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Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709
1To whom correspondence should be addressed. E-mail: Eling{at}niehs.nih.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • colorectal cancer • diallyl disulfide (DADS) • NAG-1 • p53
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and colon in animals (2
) as well as a reduction in colorectal polyps in humans (3
). Several epidemiologic studies have suggested that garlic plays an important role in the reduction of deaths by cancer (4
,5
). Garlic oil is also under consideration for the treatment of bladder cancer due to its antitumor and immune-enhancing effects (6
). Garlic oil and in particular diallyl disulfide (DADS)2
has recently become more appealing as an anticarcinogenic agent in part due to its ability to induce apoptosis in vitro (7
) and inhibit the formation and growth of tumors in animal studies in vivo (8
,9
). Like many herbal remedies, little is known about its mode of action. Garlic oil contains both organic and inorganic compounds that may be responsible for its affects. Generally, it is the lipid-soluble organic compounds from garlic that possess the most effective antiproliferative agents warranting further study as antitumorigenic agents (10
). Several studies have demonstrated the efficacy of garlic compounds as antitumorigenic agents. For example, DADS inhibits tumor growth in nude mice (8
,9
) and garlic organosulfides inhibit chemically induced carcinogenesis in hamsters (11
) and in mice (12
,13
), respectively, indicating that DADS and other garlic compounds may be useful therapeutic tools in the prevention of environmentally induced cancers. At micromolar concentrations, DADS also inhibits cell proliferation and induces apoptosis in estrogen receptor positive (KPL-1 and MCF-7) and negative (MDA-MB-231 and MKL-F) breast cell lines (14
). Constituents of garlic oil induce p53, which activate transcription of Waf1/p21 in human gastric cell lines (15
). DADS suppresses neoplastic cell growth and induces apoptosis in vitro (10
,16
). Furthermore, DADS induces apoptosis as well as the tumor suppressor gene p53 in nonsmall cell lung cancer cell lines (17
). Further elucidation of apoptosis-associated cellular proteins regulated by DADS as well as other natural products that are proapoptotic is important because of their potential benefit as anticarcinogenic agents.
The nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) is a transforming growth factor-ß superfamily member that has proapoptotic and antitumorigenic activities whose expression increases after exposure to NSAIDs (18
). NSAIDs are well known for their ability to inhibit the production of prostaglandins by inhibiting both isoforms of prostaglandin H synthase, cyclooxygenase-1 and -2. Our laboratory first identified NAG-1 as a NSAID-activated gene using subtractive hybridization of indomethacin-treated colorectal HCT-116 cells. This effort was undertaken to better understand how NSAIDs attenuate tumor growth (18
). Tumors derived from HCT-116 cells transfected to overexpress NAG-1 were reduced in number and in size as determined in athymic nude mice. These cells showed increased basal apoptosis in vitro indicating that NAG-1 is both proapoptotic and antitumorigenic (18
). NAG-1 is identical to and also known as macrophage inhibitory cytokine 1 (19
), placental bone morphogenetic protein (20
) and placental transforming growth factor-ß (PTGF-ß) (21
). PTGF-ß/NAG-1 is a secretory protein that can act in both an autocrine and paracrine fashion (21
). PTGF-ß/NAG-1 is a p53 target gene and treatment with etoposide, a known inducer of p53, increases NAG-1 protein expression (21
,22
). Induction of NAG-1 by etoposide occurs in p53 wild-type but not p53 mutant cell lines, indicating that the expression of NAG-1 is mediated by p53 (21
). NAG-1 contains two p53 target sites in its promoter, both of which bind p53; however, one is less active (21
,23
). Thus, NAG-1 induction is mediated downstream of p53 (23
). NAG-1 also contains binding sites in its promoter for Sp1, Sp3, and COUP-TF1, indicating that NAG-1 may be induced by multiple mechanisms (24
). In this study, we set out to determine whether DADS induces NAG-1 and whether that induction is p53 dependent by using a common model utilizing both p53 wild-type and p53 mutant cell lines (21
,25
,26
). We found that DADS induces p53 and NAG-1 in a time- and dose-dependent manner in p53 wild-type HCT-116, but not p53 mutant HCT-15 or PC-3 cells. We also speculate that NAG-1 expression plays a large role in the induction of apoptosis by DADS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Dimethyl sulfoxide (DMSO), DADS, propidium iodide (PI), PBS and all other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. Sulindac sulfide (SS) was from Merck (Whitehouse Station, NJ). DADS was dissolved to 4.6 mmol/L (200–1000X) in DMSO. S-Allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) were in the form of aged garlic extract (AGE) from Wakunaga Consumer Products (Mission Viejo, CA). SAC and SAMC are water-soluble allium derivatives. SAC is a major constituent of AGE, which can also be used to form SAMC when reacted with the amino acid cysteine. SAMC was freshly prepared in PBS at 4°C by combining stock solutions of SAC and cysteine followed by shaking for several hours, then mild sonication in a cold-room at 4°C. Stock solutions were then sterile filtered through 0.2-µm Millipore filter cartridges (Bedford, MA).
Cell line and reagents.
Cell lines were purchased from ATCC (Rockville, MD). Human colorectal carcinoma cells, HCT-116, were maintained in McCoy’s 5A medium. Media were supplemented with 10% fetal bovine serum (FBS) and 10 mg/L gentamicin. Human HCT-15 and PC-3 cells were maintained in RPMI-1640 plus 10% FBS, 1 mmol/L L-glutamine, 1 mmol/L sodium pyruvate and 10 mg/L gentamicin. Cell culture reagents were from Life Technologies (Rockville, MD). Vehicle treatments consisted of 0.5% DMSO. Cells were maintained at 37°C/5% CO2 and split twice weekly with 0.25% trypsin.
Cell culture experiments.
Cells were plated overnight in complete media and subsequently treated in serum-free media for various time points for protein isolation and fluorescence-activated cell sorting (FACS) analysis as indicated in the figure legends. Cells were grown to 60–70% confluency in 12-well plates and then treated with vehicle (0.5% final concentration DMSO), DADS or SS in the absence of serum.
Cell proliferation assay.
Cell proliferation was measured using the MTS colorimetric assay by Promega (Madison, WI), which estimates the number of viable cells in proliferation. Briefly, 500 cells per well were plated in 96-well tissue culture dishes overnight. Cells were then treated with various concentrations of vehicle or DADS as indicated in the figure legend in a final volume of 0.1 mL complete media containing 10% FBS. Cell viability was measured daily at 490 nm in an ELISA plate reader after the addition of 0.02 mL MTS "Aqueous One" solution per well and a 1 h incubation at 37°C/5% CO2. Each experiment was carried out in quintuplet and repeated three times. Data shown are mean OD 490 ± SEM from a representative experiment.
Protein isolation.
Protein was isolated in 1X RIPA buffer freshly made the day of the experiment with one Complete-Mini protease inhibitor tablet from Roche Diagnostics (Indianapolis, IN) per 0.01 L RIPA buffer. DNA was sheared using a 23-gauge needle; cell lysates were stored at 4°C for 30 min followed by centrifugation at 12000 x g at 4°C for 20 min to remove cellular debris.
Western blotting.
Proteins (15–20 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The blots were blocked overnight with 10% skim milk in Tris buffered saline (TBS) containing 0.1% Tween-20, and probed with anti-NAG-1 antibody (1:5000 in 1% skim milk in TBS plus 0.1% Tween-20) for 3 h at room temperature as previously described (18
). The antibody recognized both the precursor and secreted forms of NAG-1. The secondary antibody used was anti-rabbit horseradish peroxidase (HRP) Santa Cruz (Santa Cruz, CA). For p53 studies, the primary antibody for p53 was from Santa Cruz and was diluted 1:1000. The actin antibody used was a goat polyclonal immunoglobulin G diluted 1:4000 (Santa Cruz) . After washing, the blots were treated with HRP-conjugated secondary antibody for 1 h and washed several times. The signal was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) followed by autoradiography. Where necessary, blots were stripped of antibody before reuse while sealed in a plastic bag containing a solution of 62.5 mmol/L Tris-HCl, 2% SDS and 100 mmol/L ß-mercaptoethanol for 30 min with constant agitation in a 50°C water bath.
Measurement of DNA content and apoptosis.
The DNA content for vehicle, DADS and SS treated cells were determined by FACS. Cells were plated at 2.5 x 105 cells/well in 12-well plates, incubated until they reached 50% confluency, then treated in serum-free media in the presence of 23 µmol/L DADS, 10 µmol/L SS or vehicle for 48 h three or more times. After treatment, the cells were rinsed with PBS, harvested, then fixed by the slow addition of cold 70% ethanol while mixing to a total of 1 mL and stored at 4°C overnight. The fixed cells were pelleted, washed twice with PBS and stained in 1 mL of 20 mg/L PI, 1 g/L RNase in PBS for 20 min. Cells (n = 7500) were examined by flow cytometry using Becton Dickinson FACSort equipped with CellQuest (San Jose, CA) software by gating on an area vs. width dot plot to exclude cell debris and cell aggregates. Apoptosis was measured by the level of subdiploid DNA contained in cells using CellQuest software from the total gated cells. Measurements are fold-increase over matched vehicle (DMSO)-treated cells.
Densitometry measurements.
Autoradiograms from Northern and Western blots were scanned using a Umax Powerlook III scanner (Fremont, CA) equipped with a transparency adapter and scanning software. Bands were quantitated using Scion Image beta version 4.0.2 (Frederick, MD) and cut to size for publication without modification using Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA). Western blot values were first corrected using their corresponding actin levels. Values shown are fold-increase relative to vehicle-treated control as illustrated in the figure legends. All experiments were repeated at least three times using cells of different passages and freezer stocks.
Statistical analyses.
Statistical analyses were performed on a personal computer using SigmaStat (Jandel, San Raphael, CA). Values represent mean ± SEM. Differences were determined using a two-sided t test with a 0.05 level of significance or by ANOVA with Fisher’s Least Significant Difference method for multiple comparisons where there were multiple treatment groups. DADS dose curve data were normalized using a square-root transformation before ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We first determined whether DADS inhibited cell proliferation in various human colorectal (HCT-116 and HCT-15) and prostate (PC-3) cancer cell lines at various concentrations due to its ability to induce apoptosis in vitro (10
,16
). DADS inhibited growth in all cell lines tested, but the HCT-116 cells appeared to be the most sensitive (Table 1)
, warranting further study of these cells (Fig. 1A
).
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Because of the ability of DADS to inhibit cell proliferation, particularly in HCT-116 cells, we next determined whether NAG-1 protein expression was increased after treatment of HCT-116 cells (Fig. 1B
). DADS induced NAG-1 protein expression 7.8-fold ± 1.6 in HCT-116 cells following a 23 µmol/L treatment for 48 h in serum-free media (Fig. 1C
). We also tested the water-soluble garlic compounds SAC and SAMC for their ability to induce NAG-1 protein expression in HCT-116 cells because they inhibit cell growth in colorectal cancer cells (27
). At 400 µmol/L, neither SAC nor SAMC induced NAG-1 protein expression; therefore, they were not considered further (data not shown). Subsequently, a dose-response curve was performed using DADS to confirm that the induction of NAG-1 was dose dependent. DADS treatment resulted in a dose-dependent induction of NAG-1 protein expression in HCT-116 cells (Fig. 2A
). In HCT-116 cells, NAG-1 protein expression was increased 2.4-fold ± 0.6 at 4.6 µmol/L and 6.1-fold ± 1.7 at 23 µmol/L DADS, whereas p53 was induced 1.5-fold ± 0.1 and 2.3-fold ± 0.4, respectively (Fig. 2B
). Values were adjusted using their corresponding actin levels.
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). SS did not induce p53 protein expression at 10 µmol/L.
|
). SS is an NSAID known to induce NAG-1 expression and apoptosis in a p53-independent manner in HCT-116 cells; thus, it was used as a positive control for NAG-1 protein expression (18
). SS (10 µmol/L) induced NAG-1 protein expression 11- and 15-fold at 24 and 48 h, respectively. Values are adjusted for time-matched, vehicle-treated controls. Thus, the induction of p53 protein expression by DADS precedes that of NAG-1, suggesting that NAG-1 protein expression may be p53 dependent. Protein expression in p53 mutant HCT-15 and PC-3 cells.
HCT-15 cells contain a mutation in the p53 gene rendering it nonfunctional (25
,26
). PC-3 cells are an immortalized human prostate adenocarcinoma cancer cell line that also contain a mutant p53 gene (28
,29
). NAG-1 was not induced in HCT-15 or PC-3 cell lines after treatment with DADS (Fig. 4A
). In contrast, at 10 µmol/L SS, NAG-1 protein expression was induced 11.3-fold ± 0.94 (P < 0.001) in HCT-15 cells after a 48-h treatment and 12.5-fold ± 0.99 (P < 0.001) in PC-3 cells (Fig. 4B
). Expression of p53 was not changed in these cell lines after treatment with DADS or SS (Fig. 4A
).
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To confirm that the inhibition of cell growth by DADS resulted from the induction of apoptosis and to support the hypothesis that this induction was due in part to NAG-1 protein expression, HCT-116 cells were treated with 4.6 and 23 µmol/L DADS or vehicle for 48 h in serum-free media and apoptosis measured by FACS analysis. Apoptotic cells were identified as the subG1 population of 7500 gated cells. Treatment of HCT-116 cells with 4.6 µmol/L DADS resulted in a 1.9-fold increase in apoptosis, whereas 23 µmol/L DADS resulted in a 2.9-fold increase in apoptosis over vehicle according to FACS analysis. Thus, the induction of apoptosis by DADS appears to be due to NAG-1 protein expression and occurs in a dose-dependent manner (Fig. 5A
). In HCT-116 cells, 4.6 and 23 µmol/L DADS differed from the vehicle control (P < 0.03) as did 10 µmol/L SS (P < 0.02); 10 µmol/L SS was used as a positive control because it induces apoptosis in HCT-116 cells (18
,30
). To obtain additional evidence that DADS-induced apoptosis is mediated by p53-dependent NAG-1 induction, we treated p53 mutant PC-3 and HCT-15 cells with 23 µmol/L DADS or vehicle for 48 h in serum-free media and apoptosis was measured by FACS analysis. HCT-15 cells treated with DADS exhibited a 1.8-fold increase in apoptosis (P = 0.13). Furthermore, PC-3 cells did not undergo apoptosis after treatment with DADS (Fig. 5B
). SS resulted in a 2.5-fold increase in apoptosis in both HCT-15 and PC-3 cells (data not shown). Similar time courses for NAG-1 protein expression and apoptosis were observed, and the induction of apoptosis in the p53 wild-type HCT-116 cells was greater than that of the other two cell lines. These data further suggest associations among p53, DADS-induced apoptosis and NAG-1 protein expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Furthermore, SS induced apoptosis in HCT-15 (31
) and PC-3 cells, confirming the ability of the p53 mutant cell lines to undergo apoptosis mediated by NAG-1. Taken together, these data indicate that DADS induction of NAG-1 and apoptosis is in part p53 dependent. Conversely, no significant increase in apoptosis after treatment with DADS was observed in PC-3 cells and only a small and nonsignificant 1.8-fold increase was seen in HCT-15 cells, further suggesting that p53 is an essential link among DADS treatment, NAG-1 protein expression and apoptosis. However, induction of apoptosis by DADS in HCT-15 cells has been reported, but at 100–500 µmol/L DADS (7
). Furthermore, DADS-induced inhibition of cell growth was far less dramatic in both HCT-15 and PC-3 cells as measured by cell proliferation (Table 1)
. This is further evidence that similar to apoptosis, the inhibition of cell growth by DADS occurs at least in part via a p53-dependent mechanism. This is consistent with previous reports that DADS reduces cell proliferation 90% in HCT-15 cells, albeit at much higher concentrations (7
,32
). Thus, the concentration of DADS appears to be an important factor in determining the mechanism involved in inhibition of cell growth and induction of apoptosis; however, at low concentrations, NAG-1 and p53 appear to play key roles in mediating this response.
The induction of NAG-1 by DADS is dependent on p53. The induction of p53 by DADS precedes that of NAG-1, and p53 and NAG-1 are not induced in p53 mutant cell lines. The induction of apoptosis by DADS is greater in p53 wild-type than p53 null cells (17
). NAG-1 is a p53 target gene that controls cell growth (21
), and NAG-1 is a transcriptionally regulated gene that is activated by various wild-type p53 inducible systems in p53 mutant H1299 human lung cancer cells (33
). Also, NAG-1 is induced by etoposide, a known inducer of p53, in p53 wild-type but not p53 mutant cell lines (21
). NAG-1 contains two p53 binding sites as well as several other transcription binding sites in its promoter, indicating that the induction of NAG-1 expression may occur via multiple mechanisms (24
). Additionally, the p53-dependent transactivation of NAG-1 is blocked by a dominant negative p53 mutant and other p53 mutants (21
). NAG-1 is a known inducer of growth arrest and apoptosis and appears to be an important component in p53-dependent apoptosis (23
). Furthermore, HCT-116 cells transfected to overexpress NAG-1 result in decreased tumorigenicity in athymic nude mice, reduced growth in soft-agar and undergo apoptosis (18
).
Is the use of garlic (Allium sativum) and its constituents feasible as an anticarcinogenic agent rivaling that of NSAIDs? Dietary intake of garlic is associated with a reduction in a variety of cancers (4
). Garlic has been shown to have antimicrobial, antithrombotic, antitumorigenic, antiarthritic and other useful properties (34
). Garlic oil’s antitumorigenicity in gastric cell lines indicates that garlic oil or its constituents may prevent colorectal cancer (15
). Generally it is the lipid-soluble organic compounds from garlic such as DADS that possess the most effective antiproliferative effects (10
). SAC and SAMC did not induce NAG-1 in HCT-116 cells at concentrations as high as 400 µmol/L. Interestingly, in p53 wild-type HCT-116 and p53 mutant HT-29 and SW-480 cells, SAMC induced apoptosis at 200 µmol/L (27
). DADS was as effective as the anticancer compound 5-fluorouracil at reducing the growth of tumors in nude mice at equivalent doses (8
). DADS also inhibited the toxicity of benzo(a)pyrene carcinogenicity in mice (12
). Therefore, garlic oil, and DADS in particular, is toxic to cancer cells in vitro, resulting in apoptosis and cell death, suggesting that it may have therapeutic value (7
,10
,16
). Thus, DADS may be effective in the prevention of some cancers. The intake of dietary compounds such as garlic, fruits, vegetables and soybeans is inversely associated with colorectal polyp formation (3
). Thus cancer prevention may be achievable through diets rich in anticarcinogenic compounds such as garlic. The same has been said about the regular intake of NSAIDs. NAG-1 is an important link between reduced tumor growth in mice treated with or fed diets containing NSAIDs. Many food items have anticarcinogenic properties. Therefore, a diet rich in foods containing substances with anticarcinogenic properties may provide some protection against cancer development and therefore should be further investigated. Interestingly, many dietary compounds such as genistein (35
), selenium (36
), resveratrol (37
) and DADS (15
) as illustrated here act through a p53-dependent mechanism. However, the p53 gene is frequently mutated in a variety of cancers, resulting in a loss in p53’s tumor suppressor function and thereby diminishing the ability of such compounds to prevent cancers from developing through p53-dependent mechanisms. Conversely, SS exerts its proapoptotic effect in a p53-independent manner (18
,38
,39
). Therefore, it may be especially beneficial to further investigate dietary compounds that act through p53-independent mechanisms similar to that of SS because of its ability to act through both p53-dependent and -independent mechanisms. Meanwhile, dietary compounds that act in a p53-dependent manner may be better suited for the prevention of cancer before mutations arise.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 17 September 2001. Initial review completed 4 November 2001. Revision accepted 19 December 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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