Capsaicin mediates apoptosis in
human nasopharyngeal carcinoma
NPC-TW 039 cells through
mitochondrial depolarization and
endoplasmic reticulum stress
S-W Ip
1, S-H Lan
1, H-F Lu
2,3, A-C Huang
4, J-S Yang
5,
J-P Lin
6, H-Y Huang
1, J-C Lien
7, C-C Ho
8, C-F Chiu
9,
WG Wood
10and J-G Chung
11,12Abstract
Capsaicin, a pungent compound found in hot chili peppers, has been reported to have antitumor activities in many human cancer cell lines, but the induction of precise apoptosis signaling pathway in human nasopharyn-geal carcinoma (NPC) cells is unclear. Here, we investigated the molecular mechanisms of capsaicin-induced apoptosis in human NPC, NPC-TW 039, cells. Effects of capsaicin involved endoplasmic reticulum (ER) stress, caspase-3 activation and mitochondrial depolarization. Capsaicin-induced cytotoxic effects (cell death) through G0/G1 phase arrest and induction of apoptosis of NPC-TW 039 cells in a dose-dependent manner. Capsaicin treatment triggered ER stress by promoting the production of reactive oxygen species (ROS), increasing levels of inositol-requiring 1 enzyme (IRE1), growth arrest and DNA-damage-inducible 153 (GADD153) and glucose-regulated protein 78 (GRP78). Other effects included an increase in cytosolic Ca2þ, loss of the mitochondrial transmembrane potential (DCm), releases of cytochrome c and apoptosis-inducing factor (AIF), and activation
of caspase-9 and -3. Furthermore, capsaicin induced increases in the ratio of Bax/Bcl-2 and abundance of apoptosis-related protein levels. These results suggest that ER stress- and mitochondria-mediated cell death is involved in capsaicin-induced apoptosis in NPC-TW 039 cells.
Keywords
capsaicin; human nasopharyngeal carcinoma NPC-TW 039 cells; apoptosis; mitochondria; caspases-3 activation
1Department of Nutrition, China Medical University, Taichung, Taiwan 2
Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan
3
College of Human Ecology, Fu Jen Catholic University, New Taipei, Taiwan
4
Department of Nursing, St. Mary’s Medicine Nursing and Management College, Yilan, Taiwan
5
Department of Pharmacology, China Medical University, Taichung, Taiwan
6
School of Chinese Medicine, China Medical University, Taichung, Taiwan
7
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
8Department of Nursing, Central Taiwan University of Science and Technology, Taichung, Taiwan 9
Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan
10
Department of Pharmacology, University of Minnesota, School of Medicine, Geriatric Research, Education and Clinical Center, VA Medical Center, Minneapolis, Minnesota, USA
11
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
12
Department of Biotechnology, Asia University, Taichung, Taiwan Corresponding author:
Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan
Email: [email protected]
Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327111417269 het.sagepub.com
Introduction
Nasopharyngeal carcinoma (NPC) is an endemic disease in southern China and Southeast Asia, with incidence rates of 15–50 per 100,000.1Overall, NPC exists with high frequency in Asian populations, espe-cially among Chinese people.2,3The NPC can spread to lymph nodes and metastasize to other tissue, which causes a poor prognosis.4Treatment of NPC has been problematic. Eye or facial paralysis and dietary prob-lems have been demonstrated following chemother-apy and radiation therchemother-apy exposure, hence, the need for new therapeutic agents.5,6
Capsaicin (8-methyl-N-vanillyl-6-nonenamide) has been shown to act as a chemopreventive and chemotherapeutic agent in different human cancer models.7–12 In leukemia cells, capsaicin triggered apoptosis via a p53-dependent mechanism.8In human esophagus epidermoid carcinoma cells, capsaicin induced apoptosis through the elevation of intracellu-lar reactive oxygen species (ROS) and Ca2þ produc-tions and caspase-3 activation.9 Capsaicin-affected prostate cancer cells were associated with apoptosis through a p53-independent pathway.7 In colorectal cancer cells, capsaicin-induced apoptosis is elevated by cotreatment with the adenosine monophosphate-activated protein kinase activator.13 Moreover, cap-saicin treatment suppressed azoxymethane-induced aberrant cryptic foci formation in rats in vivo.14
Numerous studies have shown that capsaicin has anticarcinogen,15 antimutagenic9 and anticancer activities.16However, there is no information regard-ing the anticancer activity of capsiacin in NPC cells and its molecular mechanisms of apoptosis induction are not understood. Therefore, the aim of this study was to investigate the apoptotic action of capsaicin on human NPC, NPC-TW 039, cells in vitro.
Materials and methods
Chemicals and reagents
Capsaicin, dimethyl sulfoxide (DMSO), propidium iodide (PI), RNase and trypan blue were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), penicillin–streptomycin, trypsin-EDTA, L-glutamine, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 1-[2-Amino-5-(6-carboxy indol-2-yl)phenol]-2-(20-amino-50methylphenoxy)ethane-N,N,N0,N0-tetra acetic acid pentaacetoxymethyl ester (Indo 1/AM), 3,30-dihexyloxacarbocyanine iodide (DiOC6) were
purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Caspase-3, -8 and -9 activity assay kits were obtained from OncoImmunin, Inc. (Gaithersburg, MD, USA).
Cell culture
The human NPC cell line (NPC-TW 039) was provided by Dr Chiou-Ying Yang (Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine, 100 Units/ml penicillin and 100 mg/ml streptomycin in 75 cm2 tissue culture flasks at 37C under a humidified 5% CO2and 95% air atmosphere,
as we have previously reported.17,18
Determinations of morphology, viability and cell
cycle distribution of NPC-TW 039 cells
The NPC-TW 039 cells were plated into 12-well plates at a density of 2 105cells/well for 24 h.
Cap-saicin (0, 200, 250, 300 and 400 mM) was added to cell regimens and grown for 12, 24, 36 and 48 h. The DMSO (solvent) served as the vehicle control. For morphological examinations, cells in each well were examined and photographed under a phase contrast microscope. Cell viability was determined by a flow cytometric assay, as we have previously reported.19,20 For cell cycle and sub-G1 phase determination, cells were washed with PBS and were fixed in 70% etha-nol at –20C overnight then resuspended in PBS containing 40 mg/ml PI and 0.1 mg/ml RNase and 0.1% Triton X-100 in a dark room for 30 min at 37C, and analysed with a BD FACSCalibur (Becton-Dickinson, San Jose, CA, USA) and BD CellQuest Pro software (Macintosh) as previously described.20,21
Determinations of apoptosis, DNA damage and
fragmentation
The NPC-TW 039 cells (1 105 cells/ml) were
placed in 12-well plates and treated with 0, 200, 250 and 300 mM of capsaicin for 24 h. Cells in each well were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) and photographed using a fluorescence microscope as previously described.22,23 The NPC-TW 039 cells (1 106 cells/well) were treated with 0, 200, 250, 300 and 400 mM of capsaicin for 24 h to determine DNA fragmentation. DNA was
isolated from each sample for gel electrophoresis, visualized with ethidium bromide (EtBr) staining and then photographed under fluorescence light as previ-ously described.24,25
Determination of intracellular ROS,
Ca
2þlevels and mitochondrial membrane
potential (DC
m)
The NPC-TW 039 cells (2 105 cells/ml) were placed in 12-well plates and treated with or without 300 mM of capsaicin for 1, 2 and 3 h. The cells were harvested, washed with PBS twice, and resuspended in 500 ml of DCFH-DA (10 mM for ROS), Indo 1/AM (3 mg/ml) for Ca2þ and DiOC6(1 mmol/l) for
mitochondrial transmembrane potential in dark room for 30 min. Cells were then analysed by flow cytome-try as previously described.24,26
Assays for caspase-3, -8 and -9 activity
The NPC-TW 039 cells (2 105 cells/ml) were placed in 12-well plate and treated with 0 and 300 mM capsaicin for 24 and 48 h. Caspase-3, -8 and -9 activities were assessed using PhiPhiLuxÔ-G1D2,
CaspaLuxÔ8-L1D2 and CaspaLuxÔ9-M1D2 kits,
respectively. Cells were incubated with cell perme-able substrates for 1 h at 37C according to the man-ufacturer’s instructions and measured using flow cytometric analysis.22,23
Western blotting analysis of protein levels
associated with cell cycle and apoptosis
The NPC-TW 039 cells (1 106 cells/ml) were
placed in 6-well plates and treated with 300 mM capsaicin for 0, 12, 24, 36 and 48 h. The cells were harvested and lysed in the PRO-PREPÔ protein extraction solution (iNtRON Biotechnology, Seong-nam, Gyeonggi-Do, Korea). For protein determina-tion of each sample, the cell lysates (40 mg of each) were separated by sodium dodecyl sulfate–polyacry-lamide gel electrophoresis (SDS-PAGE) on a polya-crylamide gel followed by electrotransfer onto a sequi-blot polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA, USA). Specific polyclonal antibodies (anti-p53, p21, p16, cyclin D, cyclin E, CDK2, CDK6, apoptosis-inducing factor [AIF], cytochrome c, pro-caspase-9, pro-caspase-3, PARP, Bax, Bcl-2, ATF6-a, inositol-requiring 1 enzyme-a [IRE1-a], PERK, glucose-regulated protein 78 [GRP78], pro-caspase-12 and growth arrest and
DNA-damage-inducible 153 [GADD153]) were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat antirabbit or antimouse immunoglobulin G (IgG; Jackson Immuno-Research Laboratories, Inc., West Grove, PA, USA) was used as secondary antibodies for enhanced chemiluminescence (NEN Life Science Products, Inc., Boston, MA, USA) as previously described.24,27
RNA preparation and reverse transcriptase–
polymerase chain reaction (RT-PCR)
The NPC-TW 039 cells (1 106cells/ml) placed in
6-well plates were exposed to 300 mM of capsaicin for 6, 12, 24 and 36 h. Total RNA was extracted from the cells using a Qiagen RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) as previously described.22,28 A high-capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription at 42C for 30 min. The primer sets for the associated gene of transient receptor potential vanilloid type 1 (TRPV1) as F-CATGGCCAGTGAGAACACCATGG and R-AGCCTTTTGTTCTTGGCTTCTCCT.29 The ther-mocycler parameters were 5 min at 95C; denaturation for 1 min at 95C; annealing for 1 min at 55C; 40 cycles of 15 s at 94C, 30 s at 55C and 1 min at 72C; final extension of 10 min at 72C as described previously.29 The reaction products were analysed by electrophoresis on 1% agarose gels as described previously.30,31
Statistical analysis
Differences between the capsaicin-treated and control groups were analysed by Student’s t test and a p value < 0.05 was considered significant.
Results
Effects of capsaicin on NPC-TW 039 cell
morphology, viability, cell cycle and apoptosis
Figure 1, panels A–C, shows that effects of different doses of capsaicin on morphological changes, viabi-lity and cell cycle distribution. Increasing the dose of capsaicin and/or time of incubation led to an increase in morphological changes (Figure 1(A)) and reduced the percentage of viable cells (Figure 1(B)). Capsaicin at 400 mM significantly decreased the via-bility by almost 65%, after 48-h treatment. Data in Figure 1(C) indicate that the percentage of cells in G0/G1 also was enhanced with increasing
concentrations of capsaicin. Figure 1(C) reveals that capsaicin caused a dose-dependent increase in apop-totic cells. Further evidence in support of capsaicin-induced apoptosis can be seen in Figure 2, panels A
and B, which shows that capsaicin induced DNA condensation (Figure 2(A)) and stimulated DNA frag-mentation (Figure 2(B)). These effects were concen-tration dependent.
Effects of capsaicin on ROS, Ca
2þand
mitochondrial membrane potential (DC
m) in
NPC-TW 039 cells
The ROS production contributes to DNA damage and release of ROS into the cytoplasm.32Capsaicin treat-ment increased ROS and Ca2þas shown in Figure 3, panels B and C. In contrast, there was a decrease in the mitochondrial transmembrane potential (Figure 3(A)), suggesting that the mitochondria were depolar-ized in NPC-TW 039 cells.
Effects of capsaicin on the activities of caspase-3
and -9 in NPC-TW 039 cells
It can be seen in Figure 4, panels A and B, that 300 mM capsaicin promoted caspase-9 and -3 activi-ties in a time-dependent manner. However, capsaicin at 300 mM did not stimulate caspase-8 activity (Figure 4(C)). Based on these results, we suggest that capsaicin-triggered apoptosis is mediated through mitochondria- and caspase-dependent pathways rather than extrinsic signaling pathway.
Levels of proteins associated with cell cycle and
apoptosis
Effects of capsaicin on levels of G0/G1 phase, extrin-sic, intrinsic and ER stress-related proteins are shown in Figure 5(A–D). Protein levels of p53, p21 and p16 were increased (Figure 5(A)) but levels of cyclin D, cyclin E, CDK2 and CDK6 were reduced (Figure 5(A)). Capsaicin promoted the levels of Bax (Figure 5(B)), AIF, cytochrome c and PARP (Figure 5(C)), ATF6-a, IRE1-a, PERK, GRP78 and GADD153 (Figure 5(D)) but decreased the levels of Bcl-2 (Figure 5(B)), pro-caspase-9 and pro-caspase-3 (Figure 5(C)), and pro-caspase12 (Figure 5(D)).
Effect of capsaicin on mRNA expression
After 300 mM capsaicin treatment in NPC-TW 039 cells for 0, 6, 12, 24 and 36 h, the gene expression of the mRNA levels of TRPV1 were observed. Com-pared with 0 h, TRPV1 elevation in NPC-TW 039 cells is time dependent (Figure 6).
Figure 1. Capsaicin affected the human nasopharyngeal car-cinoma cells’ viability and cell cycle distribution of NPC-TW 039 cells. Cells were incubated with or without 0, 200, 250, 300 and 400 mM capsaicin for 24 h and 48 h then were har-vested for examining the morphological changes (A), the percentage of viable cells (B) and the distribution of cell cycle (C) by flow cytometry as described in the section on Mate-rials and methods. *p < 0.05.
Discussion
Capsaicin treatment led to ROS production and induced ER stress in NPC-TW 039 cells. It was also observed that capsaicin-induced ER stress occurred based on the GRP78 and GADD153 increases and ER stress, which elicited a rise in intracellular Ca2þ and subsequent mitochondrial membrane depolariza-tion, followed by mitochondrial release of cyto-chrome c and consequent activation of caspase-9
and -3 in NPC-TW 039 cells. Additionally, we also observed AIF release from mitochondria after capsai-cin treatment in NPC-TW 039 cells. Under normal conditions, tumor suppressor p53 is maintained at a low level33; however, after various cellular stresses, p53 is modified and activates downstream target proteins, including Bax or p21, which have been shown to be involved in apoptosis or cell cycle arrest.34Herein, we observed that capsaicin promoted
Figure 2. Capsaicin-induced DNA damage and apoptosis in NPC-TW 039. Cells were treated with 0, 200, 250, 300 or 350 mM capsaicin for 24 h or treated with 300 mM capsaicin for 0, 12, 24 and 48 h, then were harvested for 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (A) and for DNA gel electrophoresis (B) as described in the section on Materials and methods.
Correct it to "Capsaicin induced"
the levels of p53 and also its downstream target proteins p21 and p16 (Figure 5(A)). Our results are in agreement with other reports showing that capsai-cin elevates intracellular Ca2þ levels,9,34,35 suggest-ing that Ca2þ-mediated signaling is involved in capsaicin-induced apoptosis.
We found that capsaicin treatment induced the expression of ER stress-related proteins including IRE1, GADD153, and GRP78, and the activation of 12. It was previously reported that caspase-12 is activated during constitutive ER stress and is involved in ER stress-mediated apoptosis.36 The ER stress has earlier been reported to inhibit p53-dependent apoptosis.37In the present study, capsaicin treatment increased intracellular Ca2þ levels and induced the loss of the mitochondrial transmembrane potential, and these findings are in agreement with our earlier reports, showing that capsaicin causes apopto-sis by increasing intracellular Ca2þlevels and loss of the mitochondrial transmembrane potential.9
It was reported that the Bcl-2 family of proteins plays an important role in the regulation of outer mito-chondrial membrane permeability.38 An increase in the ratio of Bax/Bcl-2 can lead to an alternation in mitochondrial membrane permeability, which can cause the release of apoptotic mediators such as cyto-chrome c or AIF-producing caspase activation and DNA degradation.38,39 Our results showed that capsaicin-induced apoptosis was caspase-8 indepen-dent (capsaicin did not affect caspase-8 activity). Other reports showed that the mitochondrial outer membrane permeabilization by pro-apoptotic Bcl-2 family members is involved in the release of AIF from the mitochondria.40,41It was reported that AIF trans-locates from the mitochondria to the nucleus, then induces caspase-independent peripheral chromatin condensation, and large-scale DNA fragmentation and apoptosis are induced.39,42The TRPV1 is a non-selective, ligand-gated cationic channel that can be activated by capsaicin, noxious heat, and protons.43 We found that capsaicin promoted the gene expres-sion of TRPV1 in NPC-TW 039 cells (Figure 6). Capsaicin has been shown to bind to the TRPV1 chan-nel specifically and mediate transient Ca2þentry and increase cytosolic Ca2þ release.44 However, the role of TRPV1 in capsaicin-induced apoptosis of NPC-TW 039 cells is still unclear. The NPC-NPC-TW 039 cells were pretreated with an inhibitor of TRPV1 then treated with capsaicin. Treatment with the TRPV1 inhibitor altered the effects of capsaicin on the
Figure 3. Capsaicin affected the productions of reactive oxygen species (ROS) and Ca2þ, the levels of mitochondria membrane potential (DCm) in NPC-TW 039 cells. Cells were treated with 300 mM of capsaicin for 0, 1, 2 and 3 h and then were collected and stained by 3,3’-dihexyloxa-carbocyanine iodide (DiOC6) for the determination of mitochondrial transmembrane potential levels (A), stained with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for the determination of ROS levels (B) and stained by 1-[2-Amino-5-(6-carboxy indol-2-yl)phenol]-2-(20 -amino-50methylphenoxy)ethane-N,N,N0,N0-tetra acetic acid pentaa-cetoxymethyl ester (Indo 1/AM) for Ca2þlevels, determined (C) as described in the section on Materials and methods. *p < 0.05.
Figure 4. Capsaicin affected caspase-9 and -3 activities in NPC-TW 039 cells. Cells were treated with 300 mM of capsaicin for 24 and 48 h and were collected for determination activities of caspase-9 (A), -3 (B) and -8 (C) as described in the section on Materials and methods. Data represents mean + SD of three experiments. *p < 0.05.
percentage of viable cells, and further investigations are needed.
In conclusion, our results suggest that capsaicin induces ER stress-mediated apoptosis and mitochon-dria-dependent apoptosis as depicted in the model shown in Figure 7. Capsaicin-induced apoptosis may be regulated by multiple signaling pathways in NPC-TW 039 cells.
Funding
This study was supported by a grant DOH100-TD-C-111-005 from Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence. Figure 5. The effects of capsaicin on the associated protein levels of apoptosis in NPC-TW 039 cells. Cells were incubated with or without 300 mM capsaicin for 0, 12, 24, 36 and 48 h, and total protein from cells were collected and analysed for Western blotting as described in the section on Materials and methods. A, p53, p21, p16, cyclin D, cyclin E, CDK2 and CDK6; B, Bax and Bcl-2; C, AIF, cytochrome c, pro-caspase-9, pro-caspase-3, PARP; D, ATF6-a, inositol-requiring 1 enzyme-a (IRE1-a), PERK, glucose-regulated protein 78 (GRP78), pro-caspase-12 and growth arrest and DNA-damage-inducible 153 (GADD153).
Figure 6. Capsaicin promoted the expression of transient receptor potential vanilloid type 1 (TRPV1) in NPC-TW 039 cells. The cells were exposed to 300 mM of capsaicin for 0, 6, 12, 24 and 36 h as described in the section on Materials and methods.
References
1. Ho JH. An epidemiologic and clinical study of nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys 1978; 4: 182–198.
2. Tomatis L. The IARC program on the evaluation of the carcinogenic risk of chemicals to man. Ann N Y Acad Sci 1976; 271: 396–409.
3. Liu YH, Du CL, Lin CT, et al. Increased morbidity from nasopharyngeal carcinoma and chronic pharyngi-tis or sinusipharyngi-tis among workers at a newspaper printing company. Occup Environ Med 2002; 59: 18–22.
4. Sham JS, Choy D, and Choi PH. Nasopharyngeal carcinoma: the significance of neck node involvement in relation to the pattern of distant failure. Br J Radiol 1990; 63: 108–113.
5. Low WK. Facial palsy from metastatic nasopharyngeal carcinoma at various sites: three reports. Ear Nose Throat J 2002; 81: 99–101.
6. Ward MH, Pan WH, Cheng YJ, et al. Dietary expo-sure to nitrite and nitrosamines and risk of nasophar-yngeal carcinoma in Taiwan. Int J Cancer 2000; 86: 603–609.
Figure 7. The proposed possible signal pathways of capsaicin-induced cell cycle arrest and apoptosis in human nasophar-yngeal carcinoma NPC-TW 039 cells.
7. Sanchez AM, Sanchez MG, Malagarie-Cazenave S, et al. Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin. Apoptosis 2006; 11: 89–99. 8. Mori A, Lehmann S, O’Kelly J, et al. Capsaicin, a component of red peppers, inhibits the growth of androgen-independent, p53 mutant prostate cancer cells. Cancer Res 2006; 66: 3222–3229.
9. Wu CC, Lin JP, Yang JS, et al. Capsaicin induced cell cycle arrest and apoptosis in human esophagus epider-moid carcinoma CE 81T/VGH cells through the eleva-tion of intracellular reactive oxygen species and Ca2þ productions and caspase-3 activation. Mutat Res 2006; 601: 71–82.
10. Hail N, Jr. and Lotan R. Examining the role of mito-chondrial respiration in vanilloid-induced apoptosis. J Natl Cancer Inst 2002; 94: 1281–1292.
11. Ito K, Nakazato T, Yamato K, et al. Induction of apop-tosis in leukemic cells by homovanillic acid derivative, capsaicin, through oxidative stress: implication of phosphorylation of p53 at Ser-15 residue by reactive oxygen species. Cancer Res 2004; 64: 1071–1078. 12. Surh YJ. More than spice: capsaicin in hot chili
pep-pers makes tumor cells commit suicide. J Natl Cancer Inst 2002; 94: 1263–1265.
13. Kim YM, Hwang JT, Kwak DW, et al. Involvement of AMPK signaling cascade in capsaicin-induced apopto-sis of HT-29 colon cancer cells. Ann N Y Acad Sci 2007; 1095: 496–503.
14. Yoshitani SI, Tanaka T, Kohno H, et al. Chemopreven-tion of azoxymethane-induced rat colon carcinogenesis by dietary capsaicin and rotenone. Int J Oncol 2001; 19: 929–939.
15. Surh YJ and Lee SS. Capsaicin in hot chili pepper: car-cinogen, co-carcinogen or anticarcinogen? Food Chem Toxicol 1996; 34: 313–316.
16. Dou D, Ahmad A, Yang H, et al. Tumor cell growth inhibition is correlated with levels of capsaicin present in hot peppers. Nutr Cancer 2011; 63: 272–281. 17. Lin ML, Chen SS, Lu YC, et al. Rhein induces
apopto-sis through induction of endoplasmic reticulum stress and Ca2þ-dependent mitochondrial death pathway in human nasopharyngeal carcinoma cells. Anticancer Res 2007; 27: 3313–3322.
18. Lin ML, Chung JG, Lu YC, et al. Rhein inhibits invasion and migration of human nasopharyngeal car-cinoma cells in vitro by down-regulation of matrix metalloproteinases-9 and vascular endothelial growth factor. Oral Oncol 2009; 45: 531–537.
19. Tan TW, Tsai HR, Lu HF, et al. Curcumin-induced cell cycle arrest and apoptosis in human acute promyelocytic
leukemia HL-60 cells via MMP changes and caspase-3 activation. Anticancer Res 2006; 26: 4361–471. 20. Hsu SC, Kuo CL, Lin JP, et al. Crude extracts of
Euchresta formosana radix induce cytotoxicity and apoptosis in human hepatocellular carcinoma cell line (Hep3B). Anticancer Res 2007; 27: 2415–2425. 21. Su CC, Lin JG, Li TM, et al. Curcumin-induced
apop-tosis of human colon cancer colo 205 cells through the production of ROS, Ca2þ and the activation of caspase-3. Anticancer Res 2006; 26: 4379–4389. 22. Lu CC, Yang JS, Huang AC, et al. Chrysophanol
induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells. Mol Nutr Food Res 2010; 54: 967–976.
23. Chiang JH, Yang JS, Ma CY, et al. Danthron, an anthraquinone derivative, Induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeabil-ity transition pores and Bax-triggered pathways. Chem Res Toxicol 2010; 24: 20–29.
24. Chiu TH, Lai WW, Hsia TC, et al. Aloe-emodin induces cell death through S-phase arrest and caspase-dependent pathways in human tongue squa-mous cancer SCC-4 cells. Anticancer Res 2009; 29: 4503–4511.
25. Lin ML, Lu YC, Chung JG, et al. Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway. Cancer Lett 2010; 291: 46–58. 26. Lu HF, Wang HL, Chuang YY, et al. Danthron induced
apoptosis through mitochondria-and caspase-3-dependent pathways in human brain glioblastoma mul-tiforms GBM 8401 cells. Neurochem Res 2010; 35: 390–398.
27. Wen YF, Yang JS, Kuo SC, et al. Investigation of anti-leukemia molecular mechanism of ITR-284, a car-boxamide analog, in leukemia cells and its effects in WEHI-3 leukemia mice. Biochem Pharmacol 2010; 79: 389–398.
28. Hsia TC, Chung JG, Lu HF, et al. The effect of pacli-taxel on 2-aminofluorene-DNA adducts formation and arylamine N-acetyltransferase activity and gene expression in human lung tumor cells (A549). Food Chem Toxicol 2002; 40: 697–703.
29. Benninger F, Freund TF, and Hajos N. Control of exci-tatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice. Neuro-chem Int 2008; 52: 89–94.
30. Ji BC, Hsu WH, Yang JS, et al. Gallic acid induces apoptosis via caspase-3 and mitochondrion-dependent pathways in vitro and suppresses lung xenograft
tumor growth in vivo. J Agric Food Chem 2009; 57: 7596–7604.
31. Lu HF, Yang JS, Lin YT, et al. Diallyl disulfide induced signal transducer and activator of transcription 1 expression in human colon cancer colo 205 cells using differential display RT-PCR. Cancer Genomics Proteomics 2007; 4: 93–97.
32. Lee MG, Lee KT, Chi SG, et al. Costunolide induces apoptosis by ROS-mediated mitochondrial permeabil-ity transition and cytochrome C release. Biol Pharm Bull 2001; 24: 303–306.
33. Fuchs SY, Adler V, Buschmann T, et al. Mdm2 asso-ciation with p53 targets its ubiquitination. Oncogene 1998; 17: 2543–2547.
34. Lee YS, Kang YS, Lee JS, et al. Involvement of NADPH oxidase-mediated generation of reactive oxy-gen species in the apototic cell death by capsaicin in HepG2 human hepatoma cells. Free Radic Res 2004; 38: 405–412.
35. Huang SP, Chen JC, Wu CC, et al. Capsaicin-induced apoptosis in human hepatoma HepG2 cells. Anticancer Res 2009; 29: 165–174.
36. Morishima N, Nakanishi K, Takenouchi H, et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 2002; 277: 34287–34294.
37. Qu L, Huang S, Baltzis D, et al. Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta. Genes Dev 2004; 18: 261–277.
38. Martinou JC and Green DR. Breaking the mitochon-drial barrier. Nat Rev Mol Cell Biol 2001; 2: 63–67. 39. Susin SA, Lorenzo HK, Zamzami N, et al. Molecular
characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397: 441–446.
40. Ashktorab H, Dashwood RH, Dashwood MM, et al. H. pylori-induced apoptosis in human gastric cancer cells mediated via the release of apoptosis-inducing factor from mitochondria. Helicobacter 2008; 13: 506–517.
41. Otera H, Ohsakaya S, Nagaura Z, et al. Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. EMBO J 2005; 24: 1375–1386.
42. Ye H, Cande C, Stephanou NC, et al. DNA binding is required for the apoptogenic action of apoptosis indu-cing factor. Nat Struct Biol 2002; 9: 680–684. 43. Caterina MJ, Schumacher MA, Tominaga M, et al. The
capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997; 389: 816–824.
44. Minke B. TRP channels and Ca2þ signaling. Cell Calcium 2006; 40: 261–275.
