![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
* Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen, Germany; Interdisciplinary Research Center, Heinrich-Buff-Ring 26-32, Justus-Liebig University, D-35392 Giessen, Germany; and ** The Cancer Center, University of Minnesota, Minneapolis, MN 55455
1 To whom correspondence should be addressed. Email: toxwu{at}yahoo.com.hk.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • onion • allium • cell cycle • apoptosis • chemoprevention
Onion, a commonly consumed Allium vegetable, was found to reduce the incidence of cancers in several tissues in epidemiologic studies (1–3). For instance, onion consumption at a level of at least half an onion a day was associated with a 50% decline in stomach cancer risk in a case-control study (3). Higher onion intake was shown to be correlated with lower risk of breast cancer in a French epidemiologic study (2), and experimental studies demonstrated the anticarcinogenic effect of the onion in multiple organs of mice, rats, and hamsters (4–8).
The organosulfur compounds present in Allium vegetables are considered to be responsible for the beneficial effects of these herbs. Based on the present state of knowledge, the chemopreventive effects of the Allium family are mediated via the following mechanisms: 1) enhancement of the activity of specific mixed-function oxidases that depress the activation of carcinogens (9,10); 2) induction of phase II enzymes, which enhance detoxification and excretion of potential carcinogens and reduction of the formation of DNA adducts (11); 3) increased synthesis of glutathione (GSH),2 an endogenous tripeptide thiol that directly protects cells from damage by free radicals (12–15); and 4) induction of cell cycle arrest and apoptosis in cancer cells (16).
In recent years, the effects of chemopreventive agents on cell cycle and apoptosis have attracted considerable attention. Indeed, some organosulfur compounds or extracts derived from garlic have shown strong antiproliferative and apoptosis-inducing effects in various cancer cells (17). However, data about the effects of onion or onion-associated sulfur-containing compounds are relatively rare. To date, only one study demonstrated that onion oil inhibits the proliferation of human leukemia HL-60 cells (18). However, the mechanism responsible for the antiproliferative activity was not investigated. Hence, we investigated the effects of onion oil on the cell cycle and apoptosis, as well as the mechanisms involved in tumor cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Cell culture. A549 cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). The cells were grown in DMEM supplemented with 15% fetal calf serum, penicillin, and streptomycin and incubated at 37°C under a humidified atmosphere containing 5% CO2.
Measurement of DNA distribution and apoptosis. A549 cells (4 x 105 cells/flask) were incubated with various concentrations of onion oil for 24 h. The percentage of cells in different phases of the cell cycle was analyzed by flow cytometry after staining with propidium iodide (PI) as described (19). Additionally, an Annexin V binding assay was performed using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) as previously described (19). Annexin V positive/PI negative and Annexin V positive/PI positive cells were defined as apoptotic and necrotic cells, respectively.
Measurement of ROS production. Reactive oxygen species (ROS) were reported to act as subcellular messengers in several cellular processes including apoptosis (20). To evaluate ROS levels, A549 cells (4 x 105 cells/flask) were incubated with different concentrations of onion oil (3.1, 6.3, 12.5, and 25 mg/L) for 24 h and 12.5 mg/L onion for different time periods (0.5, 1, 2, 4, 6, and 24 h). ROS production was measured by flow cytometry as described previously (19). ROS production was expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software.
Measurement of mitochondrial membrane potential (MMP). A549 cells (4 x 105 cells/flask) treated with 12.5 mg/L onion oil for different time periods (0.5, 1, 2, 4, 6, and 24 h) were incubated for 30 min with 2 mg/L of 5,5',6,6'-tetrachloro -1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Sigma) in culture medium. The resulting cells were then washed 3 times with PBS and dislodged with trypsin-EDTA. Cells were collected in 2%-BSA PBS, washed twice by centrifugation (800 x g; 5 min), and resuspended in 0.3 mL PBS/2% BSA for analysis by a FACSCalibur flow cytometer (BD Biosciences). Cytometer settings were optimized for green (FL-1) and red (FL-2) fluorescence, and the ratio of JC-1 aggregates (red fluorescence) to and JC-1 monomers (green fluorescence) was calculated to represent MMP.
Preparation of cell lysates and Western blots. Cell pellets were resuspended in lysis buffer containing 10 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, 150 mmol/L NaCl, and 0.5% IGEPAL CA-630 (Sigma). The cytosolic protein concentration was determined by the Bradford assay (21) with BSA as the standard. Cell lysates, containing equal amounts of protein, were boiled in SDS-sample buffer for 5 min before loading on a SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane (Amersham). Membranes then were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween 20 (TBS/T) and incubated with the desired primary antibodies overnight at 4°C with slight shaking. After the triple washing with TBS/T, the appropriate horseradish peroxidase-conjugated secondary antibodies were added and incubated for 1 h at room temperature. Finally, immunoreactive proteins were visualized using enhanced chemiluminescence detection agents (Amersham). Each membrane was stripped and reprobed with an antibody against ß-actin to correct for differences in protein loading.
Statistical methods. Statistical analysis was performed using the SAS system (version 8.0, SAS Institute). For most data, if a significant effect of treatment was detected by 1-way ANOVA, Dunnett's test was conducted to compare treated cells with the control. For comparisons among groups (Fig. 2c and Fig. 4), a two-way ANOVA followed by a Student-Newman-Keuls post hoc test was used. Values are expressed as means ± SEM. Differences were considered significant at P < 0.05.
|
|
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
6.3 mg/L (Fig. 1). Onion oil at 12.5 mg/L had the strongest apoptotic effect compared with the DMSO control as indicated by the sub-G1 measurement (13.3%) and the Annexin V binding assay (9.4%) (Table 1).
|
|
Intracellular ROS production. Treatment with various concentrations of onion oil significantly elevated the intracellular ROS level (Fig. 2A). At the highest concentration (50 mg/L), onion oil caused nearly a 100% increase in ROS production compared with the control (Fig. 3A). Furthermore, 12.5 mg/L onion oil, the concentration that induced significant apoptosis and cell cycle arrest, resulted in a sustained increase of ROS production for 24 h (Fig. 2B). ROS production increased within 30 min. Pretreating cells with the putative antioxidants N-acetylcysteine (NAC) and GSH resulted in ROS production that did not differ from that of the DMSO control (Fig. 2C).
|
Cell cycle arrest and apoptosis blocked by NAC and GSH. Pretreatment of the A549 cultures with 5 mmol/L NAC and 2.5 mmol/L GSH significantly reduced the induction of apoptosis (Fig. 4A). After NAC and GSH pretreatment, the G2/M arrest caused by onion oil was also abrogated and the percentage of G2/M cells was reduced to a level even less than that of the DMSO control (Fig. 4B). Hence, the present results suggest a requirement for ROS in onion oil–mediated cell cycle arrest and cell death.
Redox-regulation of G2/M-related proteins. Onion oil treatment did not alter the expressions of non-phospho-Cdc2 and cylclinB1 but significantly reduced expression of phospho-Cdc2 and phospho-cyclinB1 after the A549 cells had been exposed to onion oil for 24 h (Fig. 5). However, 0.5–6 h of incubation with onion oil did not reduce the expression of pospho-Cdc2 and phospho-cylcinB1. This finding suggests that the regulation of Cdc2 and cyclinB1 occurs later than the oxidative burst and the loss of MMP in onion oil–induced cell cycle arrest. Furthermore, preincubation with NAC abolished the effects of onion oil on phospho-Cdc2 and cyclinB1 (Fig. 5), indicating that the expression of Cdc2 and cyclinB1 in onion oil–treated cells is redox modulated.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Seki et al. (18) first found the above-mentioned inhibition of cell proliferation and induction of cell differentiation by onion oil. Onion oil also was found to stimulate cell growth and proliferation of NIH-3T3 cells (29). In agreement with most previous studies of garlic compounds, the present results reveal that onion oil arrests the growth of human lung carcinoma cells at the G2/M phase and additionally induces programmed cell death as detected by flow cytometry. The increase in the percentage of apoptotic cells, however, was not dose dependent. The highest percentage of apoptotic cells occurred when the cells were exposed to 12.5 mg/L onion oil rather than to 25 mg/L. Results of the Annexin V assay suggest that the effect may be due to induction of necrosis instead of apoptosis at the highest concentration. GC/MS measurement suggests that the propyl sulfides, DPDS, DPTS, and MPDS, represent major sulfur-containing constituents of onion oil, which account for >53% of the content of onion oil. On the contrary, the percentage of DAS, DADS, and DATS, the major oil-soluble allyl sulfides present in garlic oil, is <2% in onion oil. Although DADS and DATS are effective inducers of cell cycle arrest and apoptosis in several tumor cell lines (17,30), it is most likely that propyl sulfides and not allyl sulfides account for the action of onion oil due to their very low concentrations of allyl sulfides. Previously, one study reported that DADS but not DPDS caused A549 cells to undergo apoptosis (31). However, there are few reports concerning the action of the other propyl sulfides.
Intracellular ROS function as trigger or signaling molecules to initiate downstream events in regulating cell cycle, cell differentiation, apoptosis and so on. (32,33). Dysfunction of the mitochondria, which is a hallmark of programmed cell death, likely produces ROS that influence numerous cell processes (33). We sought to investigate the relations among onion oil–induced decrease of mitochondrial membrane potential (MMP), increase of intracellular ROS formation, cell cycle arrest, and apoptosis. Indeed, at effective concentrations of onion oil for induction of cell cycle arrest and apoptosis, ROS production was markedly elevated and MMP was significantly reduced. Furthermore, onion oil–mediated G2/M cell cycle arrest and apoptosis was blocked by the antioxidants NAC and GSH. Overall, the present findings suggest the following: 1) cell cycle arrest at G2/M and the induction of apoptosis by onion oil is redox modulated; ROS may serve as a trigger in the signaling cascade because ROS production increased in a very short time period (in <1 h); 2) execution of apoptosis induced by onion oil may occur via a mitochondria-dependent pathway; 3) although it was reported that onion oil possesses antioxidant activity in primary mouse cells and rats (14,34), the present study suggests that onion oil increases intracellular ROS levels in human lung tumor cells.
Eukaryotic G2/M progression is regulated by the cdk1-cyclin B1 kinase complex and is maintained in an inactive form by phosphorylations at Thr14 and Thr15 of Cdk1 (35). Entry of eukaryotic cells into the M-phase of the cell cycle is regulated by activation of cdc2 kinase. Activation of cdc2 can be controlled at several steps, including cyclinB1 binding through phosphorylation of cdc2 at Thr161 and dephosphorylation of cdc2 at Thr14/Thr15 (36). Phosphorylation of cyclin B1 was also shown to be required for cdc2/cyclin B1 activation (37). Here, both phospho-cdc2 and phospho-cyclin B1 were significantly reduced by onion oil after 24 h of treatment, whereas the expressions of total endogenous cdc2 and cyclin B1 were not influenced. Hence, we postulate that accumulation of G2/M cells may be due to reduction of phospho-cyclin B1 and cdc2, indicating the critical importance of phosphorylation for G2/M transition in onion oil–treated cells. Additionally, the reduction effect of phospho-cdc2 and phospho-cyclin B1 by onion oil could be pharmacologically abolished upon pretreatment with the antioxidant NAC, suggesting that intracellular ROS triggers redox-sensitive genes and the signal is relayed to cdc2/cyclin B1 proteins, whose reduced phosphorylation resulted in the accumulation of G2/M cells in cell cycle.
Information about the bioavailability of active ingredients in onion oil is necessary for extrapolating in vitro biological action to in vivo effects in the body. However, most available reports on the bioavailability of onion constituents focused on flavonoids, which are water soluble and not present in onion oil (38). The metabolism and bioavailability of oil-soluble constituents have not been characterized in humans. However, onions are ubiquitous; they are used as an ingredient in many dishes and are accepted by almost all traditions and cultures. Onions are the second most important horticultural crop after tomatoes (38). Onion oil per se is widely used as a flavoring by the food industry and as a raw material for many medicines and functional foods. A high daily intake of onion can lead to the onion constituents and their metabolites reaching considerable concentrations in human organs. Hence, further study of the metabolism and bioavailability of onion is warranted to elucidate whether the inhibition of tumor cell proliferation and the induction of apoptosis by onion oil in vitro also occur in vivo.
Overall, the results presented in this study revealed significant inhibition of the growth of human lung cancer cells by onion oil as shown by cell cycle arrest at G2/M and the induction of apoptosis. In addition, both cell cycle arrest and the induction of apoptosis in onion oil–treated cells are ROS dependent, and dysfunction of the mitochondria is a very early event in the cascade of the induction of apoptosis. To our knowledge, this is the first report on the induction of cell cycle arrest and apoptosis in tumor cells by onion oil and the underlying mechanisms. The data presented here suggest the novel chemopreventive mechanism of onion and may inspire investigators to identify the specific constituents responsible for the observed effects.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Manuscript received 28 July 2005. Initial review completed 19 September 2005. Revision accepted 14 December 2005.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Gao CM, Takezaki T, Ding JH, Li MS, Tajima K. Protective effect of allium vegetables against both esophageal and stomach cancer: a simultaneous case-referent study of a high-epidemic area in Jiangsu Province, China. Jpn J Cancer Res. 1999;90:614–21.[Medline]
2. Challier B, Perarnau JM, Viel JF. Garlic, onion and cereal fibre as protective factors for breast cancer: a French case-control study. Eur J Epidemiol. 1998;14:737–47.[Medline]
3. Dorant E, van den Brandt PA, Goldbohm RA, Sturmans F. Consumption of onions and a reduced risk of stomach carcinoma. Gastroenterology. 1996;110:12–20.[Medline]
4. Perchellet JP, Perchellet EM, Belman S. Inhibition of DMBA-induced mouse skin tumorigenesis by garlic oil and inhibition of two tumor-promotion stages by garlic and onion oils. Nutr Cancer. 1990;14:183–93.[Medline]
5. Fukushima S, Takada N, Hori T, Wanibuchi H. Cancer prevention by organosulfur compounds from garlic and onion. J Cell Biochem Suppl. 1997;27:100–5.[Medline]
6. Niukian K, Schwartz J, Shklar G. In vitro inhibitory effect of onion extract on hamster buccal pouch carcinogenesis. Nutr Cancer. 1987;10:137–44.[Medline]
7. Ito Y, Maeda S, Sugiyama T. Suppression of 7,12-dimethylbenz[a]anthracene-induced chromosome aberrations in rat bone marrow cells by vegetable juices. Mutat Res. 1986;172:55–60.[Medline]
8. Belman S. Onion and garlic oils inhibit tumor promotion. Carcinogenesis. 1983;4:1063–5.
9. Chun HS, Kim HJ, Choi EH. Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells. Biosci Biotechnol Biochem. 2001;65:2205–12.[Medline]
10. Siess MH, Le Bon AM, Canivenc-Lavier MC, Suschetet M. Modification of hepatic drug-metabolizing enzymes in rats treated with alkyl sulfides. Cancer Lett. 1997;120:195–201.[Medline]
11. Munday R, Munday JS, Munday CM. Comparative effects of mono-, di-, tri-, and tetrasulfides derived from plants of the Allium family: redox cycling in vitro and hemolytic activity and Phase 2 enzyme induction in vivo. Free Radic Biol Med. 2003;34:1200–11.[Medline]
12. Banerjee SK, Maulik M, Mancahanda SC, Dinda AK, Gupta SK, Maulik SK. Dose-dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic. Life Sci. 2002;70:1509–18.[Medline]
13. Bose C, Guo J, Zimniak L, Srivastava SK, Singh SP, Zimniak P, Singh SV. Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic. Carcinogenesis. 2002;23:1661–5.
14. Perchellet JP, Perchellet EM, Abney NL, Zirnstein JA, Belman S. Effects of garlic and onion oils on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione and ornithine decarboxylase induction in isolated mouse epidermal cells treated with tumor promoters. Cancer Biochem Biophys. 1986;8:299–312.[Medline]
15. Scharf G, Prustomersky S, Knasmuller S, Schulte-Hermann R, Huber WW. Enhancement of glutathione and 
-glutamylcysteine synthetase, the rate limiting enzyme of glutathione synthesis, by chemoprotective plant-derived food and beverage components in the human hepatoma cell line HepG2. Nutr Cancer. 2003;45:74–83.[Medline]
16. Sun SY, Hail N Jr, Lotan R. Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst. 2004;96:662–72.
17. Wu X, Kassie F, Mersch-Sundermann V. Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds. Mutat Res. 2005;589:81–102.[Medline]
18. Seki T, Tsuji K, Hayato Y, Moritomo T, Ariga T. Garlic and onion oils inhibit proliferation and induce differentiation of HL-60 cells. Cancer Lett. 2000;160:29–35.[Medline]
19. Wu XJ, Kassie F, Mersch-Sundermann V. The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells. Mutat Res. 2005;579:115–24.[Medline]
20. Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal. 1999;11:1–14.[Medline]
21. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.[Medline]
22. Guyonnet D, Belloir C, Suschetet M, Siess MH, Le Bon AM. Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction. Mutat Res. 2001;495:135–45.[Medline]
23. Arivazhagan S, Balasenthil S, Nagini S. Garlic and neem leaf extracts enhance hepatic glutathione and glutathione dependent enzymes during N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in rats. Phytother Res. 2000;14:291–3.[Medline]
24. Munday R, Munday CM. Induction of phase II enzymes by aliphatic sulfides derived from garlic and onions: an overview. Methods Enzymol. 2004;382:449–56.[Medline]
25. Berges R, Siess MH, Arnault I, Auger J, Kahane R, Pinnert MF, Vernevaut MF, Lebon AM. Comparison of the chemopreventive efficacies of garlic powders with different alliin contents against aflatoxin B1 carcinogenicity in rats. Carcinogenesis. 2004;25:1953–9.
26. Choudhury AR, Das T, Sharma A. Mustard oil and garlic extract as inhibitors of sodium arsenite-induced chromosomal breaks in vivo. Cancer Lett. 1997;121:45–52.[Medline]
27. Dwivedi C, John LM, Schmidt DS, Engineer FN. Effects of oil-soluble organosulfur compounds from garlic on doxorubicin-induced lipid peroxidation. Anticancer Drugs. 1998;9:291–4.[Medline]
28. Herman-Antosiewicz A, Singh SV. Signal transduction pathways leading to cell cycle arrest and apoptosis induction in cancer cells by Allium vegetable-derived organosulfur compounds: a review. Mutat Res. 2004;555:121–31.[Medline]
29. Zelikoff JT, Atkins NM Jr, Belman S. Stimulation of cell growth and proliferation in NIH-3T3 cells by onion and garlic oils. Cell Biol Toxicol. 1986;2:369–78.[Medline]
30. Xiao D, Choi S, Johnson DE, Vogel VG, Johnson CS, Trump DL, Lee YJ, Singh SV. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2. Oncogene. 2004;23:5594–606.[Medline]
31. Sundaram SG, Milner JA. Diallyl disulfide induces apoptosis of human colon tumor cells. Carcinogenesis. 1996;17:669–73.
32. Shackelford RE, Kaufmann WK, Paules RS. Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med. 2000;28:1387–404.[Medline]
33. Fleury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling. Biochimie. 2002;84:131–41.[Medline]
34. Helen A, Rajasree CR, Krishnakumar K, Augusti KT, Vijayammal PL. Antioxidant role of oils isolated from garlic (Allium sativum Linn) and onion (Allium cepa Linn) on nicotine-induced lipid peroxidation. Vet Hum Toxicol. 1999;41:316–9.[Medline]
35. Molinari M. Cell cycle checkpoints and their inactivation in human cancer. Cell Prolif. 2000;33:261–74.[Medline]
36. Kohn KW. Molecular interaction map of the mammalian cell cycle control and DNA repair systems. Mol Biol Cell. 1999;10:2703–34.
37. Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature. 2001;410:215–20.[Medline]
38. Griffiths G, Trueman L, Crowther T, Thomas B, Smith B. Onions—a global benefit to health. Phytother Res. 2002;16:603–15.[Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
