Arginine enhances tumour growth. Conversely, the restriction of arginine inhibits the growth of metastatic tumours(Reference Gonzalez and Byus1, Reference Wu and Morris2) due to this amino acid being involved in several biosynthetic pathways that significantly influence carcinogenesis and tumour biology (for example, NO generation, creatine production and polyamine synthesis). More pathways are disturbed by removing arginine than by removing most other amino acids. Also, tumour cells die rapidly in an arginine-free medium, whereas diploid fibroblasts move into quiescence, a difference that clearly offers a selective advantage in targeting tumour cells(Reference Wheatley, Scott and Lamb3). Therefore, there have been attempts to use l-arginine-degrading enzymes in cancer treatment, with arginine-depletion enzyme being regarded as a potential anticancer agent(Reference Cheng, Lam and Lam4).
Most investigations of arginine deiminase (ADI; EC 3.5.3.6), which catalyses the imine hydrolysis of arginine to produce citrulline and ammonia from Mycoplasma arginini (MADI), have suggested that this inhibits cell proliferation in hepatocellular, melanoma, leukaemia and prostate cancer cell lines by inducing G0/G1 arrest(Reference Gong, Zolzer and von Recklinghausen5–Reference Kang, Kang and Park8), with these cells being auxotrophic to arginine(Reference Dillon, Prieto and Curley9). In addition, MADI has been shown to affect apoptosis, inhibit NO synthesis, exert effects against TNF-α and neutralise endotoxin(Reference Gong, Zolzer and von Recklinghausen5, Reference Noh, Kang and Shin6, Reference Beloussow, Wang and Wu10–Reference Thomas, Holtsberg and Ensor12). Recent phase I and II clinical studies have investigated the effects of the pegylated form of ADI on hepatocellular carcinoma and melanoma(Reference Ascierto, Scala and Castello13, Reference Izzo, Marra and Beneduce14).
Abnormal cell proliferation is strongly associated with carcinogenesis. The regulation of apoptosis, a process of controlled cellular death, is required for the maintenance of normal homeostasis, which represents the balance between cell proliferation and cell death in multicellular organisms(Reference Kim, Kwon and Lee15). Therefore, controlling the cell cycle and apoptosis has been considered a promising target for cancer chemoprevention(Reference Danial and Korsmeyer16). c-Myc is a proto-oncogene that acts as a transcription factor stimulating both cell-cycle progression and apoptosis, and is overexpressed in a wide range of human cancers. It plays important roles in G1-phase progression by regulating the expressions of p27Kip1(Reference Obaya, Kotenko and Cole17) and cyclin D1 protein(Reference Perez-Roger, Kim and Griffiths18). p53 is a tumour-suppressor gene that is highly mutated in many tumours. It is a key regulator of cell-cycle arrest, senescence, differentiation and apoptosis by acting as a transcription factor or interacting with other proteins such as members of the Bcl family including Bcl-2, Bcl-xL and Bax, which play a central role in apoptosis. Although members of this family have similar structures, there are two subfamilies with opposite functions: anti-apoptotic (including Bcl-2 and Bcl-xL) and pro-apoptotic (including Bax).
Lactic acid bacteria (LAB) have been used to ferment foods such as cheese, yoghurt and kimchi for at least the past 4000 years, and have recently been shown to be effective chemopreventive food ingredients against many cancer types (reviewed by de Moreno de LeBlanc et al. (Reference de Moreno de LeBlanc, Matar and Perdigon19) and Kim et al. (Reference Kim, Kim and Lee20)). However, the active components and underlying mechanisms have not been fully elucidated. We previously screened the anti-tumour capacities of various cellular components – whole cells, peptidoglycans and cytoplasmic fractions – from ten types of LAB by measuring the inhibition of proliferation in diverse human tumour cell lines which exist in the gastrointestinal tract. The cytoplasmic fraction but not the peptidoglycan fraction of Lactococcus lactis strongly suppressed the proliferation of SNU-1 human stomach cancer cells(Reference Kim, Woo and Kim21). However, the components in the cytoplasmic fraction of Lactococcus lactis (CFL) responsible for the antiproliferative activity were not determined. The present study investigated whether the antiproliferative activity of CFL in SNU-1 human stomach cancer cells is linked to the induction of cell-cycle arrest and apoptosis by ADI.
Materials and methods
Chemicals
Methyl thiazol tetrazolium (MTT), propidium iodide, ethidium bromide, 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris)-borate, EDTA and the antibody for β-actin were purchased from Sigma (St Louis, MO, USA). p53, p27Kip1, p21Cip1, c-myc, cyclin D1, Bcl-xL, Bax and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies (anti-mouse and anti-rabbit) were purchased from Zymed (San Francisco, CA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum and streptomycin were purchased from GIBCO BRL (Grand Island, NY, USA). M17 broth was obtained from Difco Laboratories (Detroit, MI, USA). All other chemicals were purchased from Sigma.
Cell culture
The SNU-1 cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). The cell lines were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with 10 % (v/v) fetal bovine serum (GIBCO BRL), penicillin (1000 U/l) and streptomycin (1 mg/l) (GIBCO BRL) in a humidified incubator (Forma Scientific, Marjetta, OH, USA) with 5 % CO2 at 37°C.
Preparation of lactic acid bacteria
LAB were used for screening antiproliferative and ADI activities. Lactococci and streptococci were cultured in M17 broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5 % (w/w) glucose, lactobacilli were cultured in deMan–Rogosa–Sharpe (MRS) broth and bifidobacteria were cultured in brain-heart-infusion broth with 0·05 % (w/w) l-cysteine. The cells were then harvested by centrifugation at 4°C, washed three times with distilled water and sonicated with a cell disruptor (Sonics & Materials, Danbury, CT, USA) for 30 min in ice. The suspension was centrifuged (Hitachi, Tokyo, Japan) at 70 000 g for 30 min and the supernatant fraction was used to obtain the cytoplasmic fraction (CFL).
Purification of arginine deiminase from cytoplasmic fraction of Lactococcus lactis
Native ADI (purified ADI; PADI) was purified from CFL. CFL was subjected to anion exchange chromatography on a Q-Sepharose Fast Flow column (5 ml; GE Healthcare, Piscataway, NJ, USA) that had previously been equilibrated with 50 mm-Tris buffer (pH 7·0). The loaded column was washed with 30 ml of the same buffer and then eluted with a linear gradient (0–1·0 m) of NaCl in 50 ml of the Tris buffer using fast protein liquid chromatography. The ADI-rich fractions were pooled, concentrated by a centrifugal concentrator (Vivaspin 15; Vivascience, Epsom, Surrey, UK) and subjected to gel permeation chromatography on a Sephacryl-S200 column (Amersham; 1·6 × 26 cm). The column was washed with 50 mm-Tris buffer (pH 7·0) containing 0·15 m-NaCl and then eluted with the same buffer using fast protein liquid chromatography. The active fractions were analysed by SDS-PAGE and Coomassie brilliant blue staining (Fig. 1).
Fig. 1 Flow chart of the procedure for purifying arginine deiminase (ADI). LADI, arginine deiminase from Lactococcus lactis, IPTG, isopropyl β-d-1-thiogalactopyranoside; E. coli, Escherichia coli. * GE Healthcare (Piscataway, NJ, USA).
Measurement of arginine deiminase enzymic activity
ADI activity was quantified based on the amount of l-citrulline produced from l-arginine as described previously(Reference Boyde and Rahmatullah22). Briefly, CFL was incubated with 10 mm-arginine and 50 mm-Tris (pH 7·0) in a final volume of 1·0 ml for 3 h at 37°C. The reaction was terminated by the addition of 0·5 ml of a 1:3 mixture (v/v) of H2SO4 and H3PO4. The amount of l-citrulline formed during the incubation was determined by measuring the coloured reaction product formed from l-citrulline and diacetyl monooxime.
Expression and purification of recombinant arginine deiminase
Recombinant ADI (ADI from L. lactis; LADI) was expressed and purified to homogeneity, and its enzyme activity was determined(Reference Kim, Jeong and Lee23). Briefly, the arcA gene of L. lactis ssp. lactis American Type Culture Collection (ATCC) 7962 (National Center for Biotechnology Information (NCBI) accession no. DQ364637) that coded LADI was cloned. We constructed pLADI, in which the arcA gene was located downstream of the T7 promoter. LADI was expressed by isopropyl β-d-1-thiogalactopyranoside (IPTG; 1 mm) in Escherichia coli BL21 (DE3) harbouring pLADI. Other purification procedures were the same as that for PADI (Fig. 1).
Measuring antiproliferative activity
The effects on cell proliferation were measured by the MTT assay, based on the ability of live cells to convert tetrazolium salt into purple formazan. In brief, the cells were seeded on ninety-six-well microplates and then treated with samples at different concentrations for 48 h, after which 20 μl MTT stock solution (5 mg/ml, Sigma) were added to each well and the plates were further incubated for 4 h at 37°C. The supernatant fraction was removed and 100 μl dimethyl sulfoxide were added to each well to solubilise the water-insoluble purple formazan crystals. The absorbance at 570 nm was measured with a microplate reader (Multiscan MCC 340; Titertek, Huntsville, AL, USA). All the measurements were performed in triplicate. Results are expressed as the percentage proliferation with respect to untreated cells.
Cell-cycle analysis
Cells (1·0 × 105) were cultured in a 10 cm dish for 24 h and then treated with LADI at 0, 0·6 or 1·2 μg/ml for 48 h. The DNA content of nuclei was then determined by staining nuclear DNA with propidium iodide (50 μg/ml; Sigma) and measuring with a flow cytometer (Becton Dickinson, San Jose, CA, USA). The proportion of nuclei in each phase of the cell cycle was determined using FlowJo DNA analysis software (Tree Star, Ashland, KT, USA).
DNA fragmentation assay
Cells (1·0 × 105) were cultured in a 10 cm dish for 24 h, treated with LADI at 1·2 μg/ml for 48 h, and then lysed in 5 mm-Tris-HCl buffer (pH 7·4) containing 0·5 % Triton X-100 and 20 mm-EDTA for 30 min at 4°C. After centrifugation at 19 000 g for 30 min, supernatant fractions were extracted with phenol–chloroform and precipitated in ethanol. Samples were subjected to electrophoresis on a 1·5 % agarose gel containing ethidium bromide (Sigma). The gel was run in TBE (Tris-borate and EDTA; Sigma) buffer at 75 V (1·5 h, room temperature) and visualised on a UV light box.
Nuclear staining with 4′,6-diamidino-2-phenylindole
Cells (1·0 × 105) were cultured in a 10 cm dish for 24 h, treated with LADI at 1·2 μg/ml for 48 h and then fixed by 4 % paraformaldehyde (Sigma). After being transferred to slides, cells were dried at room temperature and then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Sigma) for 15 min. The adhering cells were washed with PBS, air dried and mounted with 90 % glycerol. The cell nuclear morphology was observed under a fluorescence microscope (Olympus Ix70; Olympus, Tokyo, Japan).
Western blot analysis
Cells (1·0 × 105) were cultured in a 10 cm dish for 24 h and then treated with LADI at 0, 0·6 and 1·2 μg/ml for 48 h. Cell pellets obtained by centrifugation at 1000 g for 5 min were washed with and then re-suspended in PBS. Cell lysis was performed at 4°C for 30 min in cell lysis buffer (20 mm-Tris-HCl (pH 7·5), 150 mm-NaCl, 1 mm-Na2EDTA, 1 mm-ethylene glycol tetraacetic acid, 1 % Triton, 2·5 mm-sodium pyrophosphate, 1 mm-β-glycerophosphate, 1 mm-Na3VO4, leupeptin (1 μg/ml) and 1 mm-phenylmethanesulfonylfluoride). The cell lysates were centrifuged at 23 000 g for 15 min and the resulting supernatant fraction was stored at − 70°C before Western blot analysis. The protein concentration in each sample was measured by subjecting lysate protein (30 μg) to 10 % SDS-PAGE, with the protein electrophoretically transferred to a nitrocellulose membrane (Whatman, Clifton, NJ, USA). Protein bands were visualised by a chemiluminescence detection kit (GE Healthcare, Piscataway, NJ, USA) after hybridisation with the horseradish peroxidase-conjugated secondary antibody. Quantification of the bands was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
Where appropriate, data are expressed as mean values and standard deviations. Student's t test was used for single comparisons. A probability value of P < 0·05 was used as the criterion for statistical significance.
Results
Antiproliferative activity of cytoplasmic fraction of Lactococcus lactis in SNU-1 cells and its relationship with arginine deiminase activity
We first measured the antiproliferative activities of cytoplasmic fractions of six types of LAB. CFL exerted the strongest antiproliferative effects. The half-maximal inhibitory concentration (IC50) value of CFL was 17 μg/ml, and those of other LAB were more than 200 μg/ml, except for values of 107 and 98 μg/ml for L. plantarum and Bifidobacterium adolescentis, respectively (Table 1). We measured ADI activity to isolate the potential compounds responsible for these effects. CFL exerted the strongest ADI activity in the six types of LAB (Fig. 2(a)). To elucidate the active compound underlying this effect, we used an anion exchange and gel filtration column to purify PADI (Fig. 2(b)). The inhibitory effects of purified PADI on the proliferation of SNU-1 cells were determined by the MTT assay. SNU-1 cells were treated with the final ADI fractions at 31·3, 62·5, 125, 250 and 500 μg/ml. PADI significantly decreased the proliferation of SNU-1 cells in a dose-dependent manner, with an IC50 value of 2 μg/ml (Fig. 2(c)). These results indicated that the antiproliferative effects of CFL were due to ADI activity.
Table 1 Half-maximal inhibitory concentration (IC50) values of cytoplasmic fractions from lactic acid bacteria against SNU-1 cells (μg/ml)
KCTC, Korean Collection for Type Cultures; ATCC, American Type Culture Collection.
Fig. 2 Antiproliferative activity of the cytoplasmic fraction of Lactococcus lactis correlates with arginine deiminase (ADI) activity. (a) ADI enzymic activities of cytoplasmic fractions of several lactic acid bacteria (Lcas, Lactobacillus casei; Lpla, Lactobacillus plantarum; Sthe, Streptococcius thermophillus; Lbul, Lactobacillus bulgarucus; Bado, Bifidobacterium adolescentis; Llac, Lactococcus lactis; see Table 1) were determined by the amount of l-citrulline produced from l-arginine as described in the Materials and methods section. Data are means from triplicate tests, with standard deviations represented by vertical bars. ** P < 0·01. (b) SDS-PAGE analysis of purified ADI (PADI). Lanes: 1, molecular-mass marker protein; 2, total cell extract; 3, eluted fraction from Q-sepharose; 4, eluted PADI from sephacryl S-200. (c) Antiproliferative activity of PADI in SNU-1 cells as measured using the methyl thiazol tetrazolium assay. Data are means from triplicate tests, with standard deviations represented by vertical bars.
Arginine deiminase from Lactococcus lactis induces antiproliferative effects in SNU-1 cells via arginine depletion
To verify the mechanisms underlying the antiproliferative effects of ADI in SNU-1 cells, we expressed and purified LADI in an E. coli system(Reference Kim, Jeong and Lee23). The MTT assay was used to analyse the antiproliferative effects of LADI in SNU-1 cells (Fig. 3(a)). LADI dose-dependently inhibited the proliferation of SNU-1 cells with an IC50 of about 0·6 μg/ml. Subsequent experiments were performed using concentrations of 0·6 or 1·2 μg/ml. We added extra arginine to the culture medium to confirm whether the antiproliferative effect of LADI was mediated by depleting arginine. We found that the inhibition of proliferation of SNU-1 cells by LADI was dose-dependently recovered by arginine treatment (Fig. 3(b)).
Fig. 3 Antiproliferative activity of arginine deiminase from Lactococcus lactis (LADI) in SNU-1 cells by arginine depletion. (a) Antiproliferative activity of LADI. Cells were treated with LADI (0, 0·375, 0·75, 1·5, 3, 6 or 12 μg/ml) for 48 h and were measured using the methyl thiazol tetrazolium assay. (b) Arginine recovered the antiproliferative activity of LADI. Data are means from triplicate tests, with standard deviations represented by vertical bars. * P < 0·05, ** P < 0·01, *** P < 0·001.
Arginine deiminase from Lactococcus lactis induces G1-phase arrest and apoptosis in SNU-1 cells
We also examined whether the antiproliferative effects of LADI involved cell-cycle arrest and apoptosis. SNU-1 cells were treated with LADI at 0, 0·6 or 1·2 μg/ml, and the distribution of cell-cycle phases was determined using flow cytometry with propidium iodide staining. The apoptotic sub-G1-phase fraction increased from 2·54 % to 11·58 % in SNU-1 cells by LADI treatment. LADI induced cell-cycle arrest of SNU-1 cells at the G0/G1 phase and a progressive decline of G2/M-phase cells (Fig. 4). These results indicate that LADI induced apoptotic cell death concurrently with cell-cycle arrest in SNU-1 cells. We also assessed apoptosis in SNU-1 cells by examining DNA fragmentation and nuclear condensation using agarose electrophoresis and the DNA-specific fluorescent dye 4′,6-diamidino-2-phenylindole. SNU-1 cells were treated with LADI at 0 or 1·2 μg/ml for 48 h. We found that LADI increased DNA fragmentation (Fig. 5(a)) and nuclear condensation (Fig. 5(b)) in SNU-1 cells. These results provide evidence that LADI induces cell-cycle arrest and apoptosis in SNU-1 cells.
Fig. 4 Effects of arginine deiminase from Lactococcus lactis (LADI) on the cell cycle (□, sub-G1; 
, G0/G1; 
, S; 
, G2/M) in SNU-1 cells after treatment with LADI. Cells were treated with LADI (0 (a) (control), 0·6 (b) or 1·2 (c) μg/ml) for 48 h, stained with propidium iodide (PI) and analysed by fluorescence-activated cell sorting. FL2-A, PI fluorescence on channel 2. All experiments were performed in duplicate and gave similar results. In the control treatment, the percentage of cells at each stage of the cell cycle was: sub-G1, 2·54; G0/G1, 51·34; S, 46·63; G2/M, 25·97. In the 0·6 μg LADI/ml treatment, the percentage of cells at each stage of the cell cycle was: sub-G1, 10·72; G0/G1, 61·17; S, 42·72; G2/M, 4·48. In the 1·2 μg LADI/ml treatment, the percentage of cells at each stage of the cell cycle was: sub-G1, 11·58; G0/G1, 73·17; S, 40·92; G2/M, 1·50.
Fig. 5 Arginine deiminase from Lactococcus lactis (LADI) induced DNA fragmentation and chromatin condensation in SNU-1 cells. (a) LADI induced internucleosomal DNA fragmentation. SNU-1 cells were incubated with LADI at 0 or 1·2 μg/ml for 48 h. Genomic DNA was then prepared and analysed on a 1·5 % agarose gel. Lane 1 shows DNA size markers (M; 100 bp DNA ladder; Invitrogen, MI, USA). (b) LADI induced chromatin condensation and segregation. After treatment with LADI (1·2 μg/ml) for 48 h, cells were washed and fixed in 4 % paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole to assess chromatin condensation.
Effects of arginine deiminase from Lactococcus lactis on distribution of cell-cycle phases and expression of apoptosis-regulating proteins
Different regulators working in multiple pathways tightly regulate the cell cycle, including p53, cyclin and CDKi (cyclin-dependent kinase inhibitor) proteins(Reference Vermeulen, Berneman and Van Bockstaele24). The Bcl-2 family of anti- and pro-apoptotic regulators is important to life-or-death decisions(Reference Cory and Adams25). We used immunoblotting to detect proteins underlying the LADI-induced cell-cycle arrest and apoptosis in SNU-1 cells. LADI significantly increased the expressions of p53 and p27Kip1 and significantly decreased the expressions of cyclin D1 and c-myc in SNU-1 cells. However, it had no effect on the expression of p21Cip1 (Fig. 6(a)). The expression of Bcl-xL but not of Bcl-2 and Bax proteins was inhibited by LADI (Fig. 6(b)).
Fig. 6 Effects of arginine deiminase from Lactococcus lactis (LADI) on G1-phase regulation and Bcl-2-family proteins in SNU-1 cells. After treatment with LADI (0 (control), 0·6 or 1·2 μg/ml) for 48 h, cells were harvested and the levels of p53, p27Kip1, p21Cip1, c-myc, cyclin D1, Bcl-xL, Bax, Bcl-2 and β-actin proteins were determined by Western blot analysis as described in the Materials and methods using specific antibodies. The scores above the bands are percentage of control, normalised by their β-actin. All experiments were performed in duplicate and gave similar results.
Discussion
There is considerable evidence of the anticancer effects of LAB(Reference Kim, Kim and Lee20), but the underlying active compounds and mechanisms still need to be elucidated. We screened the antiproliferative effects of various species of LAB and their fractions in several cancer cell lines(Reference Kim, Woo and Kim21). We found that CFL has the lowest IC50 in SNU-1 cells. To explain this, we searched for active compounds that exhibit differential expression in these species. We hypothesised that the active compound was the enzyme expressed only by L. lacits, because it is a soluble component and is effective at a relatively low concentration.
ADI is an enzyme that has been previously described as exerting antiproliferative effects in micro-organisms. ADI underlies the death of certain cell lines infected with Mycoplasma species(Reference Sasaki, Shintani and Kihara26), and its anticancer effects have also been examined(Reference Feun and Savaraj27). It was reported that L. lactis ssp. lactis expresses ADI, and that there are important differences between the characteristics of L. lactis ssp. lactis and L. lactis ssp. cremoris (Reference Niven, Smiley and Sherman28). We previously found that whilst there was only 35 % amino acid sequence homology between LADI and MADI, all of the important sequences involving enzyme activity were conserved(Reference Kim, Jeong and Lee23). LADI also exhibits anti-inflammatory effects, inhibiting lipopolysaccharide-induced cyclo-oxygenase-2 and inducible NO synthase expression in RAW 264·7 murine macrophages(Reference Kim, Hur and Lee29). We therefore assessed ADI activity in the cytoplasmic fractions of several LAB and found a negative correlation between IC50 values and ADI activity. Due to quantity limitations, we cloned, expressed and purified LADI in E. coli in order to study the underlying mechanisms(Reference Kim, Jeong and Lee23). Both LADI and PADI inhibited cell proliferation, but the IC50 was lower for LADI, which could have been due to purity differences.
Cancer and normal cells exhibit differences in their metabolism of basic nutrients such as amino acids. Depleting nutrients that are important to cell growth kills cancer cells but not normal cells, and hence this is a potential cancer therapeutic strategy(Reference Thompson, Bauer and Lum30). Although arginine is not an essential amino acid, some tumours are auxotrophic to arginine, including melanoma and hepatocellular carcinoma. Arginine depletion arrests the cell cycle at the G0/G1 phase in these cell types(Reference Wheatley, Scott and Lamb3). In the present study we found that treatment with LADI arrested SNU-1 cells in the G0/G1 phase, and they died via apoptosis. These results indicated that SNU-1 cells are also auxotrophic to arginine because it inhibits cell growth.
c-Myc is involved in the antiproliferative effects of arginine depletion, and has previously been shown to play a key role in cell proliferation and in the inhibition of cancer cell growth by amino acid depletion(Reference Yuneva, Zamboni and Oefner31). MADI-induced arginine depletion resulted in G1-phase cell-cycle arrest and apoptosis, and inhibited c-myc expression in human T-cell acute lymphoblastic leukaemia CCRF-CEM cells(Reference Noh, Kang and Shin6). We found that LADI also inhibited c-myc in SNU-1 cells. c-Myc controls many proteins involved in the G0/G1-to-S-phase transition, such as cyclin D1, p27Kip1 and p21Cip1. It also regulates p53, Bcl-2, Bcl-xL and Bax, which are related to apoptosis(Reference Perez-Roger, Kim and Griffiths18, Reference Adhikary and Eilers32–Reference Dang, O'Donnell and Zeller34). LADI increased the expressions of p53 and p27Kip1 and decreased the expressions of cyclin D1 and Bcl-xL in SNU-1 cells. However, LADI had no effects on the expressions of p21Cip1 and Bcl-2. Previous investigations of the mechanism underlying arginine depletion showed that it inhibited cyclin D1 expression in human diploid fibroblasts(Reference Lamb and Wheatley35) and induced p27Kip1 and p21Cip1 in HepG2 human hepatoma cells(Reference Leung-Pineda, Pan and Chen36). Also, MADI reportedly inhibits Bcl-xL in human lymphoma cells(Reference Seo, Sung and Choi37). However, how arginine depletion inhibits c-myc expression remains to be elucidated.
In conclusion, the antiproliferative effects of CFL are due to ADI. LADI induces G1-phase cell-cycle arrest and apoptosis via arginine depletion by inhibiting c-myc, cyclin D1 and Bcl-xL, and inducing p53 and p27Kip1. The present paper is the first to reveal the putative active component underlying the anticancer effects of L. lactis ssp. lactis and their underlying mechanisms.
Acknowledgements
The present study was supported by the World Class University (WCU) program (no. R31-2008-00-10 056-0) and the Special Research and Development Program for Biofood Research (no. R01-2007-000-11957-0) through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology.
K. W. L. and H. J. L designed the study and prepared the paper. J. E. K. and S. Y. K. contributed to the successful execution of the experimental work.
We declare that we have no conflict of interest.
