Article pubs.acs.org/JAFC
Synergistic Anticancer Activity of Capsaicin and 3,3′Diindolylmethane in Human Colorectal Cancer Ruth Clark, Jihye Lee, and Seong-Ho Lee* Department of Nutrition and Food Science, College of Agriculture and Natural Resources, University of Maryland, College Park, Maryland 20742, United States ABSTRACT: Cancer is a leading cause of morbidity and mortality worldwide. A promising area of cancer research is focused on chemoprevention by nutritional compounds. Epidemiological studies have shown a strong negative correlation between fruit, vegetable, and spice intake and rates of cancer. Although individual active compounds have demonstrated significant anticancer activity, an emerging area of research is focusing on the combination of multiple dietary compounds that act synergistically on cancer to exert greater effects. The current study evaluated the potential synergistic effects of capsaicin, an active compound from red chili peppers, in combination with 3,3′-diindolylmethane (DIM), from cruciferous vegetables. A synergistic induction of apoptosis and inhibition of cell proliferation was observed in human colorectal cancer cells treated with the combination of capsaicin and DIM. It was also observed that these two compounds activated transcriptional activity of NF-κB and p53 synergistically. Combination treatment stabilized nuclear p53 and up- or down-regulated expression of several target genes that are downstream of NF-κB and p53. The present study suggests capsaicin and DIM work synergistically to inhibit cell proliferation and induce apoptosis in colorectal cancer through modulating transcriptional activity of NF-κB, p53, and target genes associated with apoptosis. KEYWORDS: apoptosis, colorectal cancer, capsaicin, 3,3′-diindolylmethane
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INTRODUCTION Decades of epidemiological and laboratory data indicate that cancer is linked to not only genetics but also lifestyle, including dietary intake.1 Much research has shown that individuals with higher intakes of fruits and vegetables have half the risk of cancer.2 One way in which fruit and vegetable intake can decrease the risk of cancer is through the action of the bioactive compounds that are naturally found in them. Although individual active compounds have demonstrated significant anticancer activity, an emerging area of research is focusing on the combination of multiple dietary compounds on cancer that work additively or synergistically through multiple cell-signaling pathways to exert greater effects.3−6 Recent research has been focused on investigating if combinational approaches can be used to reduce toxicity, to delay the development of cancer cells, and to reach a greater effect than with one active agent alone.5 Studying the synergism of multiple phytochemicals holds promise for enhanced anticancer capabilities because the ability of chemopreventive phytochemicals to prevent tumor development is likely the outcome of the combination of several distinct sets of intracellular effects, rather than any single biological response. Cancer is often the result of the accumulation of multiple mutations in genes that result in disruption of normal cell signaling and maintenance, so it is probable that targeting multiple actions would result in greater results.7 Many studies have suggested that phytochemicals in fruits, vegetables, and spices can have complementary and overlapping mechanisms of action, including modulation of detoxification enzymes, scavenging of oxidative agents, stimulation of the immune system, regulation of gene expression in cell proliferation and apoptosis, hormone metabolism, and antibacterial and antiviral effects.6,8,9 Thousands of phytochem© XXXX American Chemical Society
icals are present in fruits and vegetables and differ in molecular size, polarity, and solubility, which may affect the bioavailability and distribution of each phytochemical. Therefore, the use of these dietary compounds with distinct molecular mechanisms is beginning to receive attention and is considered more promising for higher efficacy in cancer research. Capsaicin is a homovanillic acid derivative (trans-8-methyl-Nvanillyl-6-nonenamide) and the spicy component of hot chili peppers that has been studied for its anticancer, 10−12 antioxidant,13,14 and anti-inflammatory and analgesic properties.15 We have previously shown that capsaicin induces apoptosis through targeting the NSAID-activated gene-1.16 3,3′-Diindolylmethane (DIM) is a major acid condensation product of indole-3-carbinol (I3C) and generated in the acidic environment of the stomach following dimerization of I3C, which is an autolysis product of glucosinolate, a naturally occurring component of Brassica species of cruciferous vegetables (cabbage, broccoli, cauliflower, and Brussels sprouts). Both I3C and DIM have demonstrated anticancer activity in a number of human cancers including breast,17 prostate,18 pancreatic,19 and colon.20 Many different combinations of phytochemicals have been shown to have additive or synergistic activity. The combination of curcumin and catechin synergistically inhibited growth and induced apoptosis of both colon adenocarcinoma and larynx carcinoma cells.21 Oral administration of a combination of green tea, phytic acid, and inositol significantly decreased the number Received: December 17, 2014 Revised: April 13, 2015 Accepted: April 15, 2015
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(2 × 105 cells/well) were plated in 12-well plates in three replicates and incubated overnight. The next day, plasmid mixtures containing 1 μg of NF-κB Luc or p53 Luc and 0.1 μg of pRL-null were transfected into the cells for 24 h. The transfected cells were treated in the compounds for 24 h. The cells were harvested in 1× luciferase lysis buffer. The luciferase activity was measured and normalized to the pRL-null luciferase activity using a dual-luciferase assay kit (Promega). Reverse Transcription (RT) Polymerase Chain Reaction (PCR). Total RNA was isolated from cells using the RNeasy Micro Kit (QIAGEN, Valencia, CA, USA), and cDNA was synthesized using a VERSO cDNA SYN RT KIT (Thermo Scientific). PCR was performed using PCR master mix (Promega) with targeted primers for p21, Bak, and GAPDH. The PCR primers were synthesized from Sigma-Aldrich. Amplification was performed under conditions of 95 °C for 10 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. Primer sequences are following: p21, forward 5′-GAGCGATGGAACTTCGACTT-3′ and reverse 5′-CAGGTCCACATGGTCTTCCT-3′; Bak, forward 5′-TCTGGCCCTACACGTCTACC-3′ and reverse 5′ACAAACTGGCCCAACAGAAC-3′; GAPDH, forward 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse 5′-TTCTAGACGGCAGGTCAGGT-3′. Reaction products were analyzed on 1% agarose gel. SDS-PAGE and Western Blot. Cells were washed with ice-cold 1× phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bioproduct Inc., Ashland, MA, USA) supplemented with protease and phosphatase inhibitor cocktail (SigmaAldrich) and centrifuged at 12000g for 10 min at 4 °C. After protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), equal amounts of proteins were subjected to 10 or 12% SDS-PAGE, and the separated protein was transferred onto nitrocellulose membranes (Osmonics, Minnetonka, MN, USA). The membranes were blocked for nonspecific binding with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then probed with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature. Chemiluminescence was detected with Pierce ECL Western Blotting substrate (Thermo Scientific) and visualized by Chemidoc MP Imaging system (Bio-Rad, Hercules, CA, USA). Isolation of Cytosol and Nucleus Fraction. Cytosol and nuclear fraction of cells were prepared according to the manufacturer’s protocols of a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). Briefly, after HCT116 cells were treated in the compounds for 24 h, the cells were washed twice with ice-cold PBS containing phosphatase inhibitors (Sigma-Aldrich). Cells were harvested with hypotonic buffer containing detergent and incubated at 4 °C for 15 min. The supernatants (cytoplasmic fraction) were collected after centrifugation at 14000g for 1 min at 4 °C and stored at −80 °C. For nuclear fractions, cell pellets were resuspended with lysis buffer and incubated at 4 °C for 30 min under shaking. After 30 min, nuclear suspensions was centrifuged at 14000g for 10 min at 4 °C, and the supernatants (nuclear fraction) were stored at −80 °C for further analysis Statistics. Statistical analysis was performed with IBM SPSS, and the data were analyzed by one-way ANOVA with Duncan’s multiplecomparison tests. Data are expressed as means ± SD, and differences were considered significant at P < 0.05.
and size of azoxymethane-induced distal tumors in Fisher 344 male rats as compared with controls.22 In humans, a combination of supplements curcumin and quercetin regressed adenomas in patients with FAP.3 DIM synergistically suppressed the proliferation of human colorectal cancer cells when combined with sulforaphane.23 DIM in combination with genistein resulted in decreased proliferation by working on estrogen and androgen receptors in vitro LNCaP PCa cells.24 These and others have shown the potential for synergistic activity of multiple phytochemicals to exert greater anticancer effects. In this study, we aimed to elucidate the anticancer properties of the combined use of capsaicin and DIM with regard to cell growth inhibition and apoptosis in human colon cancer cells. Here we report that treating cells with the combination of capsaicin and DIM results in inhibition of cell proliferation and increased apoptosis through targeting transcriptional activation of p53 and NF-κB and activation of genes downstream of these transcription factors.
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MATERIALS AND METHODS
Chemicals and Reagents. Cell culture media, Dulbecco’s modified Eagle medium (DMEM), was purchased from Lonza (Walkersville, MD, USA), and Eagle’s minimum essential medium (EMEM) was purchased from American Type Culture Collection (Manassas, VA, USA). Antibodies for p21, p27, CDK4, CDK6, Bak, Bid, Bax, Puma, Fas-L, Traf2, and Traf3 were purchased from Cell Signaling (Danvers, MA, USA). Antibodies for p53, TBP, and actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and Bcl-2 antibody was purchased from BD Bioscience (San Jose, CA, USA). Capsaicin (catalog no. E2028) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and DIM (catalog no. BML-GR207) was purchased from Enzo Life Science (Farmingdale, NY, USA). Capsaicin and DIM were dissolved in ethanol and dimethyl sulfoxide (DMSO), respectively. All chemicals were purchased from Fisher Scientific, unless otherwise specified. Cell Culture and Treatment. Human colorectal cancer cell lines, HCT116, SW480, LoVo, Caco-2, and HT-29 were purchased from American Type Culture Collection and grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ mL streptomycin. Human normal colon cells, CCD112CoN, were purchased from ATCC and grown in EMEM supplemented with 10% FBS. The cells were maintained at 37 °C under a humidified atmosphere of 5% CO2. Ethanol and DMSO were used as a vehicle and added into culture media at 0.1% (v/v). Measurement of Apoptosis. Apoptosis of HCT116 cells was measured according to the manufacturer’s instruction of propidium iodide/annexin V-FITC apoptosis detection kit (BD Biosciences) as described previously.25 Early apoptosis and late apoptosis were quantified by flow cytometry in FACS facility at the University of Maryland. Apoptosis of other human colorectal cancer cells were tested using a Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) as we described previously.26 Measurement of Cell Proliferation. Cell proliferation was assessed by using the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) method. The optical density was recorded at 490 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). Briefly, cells were plated at the concentration of 1000−2000 cells/well in 96-well tissue culture plates and then treated with capsaicin, DIM, or a combination as described in the figure captions. After 0, 24, and 48 h, MTT was added to each well and the plate was incubated for 3 h at 37 °C; absorbance was measured in an ELISA plate reader (Bio-Tek Instruments). Transient Transfection and Luciferase Assay. Transient transfection for NF-κB Luc and p53 Luc plasmid was performed using a Polyjet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturer’s instructions. HCT116 cells
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RESULTS Effect of Compounds on Apoptosis and Cell Proliferation. Previously we studied the anticancer activity of capsaicin in human colorectal cancer.16 To further extend our understanding of the anticancer activity of capsaicin, we screened several phytochemicals including EGCG, ECG, apigenin, genistein, DIM, and resveratrol for potential synergistic activity with capsaicin, based on the expression of a newly identified proapoptotic gene, NSAIDs-activated gene 1 (NAG-1). As a result, we found that capsaicin and DIM synergistically induced expression of NAG-1 gene (data not shown). Therefore, in the B
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Figure 1. Synergistic effect of capsaicin and DIM on apoptosis of multiple human colorectal cancer cell lines. Apoptosis of different human colorectal cancer cells, HCT116 (A), LoVo (B), CaCo2 (C), HT-29 (D), and SW480 (E), was tested with FACS (HCT-116 cells) and a Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instruction. Values are means ± SD, n = 3. Different letters indicate significant differences at P < 0.05.
Figure 2. Combination treatment inhibits cell proliferation in multiple human colorectal cancer cell lines, but not normal colon cells. Normal human colon CCD112CoN (A) and human colorectal cancer cell lines HCT116 (B), LoVo (C), CaCo-2 (D), HT-29 (E), and SW480 (F) were plated onto a 96-well plate and grown overnight. The cells were treated with different concentrations of capsaicin (0 or 100 μM), DIM (0 or 25 μM), or a combination (50 μM capsaicin and 12 μM DIM) in media supplemented with 1% FBS for 24 and 48 h at 37 °C under 5% CO2. Values are means ± SD, n = 3. Different letters indicate significant differences at P < 0.05. C
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Figure 3. Combination treatment synergistically increases transcriptional activity of p53 and increases expression of p53 in nucleus. (A) HCT116 cells were transfected with plasmid containing 1 μg of p53 luciferase plasmid and 0.1 μg of pRL-null vector for 24 h at 37 °C. The transfected cells were then treated with the vehicle as the control or 100 μM capsaicin and 25 μM DIM individually or in combination for 24 h in serum-free media. The cells were harvested in 1× luciferase lysis buffer, and luciferase activity was measured. Values are means ± SD, n = 3. Different letters indicate significant differences at P < 0.05. (B) HCT116 cells were treated with 100 μM capsaicin and 25 μM DIM individually or combination for 24 h in serum-free media. Cells were washed with ice-cold PBS and harvested. The cytoplasm was separated from the nucleus and cell lysates were analyzed by Western blot for p53, TATAbinding protein (TBP), and p65. TBP was used as a nuclear loading control. The graph (right panel) indicates statistics of three independent experiments. Different letters indicate significant differences at P < 0.05 (1, control; 2, 100 μM capsaicin; 3, 25 μM DIM; 4, 50 μM capsaicin + 12 μM DIM combination).
as A549 and PC-3 (data not shown), indicating that antiproliferative activity by a combination of two dietary compounds might be common in human cancers. Combination Treatment Increases Transcriptional Activity of p53 and Increases Expression in the Nucleus. The tumor suppressor gene p53 plays a major role in the regulation of apoptosis, cell cycle arrest, and DNA repair. Dysregulation of p53 leads to evasion of apoptosis and rapid growth of cancer cells;30 many genes associated with cell cycle, senescence, DNA repair, and apoptosis are transcriptional targets of p53, and they are all essential barriers to tumor initiation and progression. Therefore, targeting p53 and the downstream transcriptional targets in cancer cells offers promising therapeutic interventions for cancer prevention and treatment.31 For further study, we selected HCT116 cells, which is a p53 wild type, whereas other cell lines (CaCo-2, SW480, and HT-29) have truncated or point mutation of p53. To test if combination treatment targeted the p53 tumor suppressor, we measured the transcriptional activity of p53 in HCT116 cells transfected with the p53 luciferase plasmid. As shown in Figure 3A, treatment with the individual compounds resulted in increased transcriptional activity, but combination treatment was much more significant. To assess the level of p53 proteins in the cells, HCT116 cells were treated with the compounds for 24 h and harvested, and the cytoplasm and nucleus were separated. The fraction was analyzed by Western blotting for p53, and TATAbinding protein (TBP) and p65 were used as nuclear fraction marker and loading control, respectively. As seen in Figure 3B, there was an increase in the level of p53 in the nucleus of the cells
present study, we hypothesize that capsaicin and DIM possess combinational anticancer activity. To test this hypothesis, we treated HCT116 cells with different concentrations of the two compounds individually or combined and then measured apoptosis. As shown in Figure 1A, the combination of 50 μM capsaicin and 12 μM DIM resulted in a significant increase in the percentage of apoptotic cells. To assess if the compounds had the same effect in multiple colorectal cancer cell lines, LoVo, CaCo2, HT-29, and SW480 (Figure 1B− E) were all treated individually or in combination with the compounds. Interestingly, the combination of 50 μM capsaicin and 12 μM DIM significantly increased apoptosis in all of the cell lines. Thus, we chose this concentration for further study. To assess the effect of treatment on normal colon cancer cells, CCD112CoN (Figure 2A) was treated with the compounds individually or in combination and cell proliferation was assessed. Whereas there was a small but significant decrease seen in cell proliferation following cotreatment for 24 h, this was not seen after 48 h of treatment. This is in agreement with others that have shown that capsaicin and DIM are nontoxic to normal cells.10,27−29 However, when multiple colorectal cancer cell lines were treated with the compounds, significant inhibition of cell proliferation was noted in all cell lines (Figure 2B−F). Although the patterns of response of human colorectal cancer cells to capsaicin and DIM are different according to types of cells, the combination of low-dose capsaicin and DIM showed activity similar to that of high doses of single compounds in terms of suppressing cell proliferation. The synergistic antigrowth activity was also observed in non-colorectal cancer cell lines such D
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Journal of Agricultural and Food Chemistry incubated with the combination of both capsaicin and DIM as compared to control and the individual compounds. Combination Treatment Synergistically Increased Transcriptional Activity of NF-κB. The NF-κB family of transcription factors has been shown to play an important role in regulating apoptosis, in both inducing apoptosis and blocking it. It has also been shown to act on cell cycle regulation through sensitizing or desensitizing a cell to apoptotic signals. The dual roles of NF-κB transcription factors have led to much debate regarding its role in cancer initiation and progression.32,33 Given that both p53 and NF-κB are transcription factors stimulated with cellular stress, and the compounds already have demonstrated the ability to increase p53 transcriptional activity, we tested if capsaicin and DIM affect the transcriptional activity of NF-κB. HCT116 cells were transfected with plasmid containing 1 μg of NF-κB luciferase plasmid and 0.1 μg of pRL-null vector for 24 h at 37 °C. The transfected cells were then treated with the vehicle as the control or capsaicin and DIM individually or in combination for 24 h. The cells were harvested in 1× luciferase lysis buffer, and luciferase activity was measured (Figure 4). Interestingly, the transcriptional activity of NF-κB significantly increased with combination treatment as compared with the individual compounds.
Combination treatment resulted in down-regulation of prosurvival protein, Bcl-2 (Figure 5C). Bcl-2 inhibits apoptosis and has been shown to be overexpressed in many types of cancer cells.36 Reduced Bcl-2 expression promotes apoptotic responses to anticancer drugs, whereas increased expression leads to resistance to chemotherapeutic drugs and radiation therapy and is a target to prevent drug resistance and increased apoptosis. Tumor necrosis factor receptor-associated factors 2 and 3 (Traf2 and Traf3) have also been shown to be oncogenes that regulate the noncanonical signaling of NF-κB. Previous groups have shown that suppression of Traf2 and Traf3 results in decreased proliferation and tumorigenesis.37 Combination treatment resulted in down-regulation of these two oncogenes (Figure 5C). Following treatment with capsaicin and DIM, cells were harvested, and RT-PCR was performed to analyze levels of mRNA for p21 and Bak (Figure 5D). Increased levels of p21 and Bak mRNA were identified following treatment with both capsaicin and DIM, although significantly so only in p21. Modulation of Target Proteins in a p53-Dependent Manner. The p53 tumor suppressor protein performs a critical role in inducing apoptosis. Many of the pro-apoptotic Bcl-2 family members including Puma, Bax, and Bak have been reported to be transcriptional targets of p53.34 Also, the cell cycle regulating proteins, p27 and p21, are also transcriptional targets of p53.31 To test if the target genes modulated by combination treatment identified previously were dependent on p53, HCT116 p53 null cells were plated and treated with various concentrations of capsaicin individually (100 μM) and DIM individually (25 μM) as well as in combination (50 μM capsaicin and 12 μM DIM). The cells were harvested and the lysates subjected to analysis by Western blot. Interestingly, p21, p27, and Bak were all up-regulated in a p53-dependent manner (Figure 6).
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DISCUSSION
There is growing evidence from in vitro and in vivo studies indicating that combinations of phytochemicals can result in significant activities at concentrations where any single agent is inactive. Here, we propose two mechanisms by which a combination of capsaicin and DIM increases apoptosis and changes expression of cell growth and apoptosis-related genes. First, we observed that the compounds increased transcriptional activity (Figure 3A) as well as p53 nuclear accumulation (Figure 3B). p53 accumulation in the nucleus seems to be associated with its protein stability. Ser15 phosphorylation of p53 suppresses ubiquitination of p5338 and subsequent 26S proteasomemediated degradation by reducing the interaction of p53 with E3 ligase and MDM2 and eventually promotes the accumulation and transactivation.39 Most studies support that p53 degradation occurs exclusively on cytoplasmic proteasome by focusing on nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and many studies support proteosomal degradation of transcription factors in the nucleus. In addition, inhibition of proteasomal degradation using MG-132 leads to localization of p65 and p53 in nucleolus (fibrillar centers) compartments.40 Second, in the present study, we observed that combination of capsaicin and DIM synergistically increased the transcriptional activity of NF-κB. The transcription factor NF-κB is recognized as a key mediator of the cellular stress response upon anticancer therapy, and the activation of NF-κB can lead to a pro-death response. It is believed that increased NF-κB mediates apoptosis induced by anticancer agents.41−44 Thus, further study is
Figure 4. Combination treatment synergistically increases transcriptional activity of NF-κB. HCT116 cells were transfected with plasmid containing 1 μg of NF-κB luciferase plasmid and 0.1 μg of pRL-null vector for 24 h at 37 °C. The transfected cells were then treated with the vehicle as the control or 100 μM capsaicin and 25 μM DIM individually or combination for 24 h in serum-free media. The cells were harvested in 1× luciferase lysis buffer, and luciferase activity was measured. Values are means ± SD, n = 3. Different letters indicate significant differences at P < 0.05.
Effect of Combination Treatment on Expression of Genes Involved in Apoptosis and Cell Cycle. After observation of the effect of combination treatment on cell proliferation, apoptosis, p53, and NF-κB transcription, it was hypothesized that genes and proteins known to be regulated by p53 and NF-κB could be modulated through combination treatment. Western blot analysis was used to analyze target proteins that are involved in these processes, and RT-PCR was used to confirm mRNA levels of the target genes. As seen in Figure 5A, combination treatment resulted in increased expression of both p21 and 27 with subsequent inhibition of CDK4 and CDK 6. Treatment of HCT-116 cells with the combination of capsaicin and DIM significantly increased or tended to increase expression of multiple pro-apoptotic proteins (Figure 5B). Bak, BID, Bax, Puma, and Fas-L are all proapoptotic proteins that have been shown to be downstream of p53.30,34,35 E
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Figure 5. Analysis of protein and mRNA expressions in HCT116 cells treated with capsaicin and DIM. HCT116 cells were treated with different concentrations of capsaicin (0 or 100 μM), DIM (0 or 25 μM), or a combination (50 μM capsaicin and 12 μM DIM) for 24 h in serum-free media. The cells were harvested, and cell lysates were analyzed with Western blot for target proteins. (A) Expression of proteins regulating the cell cycle including p21, p27, CDK4, and CDK6. (B) Proteins with pro-apoptosis functions including Bak, BID, Bax, Puma, and Fas-L were analyzed. (C) Anti-apoptotic proteins including Bcl-2, Traf2, and Traf3 were analyzed. (D) HCT116 cells were treated with capsaicin (100 μM), DIM (25 μM), or a combination (50 μM capsaicin and 12 μM DIM) in serum-free media. Total mRNA was harvested and RT-PCR was performed. The graph indicates statistics of three independent experiments. Different letters indicate significant differences at P < 0.05 (1, control; 2, 100 μM capsaicin; 3, 25 μM DIM; 4, 50 μM capsaicin + 12 μM DIM combination).
signaling inputs, and NF-κB depends entirely on cooperative interactions with partner transcription factors for recruitment to some genes. NF-κB also can be regulated in the nucleus by complex mechanisms that are still obscure. They include posttranslational modification and nuclear distribution of p65, which could determine cell fate, pro-apoptotic or anti-apoptotic, and
required to investigate how compounds activate the transcriptional activity of NF-κB. Although the main control of NF-κB activation is cytoplasmic IκB, recently many studies propose additional mechanisms that are required to activate or prevent activity of NF-κB protein. In fact, NF-κB recruitment is dynamically regulated by multiple F
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because some cell lines such as SW480 and CaCo-2 are p53 mutants, and we showed in these cell lines increased apoptosis and inhibited cell proliferation with combinations (Figures 1 and 2). In summary, the data presented here show that combination treatment is able to work synergistically to mediate inhibition of cell proliferation and induction of apoptosis. Interestingly, the compounds targeted p53 and NF-κB transcriptional factors and modulated known downstream target proteins. Dietary phytochemicals offer a safe colorectal cancer preventive alternative for targeting multiple pathways.
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AUTHOR INFORMATION
Corresponding Author
*(S.-H.L.) Phone: (301) 405-4532. Fax: (301) 314-3313. E-mail:
[email protected]. Funding
This work was supported by start-up funds from the University of Maryland to S.-H.L. This work is also supported in part by grant from the National Institutes of Health (R03CA137755) to S.H.L. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Jin Boo Jeong for his technical support. We also thank Dr. Seung Joon Baek for providing p53 null HCT116 cells.
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Figure 6. Modulation of target proteins in a p53-dependent manner. HCT116 wild type (WT) and HCT116 p53 null (−/−) cells were treated with capsaicin (100 μM), DIM (25 μM), or a combination (50 μM capsaicin and 12 μM DIM) for 24 h in serum-free media. The cells were harvested, and cell lysates were analyzed with Western blot for target proteins. The graph indicates statistics of three independent experiments. Different letters indicate significant differences at P < 0.05.
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influence its transcriptional activity. For example, an increased accumulation of p65 in nucleoplasm results in apoptosis, whereas accumulation of p65 in nucleolus is a mechanism of apoptosis activation in response to apoptotic stimuli.45 Another interesting point is that p53 and NF-κB have shown an ability to interact with each other and coordinate induction of apoptosis. For example, many pro-apoptotic stimuli such as DNA-damaging agents lead to transcriptional coactivation of p53 and NF-κB. Ryan et al.46 suggested that p53 directly activates transcriptional activity of NF-κB, which is required for p53induced apoptosis. Other studies demonstrate that p53-dependent NF-κB activation could contribute to the apoptotic response,47 and the loss of p53 could abrogate NF-κB-mediated apoptotic response.48 p65 has been reported to promote apoptosis by actively repressing transcription of anti-apoptotic genes through association of p65 with histone deacetylasecontaining complexes acting as co-repressor.49 Overall, even though we did not study direct interaction between p53 and NFκB, it is possible that both NF-κB and p53 may be essential for apoptosis induced by combination treatment. Fas ligand (FasL) is believed to be a common target for p53 and NF-κB, and induction of FasL is required for p53- and NF-κB-dependent apoptosis.50−52 Therefore, FasL is a good target protein for studying p65- and p53-mediated apoptosis. Although we claim that p53 mediates capsaicin and DIM’s effect, we do not exclude the possibility that this combination suppressed cell proliferation and induced apoptosis through a p53-independent mechanism G
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Journal of Agricultural and Food Chemistry
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DOI: 10.1021/jf506098s J. Agric. Food Chem. XXXX, XXX, XXX−XXX
