BMC Cancer
`
`BioMed Central
`
`Open Access
`Research article
`Anti-proliferative effects of Bifidobacterium adolescentis SPM0212
`extract on human colon cancer cell lines
`Do Kyung Lee1, Seok Jang1, Mi Jin Kim2, Jung Hyun Kim2,
`Myung Jun Chung3, Kyung Jae Kim1 and Nam Joo Ha*1
`
`Address: 1Department of Pharmacy, Sahmyook University, Seoul 139-742, Republic of Korea, 2Department of Life Science, Sahmyook University,
`Seoul 139-742, Republic of Korea and 3Cellbiotech, Co. Ltd., Seoul 157-030, Republic of Korea
`
`Email: Do Kyung Lee - 015790@hanmail.net; Seok Jang - csclub2@hanmail.net; Mi Jin Kim - sanddalki85@hanmail.net;
`Jung Hyun Kim - lemoncursc@naver.com; Myung Jun Chung - ceo@cellbiotech.com; Kyung Jae Kim - kimkjus@yahoo.com;
`Nam Joo Ha* - hanj@syu.ac.kr
`* Corresponding author
`
`Published: 27 October 2008
`
`BMC Cancer 2008, 8:310
`
`doi:10.1186/1471-2407-8-310
`
`Received: 15 April 2008
`Accepted: 27 October 2008
`
`This article is available from: http://www.biomedcentral.com/1471-2407/8/310
`
`© 2008 Lee et al; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to
`improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as
`anti-tumor activity. The aim of the present work was to study the growth inhibition of tumor cells
`by butanol extract of Bifidobacterium adolescentis isolated from healthy young Koreans.
`Methods: The anti-proliferative activity of B. adolescentis isolates was assessed by XTT assays on
`three human colon cancer cell lines (Caco-2, HT-29, and SW480). The effects of B. adolescentis
`SPM0212 butanol extract on tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production
`were tested using the murine macrophage RAW 264.7 cell line.
`Results: The butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of
`Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, respectively, at 200 μg/mL. Additionally,
`the butanol extract of B. adolescentis SPM0212 induced macrophage activation and significantly
`increased the production of TNF-α and NO, which regulate immune modulation and are cytotoxic
`to tumor cells.
`Conclusion: The butanol extract of B. adolescentis SPM0212 increased activity of the host immune
`system and may improve human health by helping to prevent colon cancer as a biological response
`modifier.
`
`Background
`Colon cancer is a serious health problem and remains the
`leading cause of cancer mortality throughout the world
`[1]. Colon cancer incidence has rapidly increased as die-
`tary patterns have changed to contain high fat, high pro-
`tein, low carbohydrate, and low fiber [2,3]. Colon cancer
`is the second most common cancer in Korea [4]. Despite
`
`recent advances in our understanding of the biological
`processes resulting in the development of cancer, there
`remains a need for new and effective agents to control this
`disease.
`
`Microorganisms, such as Mycobacterium bovis, Streptococcus
`pyogenes, Corynebacterium parvum, and cellular compo-
`
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`nents of these bacteria have been used as biological
`response modifiers (BRM) and are beneficial adjuvants to
`cancer chemotherapy, increasing remission rates and dis-
`ease-free intervals. However, the side effect profile in clin-
`ical applications for human cancer therapy is important,
`as these bacteria are pathogens [5-7].
`
`The health and nutritional benefits of orally administered
`probiotic lactic acid bacteria, such as Lactobacillus and Bifi-
`dobacteria species, which are a gram-positive and nonpath-
`ogenic, has begun to garner an increasing amount of
`attention [8,9].
`
`Probiotics, such as lactic acid bacteria (LAB), are living
`microorganisms that affect the host in a beneficial manner
`by improving nutritional and microbial balance in the
`intestinal tract. These probiotic effects increase the
`immune response, reduce colon cancer, decrease serum
`cholesterol, and produce antimicrobial substances, such
`as bacteriocins that inhibit undesirable diarrhea- and dis-
`ease-causing pathogens in the human intestine [10-18]. In
`addition, the dietary consumption of B. lactis HN019
`enhances natural immunity in healthy elderly subjects
`[19-21]. Also, viable or heat-killed Lactobacillus and Bifido-
`bacterium species, as well as certain of their cell compo-
`nents, are capable of stimulating the production of
`hydrogen peroxide, nitric oxide (NO), and cytokines, such
`as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in
`macrophage cell lines [22-24].
`
`Further, several researchers have studied the anti-tumor
`effects exerted by lactic acid bacteria [25-38]. Sekine et al.
`detected anti-tumor activity in peptidoglycans isolated
`from the B. infantis strain, ATCC 15697, and Oda et al.
`reported anti-tumor activity in polysaccharide fractions
`originating from Lactobacillus cultures [30,32]. Glycopro-
`teins detected in the supernatants of Lactobacillus cultures
`also have anti-tumor effects [33]. Many strains, including
`L. rhamnosus GG, L. acidophilus, L. casei, B. longum, B. infan-
`tis, B. adolescentis, and B. breve, suppress experimental
`colon tumor incidence [27,32-38], but the mechanisms of
`this tumor suppression are unclear [18,28,39].
`
`Our goals were to evaluate the effects of Bifidobacterium
`adolescentis isolated from fecal samples of healthy young
`Koreans on immunostimulation and anti-proliferation of
`human colon cancer cell lines in vitro.
`
`Materials and methods
`Bacterial Culture
`Fecal samples of 20 healthy Koreans (20–30 years old)
`were collected by BBL's anaerobic sample collection and
`transport system to maintain anaerobic conditions, and
`were used within 24 hr. Fecal samples were serially diluted
`10-fold from 10-1 to 10-8, and 100 μl was spread onto
`selective BL agar containing 5% sheep blood. After 48 hr
`
`of incubation in anaerobic conditions (Bactron Anaerobic
`Chamber, Sheldon Manufacturing Inc., USA) at 37°C,
`brown or reddish-brown colonies 2–3 mm in diameter
`were selected for further identification [40].
`
`A fructose-6-phosphate phosphoketolase (F6PPK) test
`was performed [41] to ensure that the colonies selected
`were Bifidobacteria, and we analyzed the carbohydrate uti-
`lization pattern (Table 1). To identify the isolated Bifido-
`bacterium spp. at the species level, 16S rRNA sequencing
`was performed by Bioleaders (Daejeon, Korea).
`
`B. adolescentis SPM0212 was cultured at 37°C for 48 hr on
`general anaerobic medium (GAM, Nissui Pharm. Co. Ltd.,
`Japan) under anaerobic conditions (90% N2, 5% H2, 5%
`CO2).
`
`Preparation of B. adolescentis SPM0212 Extract
`For the preparation of B. adolescentis SPM0212 butanol
`extract, cultures were centrifuged (Vision, USA) at 13,000
`rpm for 10 min, then the supernatant was removed and col-
`lected bacterial cell pellets were washed with autoclaved
`phosphate-buffered saline. These cell pellets were lyophi-
`lized, and this powder (0.095 g) was suspended in 50 ml of
`distilled water. Then, it was extracted with 50 ml of n-hex-
`ane or ethyl acetate or n-butanol. The BuOH fraction was
`visibly turbid. The organic solvent of extract was concen-
`trated and removed using a rotary vacuum evaporation.
`The water, n-hexane, and EtOAc fraction was omitted
`because they showed low activity or no suppressive effect
`compared with BuOH fraction in the preliminary test.
`
`Table 1: Sugar utilization of Bifidobacterium adolescentis SPM
`
`Sugar
`
`Bifidobacterium adolescentis
`
`SPM0212
`
`SPM1005
`
`SPM1601
`
`L-Arabinose
`D-Ribose
`Xylose
`Galactose
`Fructose
`Mannose
`Mannitol
`Sorbitol
`Salicine
`Cellobiose
`Maltose
`Lactose
`Melibiose
`Saccharose
`Trehalose
`Inuline
`Melezitose
`Raffinose
`Starch
`Gluconate
`
`-
`-
`+
`+
`+
`-
`-
`-
`-
`-
`+
`+
`+
`+
`+
`-
`+
`+
`+
`+
`
`+
`-
`+
`+
`+
`-
`+
`-
`+
`-
`-
`+
`+
`+
`+
`+
`+
`-
`+
`-
`
`-
`-
`+
`+
`+
`+
`-
`-
`-
`+
`-
`-
`+
`+
`-
`-
`-
`+
`+
`-
`
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`Cell Culture
`The three human colon cancer cell lines (Table 2) and the
`murine macrophage cell line, RAW 264.7, was obtained
`from the Korean Cell Line Bank (Seoul, Korea) and the
`American Type Culture Collection (ATCC), respectively.
`Caco-2, HT-29, and SW480 cells were cultured in Roswell
`Park Memorial Institute-1640 (RPMI-1640) medium,
`including fetal bovine serum (FBS) and 1% (v/v) penicil-
`lin (10,000 U/ml)/streptomycin (10,000 U/ml) (P/S).
`RAW 264.7 cells were cultured in Dulbecco's modified
`Eagle's medium (DMEM) (with 10% FBS, 1% penicillin/
`streptomycin). All cultures were incubated at 37°C in a
`humidified atmosphere with 5% CO2. After they were
`grown to confluence in 75 cm2 tissue culture flasks
`(NunC, Denmark), cells were detached and transferred to
`new cell culture dishes in a trypsin-versene mixture (Cam-
`brex Bio Science, USA). Cell number and viability were
`assessed by the trypan blue dye-exclusion method [42].
`
`Tumor Cell Proliferation by XTT Assay
`Cell proliferation was quantified via an XTT assay
`(sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-
`bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate).
`Cells were seeded on 96-well microplates (NunC, Den-
`mark) at 3 × 103 cells/well and incubated for 72 hr with
`the test compounds. Control was only cells (no treated).
`The butanol extract (no cells) was not tested. The samples
`were then incubated with 50 μl of XTT solution (1 mg/ml)
`for 6 hrs and measured with an ELISA reader (Molecular
`Devices, USA) at 490 nm.
`
`Tumor Necrosis Factor-α (TNF-α) Quantification
`RAW 264.7 (1 × 105 cells/ml), LPS (Escherichia coli
`O127:B8 Westphal type, 100 ng/mL), and test samples
`(12.5, 25, 50, 100, 200 μg/ml) were prepared as treated
`groups and incubated for 48 hr. Following incubation,
`TNF-α secretion was assessed with an OPTEIA™ Mouse
`TNF-α kit (Pharmingen, San Diego, CA, USA) in accord-
`ance with manufacturer's protocol. Briefly, the sample
`and recombinant standards were added to antibody-
`coated plates and incubated for 2 hr. TNF-α was detected
`via the addition of horseradish peroxidase-conjugated,
`streptavidin-labeled antibodies. Color was developed
`using tetramethylbenzidine (TMB) (BD Biosciences,
`Pharmingen, USA) for 30 min and the absorbance was
`recorded at 450 nm.
`
`Nitric Oxide Assay
`RAW 264.7 cells (1 × 106 cells/ml), LPS (50 ng/ml), and
`test samples (12.5, 25, 50, 100, 200 μg/ml) were prepared
`and incubated overnight. One hundred microliters from
`the surface of cultures was transferred into a new plate and
`the equivalent amount of Griess reagent was added
`(Stock-1: 0.2% naphylendia HCl, Stock-2: 2% sulfanila-
`mide in 5% H3PO4). This plate was then incubated for 10
`min at RT and measured by an ELISA reader at 540 nm.
`Standard calibration curves were prepared using sodium
`nitrite as a standard.
`
`Effect of B. adolescentis SPM0212 on Macrophage
`Morphology
`RAW 264.7 cells (1 × 103 cells/well) were cultured in ster-
`ile glass-slide chambers for 48 hr. The culture medium
`was removed, and the cells were treated with either LPS
`(100 ng/ml) or samples of B. adolescentis SPM0212 (12.5,
`25, 50, 100, 200 μg/ml) for 48 hr. Following treatment,
`the culture supernatant was removed, and the cells were
`fixed and stained in Diff Quick Solution (Baxter, Hou-
`ston, TX). Macrophage morphology was observed using a
`light microscope (BX41, Olympus, Japan) at 400× magni-
`fication.
`
`Statistical Analysis
`All data were expressed as the mean ± standard deviation
`(SD). For statistical evaluation of data, one-way ANOVA
`was applied using the program SPSS 13.0 for Windows.
`This was followed by post hoc comparisons using the
`Tukey's test. Significant differences were considered signif-
`icant at P < 0.05.
`
`Results
`B. adolescentis Strains Inhibit the Growth of Colon
`Cancer Cell Lines
`To determine whether B. adolescentis strains inhibit the
`growth of the colon cancer cell lines, Caco-2, HT-29, and
`SW 480, cells were treated with 3 different B. adolescentis
`isolates, and XTT assays were performed. B. adolescentis
`SPM0212 exhibited the highest efficacy (data not shown).
`To further characterize the functional substances of B. ado-
`lescentis SPM0212, the cell lines were treated with the
`butanol extract of this strain. The butanol extract signifi-
`cantly inhibited proliferation of both Caco-2 and SW480
`cell lines, with inhibition of Caco-2 and SW480 growth by
`70% and 40%, respectively, at 200 μg/ml (Figure 1). Treat-
`
`Table 2: Characteristics of cell lines used in this study (KCLB, Korean Cell Line Bank)
`
`Cell line
`
`Cell type
`
`Origin
`
`Growth property
`
`KCLB (ATCC) No.
`
`Caco-2
`HT-29
`SW480
`
`Epithelioid
`Epithelioid
`Epithelioid
`
`Colonic adenocarcinoma
`Colonic adenocarcinoma
`Colonic adenocarcinoma
`
`Adherent
`Adherent
`Adherent
`
`KCLB 30037
`KCLB 3003
`KCLB 10228
`
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`**
`
`**
`
`**
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`TNF-α (pg/ml)
`
`Cell
`
`LPS
`
`25
`
`50
`
`100
`
`200
`
`(100 ng/ml) Concentration (μl/ml)
`of extract of B. adolescentis
`
`Effects of B. adolescentis SPM0212 on TNF-α production from RAW 264.7 cellsFigure 2
`
`
`Effects of B. adolescentis SPM0212 on TNF-α produc-
`tion from RAW 264.7 cells. The cells (1 × 103 cells/well)
`were treated with LPS (100 ng/ml) or butanol extract of B.
`adolescentis SPM0212 (25, 50, 100, 200 μg/ml), and incubated
`for 48 hr at 37°C and 5.5% CO2. The extracellular levels of
`TNF-α in the culture media were determined by an ELISA
`reader at 450 nm. The quantitative data were presented as
`means ± SD of three independent experiments. Control ver-
`sus B. adolescentis SPM0212 butanol extract, *p < 0.05; **p <
`0.01.
`
`ment with the same concentration of butanol extract also
`decreased proliferation of HT-29, but there was no signif-
`icant difference.
`
`Effect of B. adolescentis SPM0212 on TNF-α and NO
`Production
`Next, we examined the effects of B. adolescentis SPM0212
`butanol extract on TNF-α and NO production by the mac-
`rophage RAW 264.7 cell line (Figure 2 and 3, respec-
`tively). B. adolescentis SPM0212 butanol extract
`significantly increased TNF-α production in a dose-
`dependent manner from 25 μg/ml to 200 μg/ml (Figure
`2). Treatment with 200 μg/ml of butanol extract produced
`more TNF-α than LPS treatment, which was used as a pos-
`itive control for macrophage activation. Treatment of
`RAW 264.7 cells with B. adolescentis SPM0212 butanol
`extract also increased production of NO (Figure 3). How-
`ever, increases in TNF-α and NO production by B. adoles-
`centis SPM0212 culture supernatant were not observed
`(data not shown).
`
`Morphology of RAW 264.7 cells treated with B.
`adolescentis SPM0212
`Normal RAW 264.7 cells, when cultured in medium
`alone, look refractile and rounded morphology and do
`
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`
`
`
`
`
`
`
`
`
`Caco-2
`HT-29
`SW480
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`(% of control)
`
`Growth inhibition by XTT
`
`25
`
`50
`
`100
`
`200
`
`Concentration (μl/ml)
`of extract of B. adolescentis SPM0212
`
`Effects of growth inhibition by B. adolescentis SPM0212 on colon cancer cell lines (Caco-2, HT-29 and SW480)Figure 1
`
`
`Effects of growth inhibition by B. adolescentis
`SPM0212 on colon cancer cell lines (Caco-2, HT-29
`and SW480). The cells (1 × 103 cells/well) were treated
`with B. adolescentis SPM0212 butanol extract (25, 50, 100,
`200 μg/ml), and incubated for 72 hr at 37°C and 5.5% CO2.
`After adding 50 μl of the XTT labeling mixture, they were
`incubated for 6 hr at 37°C in 5.5% CO2. The absorbance was
`measured using an ELISA reader at 490 nm. The quantitative
`data were presented as means ± SD of three independent
`experiments. Control versus treatment groups, *p < 0.05;
`**p < 0.01.
`
`**
`
`**
`
`**
`
`**
`
`20
`
`18
`
`16
`
`14
`
`12
`
`10
`
`02468
`
`Nitrite (μM)
`
`Cell
`
`100
`50
`25
`LPS
`(50 ng/ml) Concentration (μl/ml)
`of extract of B. adolescentis
`
`200
`
`Effects of B. adolescentis SPM0212 on NO production from RAW 264.7 cellsFigure 3
`
`
`Effects of B. adolescentis SPM0212 on NO production
`from RAW 264.7 cells. The cells (1 × 103 cells/well) were
`treated with LPS (50 ng/ml) or butanol extract of B. adoles-
`centis SPM0212 (25, 50, 100, 200 μg/ml), and incubated for 22
`hr at 37°C and 5.5% CO2. Nitrite concentrations in the cul-
`ture media were determined using Griess reagent assay and
`measured by ELISA reader at 540 nm. The quantitative data
`were presented as means ± SD of three independent experi-
`ments. Control versus B. adolescentis SPM0212 butanol
`extract, *p < 0.05; **p < 0.01.
`
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`Characterization of RAW 264.7 cells in response to butanol extract of B. adolescentis SPM0212Figure 4
`
`Characterization of RAW 264.7 cells in response to butanol extract of B. adolescentis SPM0212. RAW 264.7 cells
`(1 × 104 cells/well) were cultured on cover slips in the presence of different concentrations of butanol extract of B. adolescentis
`SPM0212 for 48 hr. The cells were fixed and stained in Diff-quick and observed under a light microscope at 400×. (A) Murine
`macrophage cells. (B) LPS (50 ng/ml). (C) Butanol extract of B. adolescentis SPM0212 (25 μg/ml). (D) Butanol extract of B. ado-
`lescentis SPM0212 (50 μg/ml). (E) Butanol extract of B. adolescentis SPM0212 (100 μg/ml). (F) Butanol extract of B. adolescentis
`SPM0212 (200 μg/ml).
`
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`not spread over the surface (Figure 4A). Activated macro-
`phages usually display a distinct morphology, which is
`similar to the dendritic cell. Exposure to LPS (50 ng/ml;
`the positive control) induced morphological alteration of
`the RAW 264.7 cells (Figure 4B). Treatment with B. adoles-
`centis SPM0212 butanol extract caused RAW 264.7 cells to
`become larger and rougher in a dose-dependent manner,
`suggesting activation (Figure 4C–F).
`
`Discussion
`Bifidobacterium spp., and LAB, are probiotic organisms in
`humans and stimulate immune function and anti-tumor
`effects [19-45]. Although the precise mechanisms by
`which LAB inhibit colon cancer are not known, several
`have been proposed: (a) enhancing the host immune
`response, (b) binding and degrading potential carcino-
`gens, (c) alterations in the intestinal microflora that pro-
`duce putative carcinogens, (d) production of anti-
`tumorigenic or anti-mutagenic compounds in the colon,
`and (e) alteration of metabolic activities of intestinal
`microflora [18,46-49].
`
`LAB play an important role in the host immune system to
`produce anti-tumor effects [50-52]. Macrophages play a
`major role in the host defense against infection and tumor
`formation [53], and their function can be altered by a vari-
`ety of stimulatory and suppressive signals and environ-
`mental factors. [54,55]. The production of nitric oxide
`(NO) and tumor necrosis factors (TNF-α) by macro-
`phages mediate killing and growth inhibition of tumor
`cells, bacteria, fungi, and parasites [56]. TNF-α is a non-
`glycosylated 17 kDa protein that exists as a trimer in solu-
`tion, has receptors on almost all somatic cells, regulates
`immune modulation, and is cytotoxic to tumor cells
`[57,58]. Also, TNF-α and reactive nitrogen intermediates
`play major roles in the in vitro anti-tumor activity of
`mouse peritoneal exudates from mice stimulated with
`wall peptidoglycan from B. infantis [32]. Therefore,
`cytokine production is a good measure of macrophage
`activation and further understanding of how Bifidobacte-
`rium affects the production of macrophage mediators may
`clarify how this strain affects immune function and tumor
`cells at the cellular level [22].
`
`This study showed that the butanol extract of B. adolescen-
`tis SPM0212 increased secretion of TNF-α and NO from
`the macrophage RAW 264.7 cell line, as well as changed
`cell morphology. The butanol extract may contain key fac-
`tors for increased macrophage activation and inhibition
`of tumor cell proliferation. Moreover, the butanol extract
`of B. adolescentis SPM0212 exerted direct anti-proliferative
`activity against three human colon cancer cell lines. We
`also observed that butanol extract of B. adolescentis
`SPM0212 – caused death of Caco-2, HT-29 and SW480
`cells without any cytotoxicity to nonneoplastic epithelial
`
`cell (data not shown). Here, B. adolescentis SPM0212
`potentiated TNF-production and may be beneficial in
`human intestinal tracts for immune reinforcement [59].
`In contrast, most previously reported polysaccharides that
`exhibit anti-tumor activities did not directly inhibit the
`growth of tumor cells in vitro, but instead exerted anti-
`tumor activity by stimulating macrophages and immune
`responses. Therefore, the direct inhibitory effect by the
`butanol extract on tumor cell growth observed in this
`study is exceptional for polysaccharide biomaterials, but
`the active components remain to be elucidated. Further
`studies are needed to identify the effective components in
`the B. adolescentis SPM0212 butanol extract and will be
`required to clarify the precise mechanisms of this inhibi-
`tion.
`
`Conclusion
`Bifidobacteria strains have health-promoting effects. Our
`results showed that the butanol extract of B. adolescentis
`SPM0212, isolated from fecal samples of healthy young
`Koreans, markedly and dose-dependently decreased the
`proliferation of three human colon cancer cell lines, Caco-
`2, HT-29, and SW480. In addition, the butanol extract
`increased the production of the macrophage mediators,
`TNF-α and NO, and changed macrophage RAW 264.7 cell
`morphology. Therefore, this extract could potentially help
`to enhance the host immune system and improve human
`health by helping to prevent colon cancer as a biological
`response modifier (BRM).
`
`Competing interests
`The authors declare that they have no competing interests.
`
`Authors' contributions
`This study was conceived by NJH and designed by NJH
`and KJK. NJH and MJC were responsible for obtaining
`funding and sample collection. MJK and JHK carried out
`the extraction and separation. The cultures, XTT, TNF-α,
`and NO assay, analysis of morphology were done by DKL
`and SJ. DKL performed data analysis and wrote the draft
`of the manuscript. All authors read and approved the final
`manuscript.
`
`Acknowledgements
`This research was supported by the Sahmyook University Research Fund
`(2007). The authors are grateful to the Seoul Fellowship.
`
`2.
`
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