2011) 63–74

3. Results 3.1. Effect of Trp-P-1 on the viability of DCs In order to examine the effect of Trp-P-1 on the viability of DCs, immature DCs were treated with 0.1% DMSO as a vehicle control or various concentrations of Trp-P-1 (0, 0.1, 0.3, 1, 3, or 10 lg/ml) for 48 h. Then, the cells were stained with annexin V-FITC and PI followed by flow cytometric analysis. In general, early apoptotic cells were annexin V-FITCpositive and PI-negative and late apoptotic or necrotic cells were annexin V-FITC-positive and PI-positive. As shown in Fig. 1, Trp-P-1 did not exhibit any toxicity up to 3 lg/ml whereas there was a substantial increase in the cytotoxicity at 10 lg/ml. Thus, 1 lg/ml was the maximum concentration used in further experiments. 3.2. Trp-P-1 suppresses LPS-induced expression of maturation markers on DCs It is well known that, during maturation, DCs up-regulate the expression of co-stimulatory molecules such as CD80 and CD86 [4]. To examine the effect of Trp-P-1 on the expression of co-stimulatory molecules, immature DCs (1  106 cells/ml) were pre-treated with Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for an additional 48 h. Upon exposure to LPS alone, DCs highly expressed the co-stimulatory molecules CD80 and CD86, and MHC class I and II (Fig. 2). Trp-P-1 did down-regulate the expression of these maturation markers in a dose-dependent manner under identical conditions (Fig. 2). 3.3. Trp-P-1 prevents the attenuation of phagocytic capacity in mature DCs DCs are highly phagocytic in an immature state but their phagocytic capacity is reduced in mature state [25]. To examine the effect of Trp-P-1 on the phagocytic capacity of DC, immature DCs (1  106 cells/ml) were pre-treated with Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for an additional 48 h. As expected, immature DCs showed high phagocytic capability (MFI at 37 °C = 76) whereas LPSstimulated DCs showed markedly reduced phagocytic activity (MFI at 37 °C = 4) (Fig. 3A). In contrast, pre-treatment with Trp-P-1 elicited an increase in the phagocytic activity of stimulated cells (MFIs of 0.1, 0.3, and 1 lg/ml Trp-P-1 groups at 37 °C were 7, 8, and 14, respectively) (Fig. 3A). Since Trp-P-1 markedly induced the phagocytic activity of DCs that is typically decreased by LPS stimulation, we next examined expression of the phagocytosis-related receptors, CD205 and CD206. Increased expression of CD205 and decreased expression of CD206 were observed on DCs matured with LPS (Fig. 3B and C), which are typical changes during the maturation of DCs. However, as expected, these changes in the expression of CD205 and CD206 were reversed in the presence of Trp-P-1 (Fig. 3B and C). 3.4. Trp-P-1 decreases the production of cytokines IL-12 and TNF-a by LPSmatured DCs Since LPS-matured DCs produce cytokines such as TNF-a and IL-12, resulting in regulation of the subsequent adaptive immunity [26], we investigated the effect of Trp-P-1 on cytokine expression in DCs. Robust induction of cytokine expression was observed in LPS-stimulated DCs (Fig. 4A and B). Upon exposure of LPS-stimulated DCs to Trp-P-1, expression of IL-12 and TNF-a was down-regulated in a dose-dependent manner (Fig. 4A and B). 3.5. Trp-P-1 attenuates T-cell proliferation and activation induced by mature DCs Mature DCs mediate the activation of adaptive immunity through induction of T lymphocyte activation and proliferation [1]. To examine whether Trp-P-1 affects the ability of DCs to stimulate proliferation and activation of autologous T-lymphocytes, CFSE-labeled PBMC were co-cultured with mature DCs that were pre-treated with or without Trp-P-1. Co-culture with LPS-matured DCs induced T-lymphocyte proliferation

comparable to that induced by treatment with a T-cell mitogen, concanavalin A (Con A) (Fig. 5A). However, Trp-P-1 reduced the ability of DCs to stimulate T-lymphocyte proliferation in a dose-dependent fashion (Fig. 5A). Concomitantly, Trp-P-1 also inhibited the capacity of mature DC to activate T-lymphocytes as evidenced by a decrease in the expression of T-cell activation markers CD25 (Fig. 5B) and CD69 (Fig. 5C). These results suggest that Trp-P-1 impairs DC maturation and activation leading to reduction of their potential to activate T-lymphocytes. 3.6. Trp-P-1 decreases the cancer cell-killing activity of DCs DCs not only mediate the regulation of adaptive immunity against cancer cells but also directly kill them through various mechanisms [27–29]. To determine whether Trp-P-1 alters the cancer cell-killing activity of DCs, DCs were stimulated with LPS in the presence or absence of Trp-P-1 and co-cultured with human T-cell lymphoma Jurkat cells. As shown in Fig. 6, mature DCs exhibit superior killing activity against Jurkat cells compared with immature DCs. When exposed to Trp-P-1, the mature DCs showed a diminished killing activity against Jurkat cells (Fig. 6), suggesting that Trp-P-1 negatively affects the cancer cell-killing activity of DCs.

A

4

With t LPS Without

3

IL--12 (ng g/ml)

Comparative data were analyzed using Student’s t-test and considered statistically significant when the P value was less than 0.05.

With LPS

*

* *

2

1

0 -

DMSO

0.1

0.3

1

Trp-P-1 Trp P 1 (μg/ml)

B

4

Without LPS

With LPS

*

3

TNF F-α (ng g/ml)

68

*

*

2

1

0 -

DMSO

0.1

0.3

1

Trp Trp-P-1 P 1 (μg/ml) Fig. 4. Trp-P-1 down-regulates IL-12 and TNF-a in DCs matured with LPS. Immature DCs were pre-treated with 0.1% DMSO as the vehicle control or with various concentrations of Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h, followed by stimulation with 0.1 lg/ml LPS for an additional 48 h. At the end of the stimulation period, the culture supernatants were collected and subjected to ELISA for determination of IL-12 and TNF-a. Values are mean ± SD of triplicate assays. Indicates statistical significance at P < 0.05. The results shown are representative of three separate experiments with similar results.

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CD3

A

No treatment

ConA

1.5

5.8

PBMC co-cultured with DC matured in the presence of DMSO

5.1

5.1

Trp-P-1 (0.1 µg/ml) 4.1

Trp-P-1 (0.3 µg/ml) 3.5

Trp-P-1 (1.0 µg/ml) 2.4

CFSE

B CD3

Isotype control

No treatment

6.4

14.5

ConA 23.9

PBMC co-cultured with DC matured in the presence of DMSO

12.9

13.4

Trp-P-1 (0.1 µg/ml)

Trp-P-1 (0.3 µg/ml)

Trp-P-1 (1.0 µg/ml)

12.1

11.7

10.0

CD25

CD3

C

Isotype control

No treatment

9.1

20.7

ConA 51.4

PBMC co-cultured with DC matured in the presence of

-

DMSO 27.8

28.1

Trp-P-1 (0.1 µg/ml)

Trp-P-1 (0.3 µg/ml)

Trp-P-1 (1.0 µg/ml)

26.3

25.0

25.9

CD69 Fig. 5. Trp-P-1 decreases the ability of mature DCs to induce T-cell proliferation and activation. Immature DCs were pre-treated with 0.1% DMSO as the vehicle control or with various concentrations of Trp-P-1 (0, 0.1, 0.3, or 1 lg/ml) for 1 h, followed by stimulation with 0.1 lg/ml LPS for an additional 48 h. For analysis of T-cell proliferation capacity, the DCs were co-cultured with CFSE-labeled autologous PBMC at a 1:10 ratio for an additional 4 days followed by flow cytometric analysis (A). To examine the capacity for T-cell activation, the DCs were co-cultured with autologous PBMC at a 1:10 ratio for an additional 4 days then stained with APC-labeled anti-human CD3 together with FITC-labeled anti-human CD25 (B) or PE-labeled anti-human CD69 (C) antibodies followed by flow cytometric analysis. One of three similar results is shown.

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Immature DC Mature DC M t Mature DC with ith DMSO Mature DC with Trp-P-1 p = 0.017

0 047 p = 0.047

p = 0.054

p = 0.007

1:10

1:5

Cy ytotox xicity (%)

40 30 20 10 0

Jurkat to DC ratio Fig. 6. Trp-P-1 attenuates the cancer cell-killing activity of DC matured with LPS. Jurkat cells were labeled with [3H]-thymidine and plated at 1  104 cells/well in a U-bottom 96-well plate. Immature DCs (1  106 cells/ml) were stimulated with LPS for 24 h in the presence or absence of Trp-P-1, then washed and co-cultured with the radiolabeled Jurkat cells at a ratio of 5:1 or 10:1 for an additional 18 h. At the end of the culture period, the cells were harvested on a glass fiber filter and the radioactivity was measured using a scintillation counting system. Cancer cell-killing activity was calculated as described in Section 2.

3.7. Trp-P-1 suppresses the phosphorylation of p38, but not ERK1/2 or JNK kinase LPS is known to be recognized by TLR4 for triggering intracellular signaling cascades leading to activation of MAP kinases that play an important role in DC maturation and activation [30]. Therefore, we hypothesized that Trp-P-1 interfered with LPS-binding to TLR4 and/or the signaling pathways. Flow cytometric analysis showed that Trp-P-1 did not affect binding of LPS to TLR4 on DCs (Fig. 7A). Western blot analysis demonstrated that LPS treatment maximally increased phosphorylation of the MAP kinases at 15 min after stimulation (Fig. 7B) and pre-treatment with Trp-P-1 inhibited the LPS-induced phosphorylation of p38 kinase, but not those of ERK1/2 and JNK (Fig. 7C). Furthermore, LPS-induced DC maturation was remarkably suppressed by the p38 kinase inhibitors SB203580 and SB202190 at the concentrations with no observed cytotoxicity (Fig. 8A and B). These results suggest that Trp-P-1 inhibited the LPS-induced maturation and activation of DC through the inhibition of p38 signaling pathways.

4. Discussion Although transformed cells are occasionally generated in our body, cancer cells rarely survive in healthy conditions due to their elimination by the host immune system. Carcinogens that promote the initiation and progression of tumor growth often induce immunosuppression, allowing cancer cells to resist the host defenses and survive. In this study, we demonstrated an inhibitory effect of Trp-P-1 on the maturation and activation of human DCs based on the findings that Trp-P-1 inhibited the following: (i) augmentation of expression of co-stimulatory receptors, (ii) induction of MHC protein expression, (iii) decreased phagocytic capacity of DCs, (iv) the production of cytokines TNF-a and IL-12, (v) the ability of DCs to activate T-cell proliferation and activation, and (vi) the ability of DCs to kill cancer

cells. In light of the fact that DCs play a central role in anti-cancer immunity, not only by directly killing the transformed cells but also by activating cancer-specific adaptive immunity, our results showing the inhibitory effects of Trp-P-1 on DC function suggests a novel mechanism for the immunosuppressive effect of HCAs. The type, intensity, and duration of adaptive immunity are determined by three kinds of signals provided by DCs, i.e., recognition of MHC/antigen complex by T cell receptor, co-stimulation, and cytokines [31]. Interestingly, Trp-P-1 appears to inhibit activation of adaptive immunity at multiple steps, based on the following findings: Firstly, it reduced LPS-induced expression of MHC proteins, which are essential for the presentation of cancer antigens. Secondly, Trp-P1 diminished the expression of co-stimulatory molecules, CD80 and CD86. Thirdly, it reduced cytokine production in LPS-stimulated DCs. Remarkably, the decreased production of IL-12 by Trp-P-1 could potentially weaken the effective removal of cancer cells since IL-12 facilitates the polarization of immune responses into a Th-1 type, leading to the preferential activation of cytotoxic T-lymphocytes, which are major killers of cancer cells [32]. Although the inhibitory effect of Trp-P-1 on DCs is likely to primarily affect adaptive immunity by inhibiting T-lymphocytes, Trp-P-1 could also attenuate the ability of DCs to kill cancer cells as demonstrated by the JAM experiment using Jurkat cells. Accumulating evidence suggests two potential mechanisms for the direct killing of cancer cells by DCs (also called killer DCs). Firstly, DCs secrete soluble mediators such as nitric oxide, TNF-a, perforin, and granzymes, all of which are involved in the killing of target cells [28,29,33]. Secondly, DCs can activate death receptors such as receptors for TNF-related apoptosis-inducing ligand (TRAIL) on cancer cells, leading to apoptotic death [34]. Thus, Trp-P-1 may attenuate the cancer killing activity of DCs through interfering with one of these two mechanisms, or with both in concert. Mechanism studies using flow cytometry and Western blotting demonstrated that Trp-P-1 did not affect LPSbinding to TLR4 but decreased p38 phosphorylation. This mechanism seems to be due to the molecular structure of Trp-P-1. In fact, there are two classes of HCAs according to polarity: polar HCAs and non-polar HCAs. Trp-P-1 belongs to the non-polar HCAs because of its pyridoindole moiety [35]. Notably, the polarity influences molecular absorption, distribution, metabolism, and excretion [36]. In general, non-polar molecules are easily absorbed into the cells. More interestingly, Trp-P-1 structure resembles the p38 kinase inhibitors such as SB203580 and 202190 which possess a pyridylimidazole moiety [37]. Indeed, selective inhibition of p38 kinase among MAP kinases often results in the attenuation of maturation and cytokine production of DCs [38–40]. Therefore, Trp-P-1 is likely to penetrate the cell membrane and directly inhibits the phosphorylation of p38 MAP kinase through a mechanism mimicking the inhibitors of p38 kinase, consequently leading to the attenuation of DC maturation and activation. Previous reports suggest that most HCAs are able to induce mutagenesis and carcinogenesis after metabolic activation by cytochrome P450s rather than in their native forms [15,16]. Although HCAs are mainly metabolized by

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A

FITC-LPS C S 0 μg μg/ml _

0.3 μg/ml μg

0.1 μg μg/ml

1 μg/ml μg

3 μg/ml μg

5.2

7.1

11.7

29.9

73.1

50 5.0

68 6.8

13 4 13.4

28 28.7 7

73.5 73 5

5.2

6.6

13.3

30.8

74.1

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Trp P 1 Trp-P-1

FITC

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120

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1 0 1.0

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22 2.2

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p-ERK/ERK 1.0

2.7

0.9

3.1

1.1

3.4

4.9

1.2

4.7

0.9

4.9

p38

p38 3 3 3.3

3 7 3.7

2 4 2.4

2 2 2.2

10 1.0

p-p38/p38

p ERK p-ERK

p ERK p-ERK

ERK

ERK

p-ERK/ERK 1.0

12.3 13.6

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p-JNK

JNK

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p-p38

1 0 1.0

DMSO

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p-p38/p38

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p-JNK/JNK

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β-actin

Fig. 7. Trp-P-1 does not affect LPS-binding to TLR4 but decreases the phosphorylation of p38 kinase. (A) Immature DCs (1  106 cells/ml) were pre-treated with DMSO (0.1%) as a vehicle control or Trp-P-1 (1 lg/ml) for 1 h followed by incubation with FITC-conjugated LPS for additional 1 h at 37 °C. Binding of LPS to TLR4 on DC was analyzed by flow cytometry. (B) Immature DCs (2  106 cells/ml) were stimulated with LPS (0.1 lg/ml) for the indicated time periods and subjected to Western blot analysis to determine the intracellular levels of p38 kinase, ERK1/2, JNK, and their phosphorylated forms. Relative values of phosphorylated forms to nonphosphorylated forms were determined by densitometry. (C) Immature DCs (2  106 cells/ml) were pre-treated with DMSO (0.1%) or Trp-P-1(1 lg/ml) for 1 h followed by stimulation with LPS (0.1 lg/ml) for an additional 15 min. Then, the cells were subjected to Western blot analysis to determine the intracellular levels of p38 kinase, ERK1/2, JNK, and their phosphorylated forms. Relative values of phosphorylated forms to nonphosphorylated forms were determined by densitometry.

cytochrome P450 1A2 leading to formation of DNA adducts in the liver, other P450 subtypes such as 1A1, 1B1, and 3A4 are also involved in HCA metabolism [41,42]. Interestingly, human DCs express cytochrome P450 1A1, 1A2, and 1B1 subtypes [43]. On the other hand, we found that HCAs could inhibit the in vitro proliferation and activation of

lymphocytes that barely express the major drug metabolizing enzymes such as cytochrome P450s [18–20]. These results imply that the parent structures are also able to provoke the immunosuppressive effect. Thus, it would be intriguing to further determine whether the inhibitory effect of Trp-P-1 on human DCs is caused by the parent

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LPS

DMSO + LPS

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90.5

7.8

92.9

3.8

93 2 93.2

3.5

LPS SB203580 ((2.5 μ μM))

SB203580 ((5 μ μM))

SB203580 (10 μM)

1.1

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94.2 94 2

3.8 38

92.2 92 2

58 5.8

87 4 87.4

10 2 10.2

LPS

PI

SB202190 (0.5 μM)

SB202190 (1 μM)) ( μ

SB202190 μM)) S 0 90 ((2 μ

1.21

0.7

1.4

1.0

1.1

0.6

95 7 95.7

25 2.5

94 3 94.3

3.4 34

94 6 94.6

35 3.5

A Annexin i V

B No treatment

LPS _

DMSO

SB203580 SB203580 SB203580 SB202190 SB202190 SB202190 2 5 μM 5 μM 10 μM 0 5 μM 1 μM 2 μM 2.5 0.5

CD80

CD86 15

MHC I

MHC II Fig. 8. Inhibitors for p38 kinase decrease the expression of co-stimulatory molecules and MHC proteins on DCs stimulated with LPS. Immature DCs (1  106 cells/ml) were pre-treated with 0.1% DMSO, SB203580 (2.5, 5, or 10 lM), or SB202190 (0.5, 1, or 2 lM) for 1 h followed by stimulation with LPS at 0.1 lg/ml for additional 48 h. Then, the cells were stained with (A) annexin V-FITC and PI, or (B) PE-labeled anti-CD80 antibody, APC-labeled anti-CD86 antibody, PECy5-labeled anti-MHC class I antibody and FITC-labeled anti-MHC class II antibody. Then, the cells were subjected to the flow cytometric analysis.

structure, its metabolites generated within the DCs themselves, or by both. HCAs are a dietary carcinogen highly-threatening human health since they are continuously ingested with overcooked foods. Our results show that Trp-P-1 impairs the maturation and activation of human DCs resulting in

attenuation of their capacity to kill cancer cells and stimulate T-cell activation and proliferation. Ultimately, our findings indicate that Trp-P-1 provides a favorable situation to the generation and promotion of cancers by inhibiting the host immune system and thereby allowing cancer cells to resist the host defenses.

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Conflicts of interest None declared. Acknowledgements This study was supported by grants from the Expansion of Nuclear R&D Infrastructure Program through the Korea Science and Engineering Foundation (2008-01571), from the Basic Science Research Program through the National Research Foundation of Korea (NRF, 2009-0075529), and from the Science Research Center to the Bone Metabolism Research Center (2009-0063271) funded by the Korean Ministry of Education, Science and Technology, Republic of Korea. References [1] J. Banchereau, R.M. Steinman, Dendritic cells and the control of immunity, Nature 392 (1998) 245–252. [2] R.S. Goldszmid, J. Idoyaga, A.I. Bravo, R. Steinman, J. Mordoh, R. Wainstok, Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma, J. Immunol. 171 (2003) 5940–5947. [3] M. Rescigno, F. Granucci, S. Citterio, M. Foti, P. Ricciardi-Castagnoli, Coordinated events during bacteria-induced DC maturation, Immunol. Today 20 (1999) 200–203. [4] F. Sallusto, M. Cella, C. Danieli, A. Lanzavecchia, Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products, J. Exp. Med. 182 (1995) 389–400. [5] J.S. Felton, M.G. Knize, N.H. Shen, P.R. Lewis, B.D. Andresen, J. Happe, F.T. Hatch, The isolation and identification of a new mutagen from fried ground beef: 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), Carcinogenesis 7 (1986) 1081–1086. [6] Z. Yamaizumi, T. Shiomi, H. Kasai, S. Nishimura, Y. Takahashi, M. Nagao, T. Sugimura, Detection of potent mutagens, Trp-P-1 and TrpP-2, in broiled fish, Cancer Lett. 9 (1980) 75–83. [7] H. Esumi, H. Ohgaki, E. Kohzen, S. Takayama, T. Sugimura, Induction of lymphoma in CDF1 mice by the food mutagen, 2-amino-1-methyl6-phenylimidazo[4,5-b]pyridine, Jpn. J. Cancer Res. 80 (1989) 1176– 1178. [8] T. Kato, H. Migita, H. Ohgaki, S. Sato, S. Takayama, T. Sugimura, Induction of tumors in the Zymbal gland, oral cavity, colon, skin and mammary gland of F344 rats by a mutagenic compound, 2-amino3,4-dimethylimidazo[4,5-f]quinoline, Carcinogenesis 10 (1989) 601– 603. [9] T. Shirai, M. Sano, S. Tamano, S. Takahashi, M. Hirose, M. Futakuchi, R. Hasegawa, K. Imaida, K. Matsumoto, K. Wakabayashi, T. Sugimura, N. Ito, The prostate: a target for carcinogenicity of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods, Cancer Res. 57 (1997) 195–198. [10] S. Takayama, Y. Nakatsuru, H. Ohgaki, S. Sato, T. Sugimura, Carcinogenicity in rats of a mutagenic compound, 3-amino-1,4dimethyl-5H-pyrido[4,3-b]indole, from tryptophan pyrolysate, Jpn. J. Cancer Res. 76 (1985) 815–817. [11] L.M. Butler, R. Sinha, R.C. Millikan, C.F. Martin, B. Newman, M.D. Gammon, A.S. Ammerman, R.S. Sandler, Heterocyclic amines, meat intake, and association with colon cancer in a population-based study, Am. J. Epidemiol. 157 (2003) 434–445. [12] S. Nowell, B. Coles, R. Sinha, S. MacLeod, D. Luke Ratnasinghe, C. Stotts, F.F. Kadlubar, C.B. Ambrosone, N.P. Lang, Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer: contribution of metabolic variation to risk, Mutat. Res. 506–507 (2002) 175–185. [13] P. Baranczewski, J.A. Gustafsson, L. Moller, DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC, Biomarkers 9 (2004) 243–257. [14] Y. Totsuka, K. Fukutome, M. Takahashi, S. Takahashi, A. Tada, T. Sugimura, K. Wakabayashi, Presence of N2-(deoxyguanosin-8-yl)-

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

73

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) in human tissues, Carcinogenesis 17 (1996) 1029–1034. W. Pfau, H. Marquardt, Cell transformation in vitro by food-derived heterocyclic amines Trp-P-1, Trp-P-2 and N(2)-OH-PhIP, Toxicology 166 (2001) 25–30. E. Wolz, W. Pfau, G.H. Degen, Bioactivation of the food mutagen 2amino-3-methyl-imidazo[4,5-f]quinoline (IQ) by prostaglandin-H synthase and by monooxygenases: DNA adduct analysis, Food Chem. Toxicol. 38 (2000) 513–522. H. Yanagisawa, O. Wada, Inhibitory effects of carcinogenic tryptophan pyrolysis products on phytohemagglutinin-induced blast transformation of human lymphocytes, Toxicol. Lett. 51 (1990) 227–231. C.H. Yun, D.K. Chung, K. Yoon, S.H. Han, Involvement of reactive oxygen species in the immunosuppressive effect of 3-amino-1,4dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), a food-born carcinogenic heterocyclic amine, Toxicol. Lett. 164 (2006) 37–43. Y.W. Lee, S.H. Han, J.H. Suh, Y.J. Jeon, K.H. Yang, Down-regulation of protein kinase C: a potential mechanism for 2-amino-3methylimidazo[4,5-f]quinoline-mediated immunosuppression, Toxicol. Lett. 102–103 (1998) 79–83. C.H. Yun, C.G. Son, U. Jung, S.H. Han, Immunosuppressive effect of 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through the inhibition of T-lymphocyte proliferation and IL-2 production, Toxicology 217 (2006) 31–38. J. Im, S.S. Kang, J.S. Yang, C.H. Yun, Y. Yang, S.H. Han, 3-Amino-1,4dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) attenuates LPS-induced IL-8 expression by decreasing mRNA stability in THP-1 cells, Toxicol. Lett. 177 (2008) 108–115. J. Im, H.S. Choi, S.K. Kim, S.S. Woo, Y.H. Ryu, S.S. Kang, C.H. Yun, S.H. Han, A food-born heterocyclic amine, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP), suppresses tumor necrosis factor-alpha expression in lipoteichoic acid-stimulated RAW 264.7 cells, Cancer Lett. 274 (2009) 109–117. D. Usharauli, A. Perez-Diez, P. Matzinger, The JAM Test and its daughter P-JAM: simple tests of DNA fragmentation to measure cell death and stasis, Nat. Protoc. 1 (2006) 672–682. A. Troelstra, P. Antal-Szalmas, L.A. de Graaf-Miltenburg, A.J. Weersink, J. Verhoef, K.P. Van Kessel, J.A. Van Strijp, Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes, Infect. Immun. 65 (1997) 2272–2277. C. Reis e Sousa, A. Sher, P. Kaye, The role of dendritic cells in the induction and regulation of immunity to microbial infection, Curr. Opin. Immunol. 11 (1999) 392–399. T. Kaisho, O. Takeuchi, T. Kawai, K. Hoshino, S. Akira, Endotoxininduced maturation of MyD88-deficient dendritic cells, J. Immunol. 166 (2001) 5688–5694. N.A. Fanger, C.R. Maliszewski, K. Schooley, T.S. Griffith, Human dendritic cells mediate cellular apoptosis via tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL), J. Exp. Med. 190 (1999) 1155–1164. M. Schmitz, S. Zhao, Y. Deuse, K. Schakel, R. Wehner, H. Wohner, K. Holig, F. Wienforth, A. Kiessling, M. Bornhauser, A. Temme, M.A. Rieger, B. Weigle, M. Bachmann, E.P. Rieber, Tumoricidal potential of native blood dendritic cells: direct tumor cell killing and activation of NK cell-mediated cytotoxicity, J. Immunol. 174 (2005) 4127– 4134. G. Stary, C. Bangert, M. Tauber, R. Strohal, T. Kopp, G. Stingl, Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells, J. Exp. Med. 204 (2007) 1441–1451. D. Dowling, C.M. Hamilton, S.M. O’Neill, A comparative analysis of cytokine responses, cell surface marker expression and MAPKs in DCs matured with LPS compared with a panel of TLR ligands, Cytokine 41 (2008) 254–262. M.L. Kapsenberg, Dendritic-cell control of pathogen-driven T-cell polarization, Nat. Rev. Immunol. 3 (2003) 984–993. A.S. Giermasz, J.A. Urban, Y. Nakamura, P. Watchmaker, R.L. Cumberland, W. Gooding, P. Kalinski, Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines, Cancer Immunol. Immunother. 58 (2009) 1329– 1336. A. Nicolas, D. Cathelin, N. Larmonier, J. Fraszczak, P.E. Puig, A. Bouchot, A. Bateman, E. Solary, B. Bonnotte, Dendritic cells trigger tumor cell death by a nitric oxide-dependent mechanism, J. Immunol. 179 (2007) 812–818. J. Shi, K. Ikeda, N. Fujii, E. Kondo, K. Shinagawa, F. Ishimaru, K. Kaneda, M. Tanimoto, X. Li, Q. Pu, Activated human umbilical cord blood dendritic cells kill tumor cells without damaging normal hematological progenitor cells, Cancer Sci. 96 (2005) 127–133.

74

J.H. Jeon et al. / Cancer Letters 301 (2011) 63–74

[35] M. Murkovic, Formation of heterocyclic aromatic amines in model systems, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 802 (2004) 3–10. [36] J.J. Rafter, J.A. Gustafsson, Metabolism of the dietary carcinogen TrpP-1 in rats, Carcinogenesis 7 (1986) 1291–1295. [37] K.W. Cheng, F. Chen, M. Wang, Heterocyclic amines: chemistry and health, Mol. Nutr. Food Res. 50 (2006) 1150–1170. [38] Y.L. Lin, Y.C. Liang, S.S. Lee, B.L. Chiang, Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways, J. Leukoc. Biol. 78 (2005) 533–543. [39] L. Palova-Jelinkova, D. Rozkova, B. Pecharova, J. Bartova, A. Sediva, H. Tlaskalova-Hogenova, R. Spisek, L. Tuckova, Gliadin fragments induce phenotypic and functional maturation of human dendritic cells, J. Immunol. 175 (2005) 7038–7045.

[40] K. Pan, H. Wang, W.L. Liu, H.K. Zhang, J. Zhou, J.J. Li, D.S. Weng, W. Huang, J.C. Sun, X.T. Liang, J.C. Xia, The pivotal role of p38 and NFkappaB signal pathways in the maturation of human monocytederived dendritic cells stimulated by streptococcal agent OK-432, Immunobiology 214 (2009) 350–358. [41] G.J. Hammons, D. Milton, K. Stepps, F.P. Guengerich, R.H. Tukey, F.F. Kadlubar, Metabolism of carcinogenic heterocyclic and aromatic amines by recombinant human cytochrome P450 enzymes, Carcinogenesis 18 (1997) 851–854. [42] T. Shimada, C.L. Hayes, H. Yamazaki, S. Amin, S.S. Hecht, F.P. Guengerich, T.R. Sutter, Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1, Cancer Res. 56 (1996) 2979–2984. [43] S. Sieben, J.M. Baron, B. Blomeke, H.F. Merk, Multiple cytochrome P450-isoenzymes mRNA are expressed in dendritic cells, Int. Arch. Allergy Immunol. 118 (1999) 358–361.