Arch Pharm Res Vol 30, No 11, 1455-1463, 2007

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http://apr.psk.or.kr

Anti-proliferate and Pro-apoptotic Effects of 2,3-Dihydro-3,5- dihydroxy-6-methyl-4H-pyranone through Inactivation of NF-KB in Human Colon Cancer Cells

Jung Ok Ban, In Guk Hwang 1, Tae Myoung Kim 2, Bang Yeon Hwang, Ung Soo Lee 3, Heon-Sang Jeong 1, Young Won Yoon 2, Dae Joong Kim =, and Jin Tae Hong College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea, ~College of Agnculture, Life and Environments Sciences, Chungbuk National University, Cheongju 361-763, Korea, 2College of Vetemary Medicine, Chungbuk National University, Cheongju 361-763, Korea, and 3Department of Food and Biotechnology, Chungju National University, Chungju 380-702, Korea

(Received June 7, 2007)

Many natural compounds have been shown to prevent cancer cell growth through the redox regulation of transcription factors. NF-KB, a redox transcription factor, has been implicated in the apoptotic cell death of several cancer cells. This study examined whether or nor 2,3-dihy- dro-3,5-dihydroxy-6-methyl-4H-pyranone (DDMP) isolated from onions can modulate the activ- ity of NF-KB, thereby induce the apoptotic cell death of colon cancer cells. Treatment with different DDMP concentrations (0.5-1.5 mg/mL) for various periods (0-48 h) inhibited the growth of colon cancer cells (SW620 and HCT116) followed by the induction of apoptosis in a dose dependent manner. It was also found that DDMP modulated tumor necrosis factor-c~ (TNF-cQ and tetradeanoyl phorbol acetate (TPA)-induced NF-KB transcriptional and DNA bind- ing activity. Moreover, DDMP suppressed the NF-~<B target anti-apoptotic genes (Bcl-2), whereas it induced the expression of the apoptotic genes (Bax, cleaved caspase-3 and cleaved PARP). These results suggest that DDMP from onions inhibit colon cancer cell growth by inducing apoptotic cell death through the inhibition of NF-KB.

Key words: Onion (Allium cepa L.), 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyranone, Nuclear transcription factor-KB, SW620, HCT116, Apoptosis

INTRODUCTION

The role of dietary compounds in cancer prevention and treatment has been discussed widely (Niho eta/., 2006; Wang et al., 2006; Kim eta/., 2004). In this regard, the potential chemopreventive effects of onions have attracted considerable interested because onions contain a wide variety of phytochemicals and microconstituents such as trace elements, vitamins, fructans, flavonoids and sulphur compounds, which may have protective effects against cancer. Onions also contain alk(en)yl polysulphides and glycosides of flavonoids, which can modulate the hepatic drug-metabolising enzymes in rats, and reduce the carcin-

Correspondence to: Jin Tae Hong, Collage of Pharmacy, Chung- buk National University 48, Gaesin-dong, Heunduk-gu, Cheungju, Chungbuk 361-763, Korea Tel: 82-43-261-1813, Fax: 82-43-268-2732 E-mail: jinthong@chungbuk.ac.kr

ogenicity of environmental chemicals (Fukushima et al., 1997; Teyssier et aL, 2001). Even though several compounds isolated from onion have demonstrated chemopreventive effects, the identification of the new compound (DDMP), which is major contributor to the anti-cancer effect of onions, and molecular mechanisms for its action are not completely understood.

Recent evidence suggests that NF-KB activation is related to the multiple aspects of oncogenesis, including the control of apoptosis, proliferation, differentiation, and the migration of the cells (Karin et al., 2002; Young et aL, 2003; Shishodia et al., 2004). An association between colorectal cancer development and the activation of NF- ~<B has been demonstrated. The NF-•B activity was re- ported to be higher in the colon cancer cell lines and human tumor samples as well as the nuclei of stromal macro- pharges in sporadic adenomatous polyps (Lind et aL, 2001; Hardwick et aL, 2001). NF-KB can lead to the further pro-

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liferation of transformed cells through the enhanced pro- duction of growth factors and cytokines (Burkhard et aL, 2002). Therefore, the inactivation of NF-KB in many cancer cells by the chemotherapeutic agents has been demonstrat- ed to inhibit the cancer cell growth through the antiapoptotic function of NF-KB in mammalian cells including colon cancer cells (Lee et aL, 2005; Jiang et aL, 2006; Liang et aL, 2006; Aggarwal et aL, 2006). Recently, a single com- pound (named as DDMP) was isolated and identified from onions. DDMP was first identified from Phaseolus co- ccineus by Yoshiki et al. (1994), and was found to have a variety of important biological activities, such as reactive oxygen-scavenging activity (Yoshiki and Okubo, 1995; Takara et al., 2007), anti alpha-glucosidase activity in patients with diabetes mellitus (Quan et aL, 2003) and anti mutagenic activity against arylamine and 2-acetoxyacety- laminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells (Plewa et al., 1999; Berhow et al., 2000). In order to investigate the anti-tumor activity of DDMP, and its action mechanism, this study examined the effect of DDMP on the activity of NF-•B in colon car- cinoma cell lines in parallel with the effect of this com- pound on colon cell growth and apoptosis.

MATERIALS A N D METHODS

Chemicals A novel isolated chemical was characterized, and found

to be 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone, which is the same compound identified in the hypocotyls of soybean by Yoshiki and Okubo (1994). Fig. 1 show the structure. DDMP was resolved in 0.1% DMSO, and the cells were treated with 0.5-1.5 mg/mL.

o

Fig. 1. Structure of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone, a novel compound isolated from onions

Cell culture The SW620 and HCT116 human colon cancer cells

were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.). The SW620 and HCT116 human colon cancer cells were grown in RPMI1640 containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 ~g/ mL streptomycin at 37~ in 5% CO2 humidified air.

Cell viability assay The SW620 and HCT116 colon cancer cells were

plated at a density of 104 cells/well in 96 well plates per 100 ~L of medium. In order to determine the appropriate concentration that is not cytotoxic to the cells, the cytotoxic effect in the cells cultured for 12, 24, 36 and 48 h was evaluated using a cell counting kit-8 assay kit according to the manufacture's instructions (Dojindo, Maryland, U.S.A.). Briefly, 10 ~L of a CCK-8 solution was added to the cells cultured for the designed time. The plates were incubated for 1-4 h in an incubator. The resulting color was assayed at 450 nM using a microplate absorbance reader (Sunrise, Tecan, Switzerland). Each assay was carried out in triplicate.

Gel electromobility shift assay A gel shift assay was performed according to the manu-

facturer's recommendations (Promega, Madison, WI, U.S.A.). Briefly, 1• cells/ml was washed twice with 1• PBS, which was followed by the addition of 1 mL PBS. The cells were scraped into a cold Eppendorf tube, which was then spun down at 15,000 g for 5 min. The resulting supernatant was removed. Solution A (50 mM HEPES, pH 7.4, 10 mM KCI, 1 mM EDTA, 1 mM EGTA, 1 mM di- thiothreitol, 0.1 mg/mL phenylmethylsulfonyl fluoride, 1 ~g/ mL pepstatin A, 1 ~g/mL leupeptin, 10 ~g/mL soybean trypsin inhibitor, 10 ~g/mL aprotinin, and 0.5% Nonidet P- 40) was added to the pellet in a 2:1 ratio (v/v) and in- cubated on ice for 10 min. Solution C (solution A + 10% glycerol and 400 mM KCI) was then added to the pellet in a 2:1 ratio (v/v), and vortexed on ice for 20 min. The cells were centrifuged at 15,000 g for 7 min, and the resulting nuclear extract supernatant was collected in a chilled Eppendorf tube. The consensus oligonucieotides were end- labeled using T4 polynucleotide kinase and [~,_p32] ATP for 10 min at 37~ The gel shift reactions were assembled and allowed to incubate at room temperature for 10 min, which was followed by the addition of 1 I~L (50,000- 200,000 cpm) of 32p-labeled oligonucleotide and another 20 min of incubation at room temperature. Subsequently 1 ~L of a gel loading buffer was added to each reaction and loaded onto a 4% nondenaturing gel and electro- phoresis until the dye was three-fourths of the way down the gel. In the EMSA competition studies, a 100-fold excess of the unlabeled competitor oligonucleotide, NF-KB, was incubated with the nuclear extract for 30 min before add- ing the labeled probe. The mixture with the labeled probe was incubated for a further 30 min on ice. For the supershift assay, 0.5 ~g of the indicated antibodies (p65 and p50) were added and the mixture was incubated for an addi- tional 30 min on ice, and subjected to gel electrophoresis using 6% native polyacrylamide gels in 1 • Tris-borate- EDTA buffer for 2 h. The gel was dried at 80~ for 50 min and exposed to film overnight at -70~ The relative density of the DNA-protein binding bands was scanned by densitometry using Mylmage (SLB, Seoul, Korea), and

Modulating the Activity of NF-•B by DDMP 1457

quantified using Labworks 4.0 software (UVP Inc, Upland, CA, U.S.A.).

Western blot analysis The cultured cells were washed twice with lxPBS. 1 mL

of PBS was then added, and the cells were scraped into a cold Eppendorf tube. The cells were homogenized with a lysis buffer [50 mM Tris pH 8.0, 150 mM NaCI, 0.02% sodium azide, 0.2% SDS, 1 mM PMFS, 10 pL/mL aprotinin, 1% igapel 630 (Sigma-Aldrich, St. Louis, MO, U.S.A.), 10 mM NaF, 0.5 mM EDTA, 0.1 mM EGTA and 0.5% sodium deoxycholate], and centrifuged at 23,000 g for 15 min. The protein concentration was measured using the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories Inc., Hercules, CA), and an equal amount of protein (50 I~g) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech Inc., Piscataway, N J, U.S.A.). The blots were blocked for 2 h at room tem- perature with 5% (w/v) non-fat dried milk in a Tris-buffered saline [10 mM Tris (pH 8.0) and 150 mM NaCI] solution containing 0.05% tween-20. The membrane was incubated for 5 h at room temperature with the specific antibodies: rabbit polyclonal for Bax and Bcl-2 (1:500 dilution, Santa Cruz Biotechnology Inc. Santa Cruz, CA, U.S.A.); and cleaved Caspase-3 and cleaved PARP (1:1000 dilution, Cell Signaling Technology, Inc. Beverly, MA, U.S.A.)for Caspase-3. The blot was then incubated with the corres- ponding conjugated anti-rabbit and anti-mouse immuno- globulin G-horseradish peroxidase (1:4,000 dilution, Santa Cruz Biotechnology Inc.). The immunoreactive proteins were detected using an ECL western blotting detection system. The relative density of the protein bands was scanned by densitometry using Mylmage (SLB, Seoul, Korea), and quantified using Labworks 4.0 software (UVP Inc., Upland, CA, U.S.A.).

Transfection and assay of luciferase activity The SW620 and HCT116 human colon cells (2.5x10 s

cells/cm 2) were plated in 24-well plates and transiently transfected with the pNF-KB-Luc plasmid (5x NF-KB; Stratagene, CA, U.S.A.) using a mixture of the plasmid and lipofectAMINE PLUS in OPTI-MEN according to the manufacture's specification (Invitrogen, Carlsbad, CA, U.S.A.). The transfected cells were treated with TNF-~ (10 ng/mL) or TPA (50 nM) and different DDMP concentrations (0-1.5 mg/mL) for 8 h. The luciferase activity was measured using a luciferase assay kit (Promega, Madison, Wl, U.S.A.) according to the manufacturer's instructions (WinGIow, Bad Wildbad, Germany).

Detection of apoptosis The apoptosis assay was first performed using 4,6-

diamino-2-phenylindole (DAPI) staining. The SW620 and HCT116 human colon cancer cells were cultured in the presence or absence of increasing DDMP concentrations, and the induction of apoptotic cell death was evaluated after 24 h. The apoptotic cells were determined by the morphological changes after DAPI staining using fluores- cence microscopy (DAS microscope, 200x: Leica Micro- systems, Inc., Deefield, IL). Apoptosis was also evaluated using a TUNEL staining assay. In short, the cells were cultured on 8-chamber slides. After treatment with DDMP (0-1.5 mg/mL) for 24 h, the cells were washed twice with PBS and fixed by incubation in 4% paraformaldehyde in PBS for I h at room temperature. The TUNEL assays were performed using an in situ Cell Death Detection Kit (Roche Diagonostics GmbH, Mannheim, Germany) according to the manufacture's instructions. The total number of cells in a given area was determined by DAPI and TUNEL staining. The apoptotic index was determined as the number of DAPI-stained TUNEL-positive cells divided by the total cell number of cells xl00.

Data analysis The data was analyzed using one-way analysis of

variance followed by Tuckey test as a post hoc test. A p value <0.05 was considered significant.

RESULTS

DDMP inhibited NF-~:B activation NF-KB has been implicated in the apoptotic cell death of

several cancer cells. In order to determine if the inhibition of NF-KB is important in DDMP-induced apoptotic cell death, the DNA binding activity of NF-KB was determined using an electrophoretic gel mobility shift assay. It was found that a high DNA binding activity of NF-KB was de- tected in the untreated SW620 and HCT116 colon cancer cells, whereas the DDMP treatment decreased the DNA binding activity of NF-KB in a dose dependent manner (0- 1.5 mg/mL) in both SW620 (Fig. 2Aa) and HCT116 (Fig. 2Ab) colon cancer cells, the results shown in Fig. 2Aa and Ab show an almost 1-fold increase in the DNA binding activity of NF-~B. The DNA binding activity of NF-KB was verified by a competition assay using unlabelled NF-~:B as well as by a super shift assay using the antibody treat- ment of the NF-KB subunits (p65 and p50). The addition of the anti-p65 antibody clearly shifted the DNA binding of NF-KB indicating that the p65 subunit might be a major target of DDMP (Fig. 2Ac). In order to determine the effect of DDMP on the TNF-o~-induced NF-KB-dependent reporter gene expression, the cells were transiently transfected with the NF-~:B-regulated luciferase reporter construct, and the transfected cells were then stimulated with TNF-~ alone or with a combination of TNF-~ and DDMP. Con-

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Fig. 2. Effect of DDMP on the activation of NF-KB in both SW620 and HCT116 colon cancer cells. (A), The nuclear extract from SW620 (Aa) and HCT116 (Ab) colon cancer cells treated with DDMP (0-1.5 mg/mL) for 1 h and incubated in the binding reactions of 32P-end-labeled oligonucleotide containing the kB sequence. The activation of NF-~<B was examined using EMSA as described in Materials and Methods. For the competition assays, the nuclear extracts from the cells treated with TNF-c( were incubated for 1 h before EMSA with the unlabeled or labeled NF-KB oligonucleotide. For the supershift assays, the nuclear extracts from the cells treated with TNF-c( were incubated for 1 h with specific antibodies against the p50 and p65 NF-•B isoforms before EMSA. SS indicates the supershift assay using the p65 antibody (Ac). Quantification of the band intensities from three independent experimental results was determined by densitometry (Imaging System), and the value under the band indicates the fold difference from the untreated control group. (B), Colon cancer cells were transfected with the pNF-KB-Luc plasmid (5 • NF•kB) and then activated with TNF-c( (10 ng/mL) and TPA (50 nM) alone or TNF-ct and TPA plus different DDMP concentrations (0-1.5 mg/mL) at 37~ in both SW620 (Ba) and HCT116 (Bb) colon cancer cells, and the luciferase activity was then determined. All the values represent the means + S.D. of three independent experiments performed in triplicate. RLU is relative to the luciferase activity of the transfected unstimulated cells. *P<0.05 indicates statistically significant differences from the TNF-(~ and TPA treated group. (C), The cells were treated with different concentrations (0-1.5 mg/mL) of DDMP at 37~ for 1 h. The nuclear or cytosolic proteins (50 ~Lg) extracted after treatment were subjected to 12% SDS-PAGE. The expression of p50, p65, I~:B-c~, p-hcB-(~, Histone H1 and J3-actin proteins were detected by western blotting using the specific anti-bodies. The Histone H1 and b-actin protein here were used as an internal control for the nuclear or cytosolic extracts, respectively. NE, nuclear extract; CE, cytosolic extract, a, SW620 colon cancer cells; b, HCT116 colon cancer cells.

sistent with the inhibitory effect on NF-KB DNA binding activity, DDMP (0-1.5 mg/mL dose dependently) inhibited the TNF-ot and TPA induced NF-KB luciferase activity in both the SW620 (Fig. 2Ba) and HCT116 (Fig. 2Bb) colon cancer cells. It was also found that DDMP concentration dependently inhibited the phosphorylation of IKB in the cytosol, and the nuclear translocation of p50 and p65 in

both the SW620 (Fig. 2Ca) and HCT116 (Fig. 2Cb) colon cancer cells.

DDMP inhibited in SW620 and HCT116 human colon cancer cell growth

The morphological observations showed that the cells gradually decreased in size, and changed to a round single

Modulating the Activity of NF-KB by DDMP 1459

Fig. 3. Morphological changes and cell viability of colon cancer cells by DDMP. (A), The cell morphology of the SW620, HCT116 and Caco-2 cells treated or untreated with DDMP for 24 h. (B), The cell viability was determined using a CCK-8 assay, as described in Materials and Methods. The SW620, HCT116 and Caco-2 were treated with various doses (0-1.5 mg/mL) of DDMP for different times (12-48 h). The values represent the mean + S.D. of three experiments performed in triplicate, a, SW620 colon cancer cells; b, HCT116 colon cancer cells; c, Caco-2 cells. Scale bar: 100 I~M.

cell shape in a dose manner through the treatment of DDMP in both SW620 (Fig. 3Aa) and HCT116 (Fig. 3Ab) colon cancer cells. In order to determine the inhibitory effect of DDMP on SW620 and HCT116 human colon cancer cell growth, call growth was analyzed using a CCK-8 assay. The cancer cell growth inhibitory effects in both SW620 (Fig. 3Ba) and HCT116 (Fig. 3Bb) human colon cancer calls were dose (0-1.5 mg/mL) and time (12- 48 h) dependent. The IC~o values (48 h) of cell growth inhibition were 0.7 mg/mL and 0.9 mg/mL in the SW620 and HCT116 colon cancer cells, respectively. However, they had little effect on the normal colon Caco-2 cells (Fig. 3Ac and 3Bc). Therefore, DDMP appears to exert a cyto- toxic effect on colon cancer cells without incurring cyto- toxicity to the normal colon cells under these experimental conditions.

DDMP induced apoptotic cell death The induction of apoptotic call death is an important

way of blunting cancer cell growth. In order to determine if the inhibition of cell growth by DDMP was due to the in- duction of apoptotic call death, the changes in the chromatin

morphology of the cells were evaluated by DAPI staining. TUNEL staining assays were performed to further charac- terize the apoptotic cell death by DDMP. The labeled cells were then analyzed by fluorescence microscopy. The DDMP treated cells were labeled with a TUNEL assay, and the fluorescence intensity increased in both SW620 (Fig. 4Aa) and HCT116 (Fig. 4Ab) colon cancer cells. The number of apoptotic cells (DAPI-positive TUNEL stained calls) in the SW620 colon cancer cells increased to 0, 3, 42 and 79% by the addition of 0, 0.5, 1 and 1.5 mg/mL DDMP, respectively (Fig. 4Ba). The number of apoptotic cells in the HCT116 colon cancer call increased to 0, 5, 68 and 87% by the addition of 0, 0.5, 1 and 1.5 mg/mL DDMP, respectively (Fig. 4Bb).

DDMP induced the expression of apoptotic regu- latory proteins

NF-KB can regulate the genes that control apoptotic cell death. NF-kB activation in colon cancer cells correlates with their resistance to apoptosis and increased levels of anti-apoptotic proteins. The anticancer drugs are believed to mediate cell death by activating the key elements in the

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Fig. 4. Morphological changes and apoptotic cell death of the SW620 and HCT116 colon cancer cells by DDMP. (A), The cell morphology of SW620 and HCT116 treated or not treated with DDMP for 24 h was examined by optical microscopy. The apoptotic cells were examined by fluorescence microscopy after DAPI and TUNEL staining. (B), The apoptotic index was determined as the number of DAPI-stained TUNEL-positive cells counted. The values are the mean + S.E. of three experiments performed in triplicate. *P<0.05 indicates statistically significant differences from the untreated group, a, SW620 colon cancer cells; b, HCT116 colon cancer cells. Scale bar: 100 #M.

apoptosis program relating NF-KB. The expression of the apoptosis related proteins was investigated in order to de- termine the relationship between the induction of apoptosis by DDMP and the expression of the NF-~:B target apoptotic gene. In both SW620 (Fig. 5a) and HCT116 (Fig. 5b) colon cancer cells, there was increased expression of the apop- totic proteins such as Bax, cleaved caspase 3 and cleaved PARP but decreased expression of the anti-apoptotic protein Bcl-2. Moreover, the ratios of Bax/Bcl-2, cleaved caspase3/caspase3 and cleaved PARP/PARP were in- creased significantly in both the SW620 and HCT116 colon cancer cells aRer the DDMP treatment in a dose-depen- dent manner.

DISCUSSION

There has been considerable interest in the potential benefits of allium vegetables since antiquity but the details of these benefits are not completely understood. The allium genus includes approximately 500 species. The commonly

used allium vegetables include garlic, onion, leeks, ginger, and red pepper, which are used all over the world in differ- ent delicacies. There is increasing evidence that dietary phytochemicals, in particular, allium vegetables, may play important roles as chemopreventive or chemotherapeutic agents in prevention of many diseases, including colon cancer. Onion has also been shown to have significant anti-cancer effects (Leighton et aL, 1992; Galeone et aL, 2006). However, the precise molecular mechanisms and the actual components of onion that reduce the incidence of tumors are not fully understood.

In this study, it was found that a treatment with DDMP isolated from onions resulted in the inhibition of NF-KB activation in SW620 and HCT116 human colon cancer cells. This effect was paralleled with its effect on cell growth inhibition and the induction of apoptosis. Therefore, DDMP might be an important component in the anti-cancer effect of onion. Recent evidence has indicated that the trans- cription factor, NF-KB, is involved in tumor development (Lee et aL, 2005; Jiang et aL, 2006; Liang et aL, 2006;

Modulating the Activity of NF-KB by DDMP 1461

Fig. 5. Effect of DDMP on the expression of the apoptosis regulatory proteins. The SW620 (a) and HCT116 (b) colon cancer cells were treated with different concentrations (0-1.5 mg/mL) of DDMP at 37~ for 24 h. Equal amounts of the total proteins (50 pg/lane) were subjected to 12% SDS-PAGE. The expression of Bax, cleaved caspase-3, cleaved PARP, Bcl-2 and and b-actin was detected by western blotting using the specific antibodies. The I}-actin protein was used as the internal control. Quantification of the band intensities from the three independent experimental results was determined by densitometry (Imaging System). The data is reported as the mean _+S.D. from three experiments performed in triplicate for Bax/Bcl-2, cleaved caspase3/caspase3 and cleaved PARP/PARP. a, SW620 colon cancer cells; b, HCT116 colon cancer cells.

Aggarwal et aL, 2006). The activation of NF-KB is asso- ciated with several aspects of tumorigenesis, including cancer cell proliferation, the prevention of apoptosis, and the increase in angiogenesis and the metastasis potential (Karin et aL, 2002; Lee et aL, 2005). NF-KB is constitutively activated in human pancreatic breast, prostate and lung cancer cells as well as in colorectal carcinomas to confer favorable circumstances for cancer cell growth (Lee et al., 2005; Jiang et aL, 2006; Saccani et aL, 2006; Yu et aL, 2004; Huang et al., 2001; Tang et aL, 2006). In particular, NF-kB is also activated in human colon tumor samples and adenomatous polyps (Hardwick et aL, 2001). Therefore, these results suggest that the inhibition of NF-KB by DDMP might be a critical mechanism in the inhibition of SW620 and HCT116 human colon cancer cell growth. Several studies have demonstrated that the inhibition of NF-KB can contribute to the arrest of cancer cell growth. The epoxyquinone A monomer, a synthetic derivative of the natural product, epoquinol A, inhibited NF-KB by bind- ing to the p65 subunit, and NF-~:B inhibition was corre- lated with its inhibitory effect on colon cancer cell growth (Liang et aL, 2006). Aggarwal et al also reported that curcumin inhibited colon cancer cell growth through the inactivation of NF-KB (Aggarwal et aL, 2006). Recently, it was also found that the inhibition of NF-KB by 2'-hydroxy- namaldehyde resulted in the inhibition of colon cancer cell

growth through the induction of apoptotic cell death of human colon cancer cells (Lee et aL, 2005). These results suggest that the constitutive activation of the NF-KB signaling pathway might be an important step in develop- ment of colon tumors. Hence, the inhibition of NF-KB activa- tion might be one mechanism for how DDMP mediates the prevention of colon cancer cell growth.

The precise mechanism for how DDMP inhibits colon cancer cell growth is unclear. However, it might be related to the role of NF-KB in cancer development through the regulation of the cell death related genes. NF-KB plays an important role in cell protection against a wide variety of apoptotic stimuli including chemotherapeutic treatments through the inactivation of the antiapoptotic gene program in cells (Lee et aL, 2005; Yemelyanov et aL, 2006). The pro- and anti-apoptotic genes regulated by NF-KB include the inhibitor of apoptosis proteins such as the inhibitor of the apoptosis protein Bcl-2, and the activator of the apoptosis proteins such as Bax, Cleaved Caspase-3 and -9, and Poly (ADP-ribose) polymerase (PARP) (Lee et aL, 2005; Yemelyanov et al., 2006). Apoptosis is an important mechanism for eliminating unwanted cells in a wide variety of physical processes, and the regulation of this process has been implicated in the pathogenesis of many chronic diseases, including cancer development (Pierce et aL, 2007). The DDMP treatment in SW620 and HCT116 human colon cancer cells alters the expression of the apoptosis regulatory proteins. DDMP inhibited the expres- sion of the anti-apoptotic proteins (Bcl-2), whereas it in- creased the expression of the proapoptotic proteins (Bax, cleaved caspase-3 and cleaved PARP). Therefore, the change in the expression of the apoptosis and anti- apoptosis regulatory proteins by DDMP can produce favorable circumstances for cancer cells death. In addition, there was no cytotoxic effect observed in the normal colon cells treated with DDMP (up to 1.5 mg/mL). Overall, these results show that DDMP might be an important component in the anti-cancer effects of onion, and the induction of apoptosis might be involved in the DDMP-induced inhibi- tion of cell growth through the inactivation of NF-KB.

ACKNOWLEDGEMENTS

This work was supported by a grant (20050401-034- 063-007-03-00) from BioGreen 21 Program, Rural Develop- ment Administration, Republic of Korea.

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