Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines

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 dietary patterns have changed to contain high fat, high protein, 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-nents of these bacteria have been used as biological response modifiers (BRM) and are beneficial adjuvants to cancer chemotherapy, increasing remission rates and disease-free intervals. However, the side effect profile in clinical applications for human cancer therapy is important, as these bacteria are pathogens [5][6][7].
The health and nutritional benefits of orally administered probiotic lactic acid bacteria, such as Lactobacillus and Bifidobacteria species, which are a gram-positive and nonpathogenic, 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 disease-causing pathogens in the human intestine [10][11][12][13][14][15][16][17][18]. In addition, the dietary consumption of B. lactis HN019 enhances natural immunity in healthy elderly subjects [19][20][21]. Also, viable or heat-killed Lactobacillus and Bifidobacterium species, as well as certain of their cell components, 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][23][24].
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.

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 utilization pattern (Table 1). To identify the isolated Bifidobacterium spp. at the species level, 16S rRNA sequencing was performed by Bioleaders (Daejeon, Korea).

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 collected bacterial cell pellets were washed with autoclaved phosphate-buffered saline. These cell pellets were lyophilized, and this powder (0.095 g) was suspended in 50 ml of distilled water. Then, it was extracted with 50 ml of n-hexane or ethyl acetate or n-butanol. The BuOH fraction was visibly turbid. The organic solvent of extract was concentrated 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.

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) penicillin (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% CO 2 . After they were grown to confluence in 75 cm 2 tissue culture flasks (NunC, Denmark), cells were detached and transferred to new cell culture dishes in a trypsin-versene mixture (Cambrex Bio Science, USA). Cell number and viability were assessed by the trypan blue dye-exclusion method [42].

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 significant at P < 0.05.

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. adolescentis SPM0212, the cell lines were treated with the butanol extract of this strain. The butanol extract significantly 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- ment with the same concentration of butanol extract also decreased proliferation of HT-29, but there was no significant 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 macrophage RAW 264.7 cell line (Figure 2 and 3, respectively). B. adolescentis SPM0212 butanol extract significantly increased TNF-α production in a dosedependent 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 positive control for macrophage activation. Treatment of RAW 264.7 cells with B. adolescentis SPM0212 butanol extract also increased production of NO ( Figure 3). However, increases in TNF-α and NO production by B. adolescentis 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 After adding 50 μl of the XTT labeling mixture, they were incubated for 6 hr at 37°C in 5.5% CO 2 . 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. Effects of B. adolescentis SPM0212 on NO production from RAW 264.7 cells Figure 3 Effects of B. adolescentis SPM0212 on NO production from RAW 264.7 cells. The cells (1 × 10 3 cells/well) were treated with LPS (50 ng/ml) or butanol extract of B. adolescentis SPM0212 (25, 50, 100, 200 μg/ml), and incubated for 22 hr at 37°C and 5.5% CO 2 . Nitrite concentrations in the culture 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 experiments. Control versus B. adolescentis SPM0212 butanol extract, *p < 0.05; **p < 0.01. G not spread over the surface ( Figure 4A). Activated macrophages 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. adolescentis 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 . 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 carcinogens, (c) alterations in the intestinal microflora that produce putative carcinogens, (d) production of antitumorigenic or anti-mutagenic compounds in the colon, and (e) alteration of metabolic activities of intestinal microflora [18,[46][47][48][49].
LAB play an important role in the host immune system to produce anti-tumor effects [50][51][52]. Macrophages play a major role in the host defense against infection and tumor formation [53], and their function can be altered by a variety of stimulatory and suppressive signals and environmental factors. [54,55]. The production of nitric oxide (NO) and tumor necrosis factors (TNF-α) by macrophages mediate killing and growth inhibition of tumor cells, bacteria, fungi, and parasites [56]. TNF-α is a nonglycosylated 17 kDa protein that exists as a trimer in solution, 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 Bifidobacterium 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. adolescentis 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 factors 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 antitumor 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 inhibition.

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).
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