Thirabunyanon & Hongwittayakorn, 2013 [cancer]

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Potential Probiotic Lactic Acid Bacteria of Human Origin
Induce Antiproliferation of Colon Cancer Cells
via Synergic Actions in Adhesion to Cancer Cells
and Short-Chain Fatty Acid Bioproduction
Mongkol Thirabunyanon & Penrat Hongwittayakorn
Received: 3 August 2012 /Accepted: 28 November 2012 /
Published online: 13 December 2012
# Springer Science+Business Media New York 2012
Abstract The activities and modes of probiotic action of lactic acid bacteria isolated from
infant feces were investigated for alternative application in the prevention and biotherapy of
colon cancer. From a total of 81 isolates of Gram-positive rod and cocci bacteria obtained
from healthy infants, only 15 isolates had the probiotic criteria which included growth
inhibition against eight food-borne pathogens, no blood hemolysis, and tolerance to gastro-
intestinal tract properties such as pH2.5 and 0.3 % bile salt. Four probiotic bacteria showed
antiproliferation of colon cancer cells with the use of MTT and Trypan blue exclusion assay
at the rates of 17–35 %. Through comparison of probiotic 16S rRNA sequences, they were
identified as Pediococcus pentosaceus FP3, Lactobacillus salivarius FP25, L. salivarius
FP35, and Enterococcus faecium FP51. Finding the mechanism of proliferative inhibition of
colon cancer cells in this study indicated synergic induction by probiotic bacteria directly
adhered to these cancer cells and triggered the bioproduction of short-chain fatty acids,
mainly butyric and propionic acids. This study suggested that the use of these probiotics may
be suitable as an alternative bioprophylactic and biotherapeutic strategy for colon cancer.
Keywords Caco-2 . Colon cancer . Food-borne pathogens . Lactic acid bacteria . Probiotic .
Short-chain fatty acids
Introduction
Microbiota in the human intestines are generally implicated as the causes of human diseases
such as colon cancer and promoters of good health as protectors against diseases. These
microbiota have been assumed as amounting from 1012 to 1014colony-forming units (CFU)/
g of luminal content [1], with large communities that include more than 2,000 different
species of aerobic, facultative, and anaerobic bacteria [2]. There are many interacting
functions of microbiota and/or intestinal epithelial cells: nutritional metabolism, vitamin
and short-chain fatty acid (SCFA) production, pathogenic infection site, and for protection
Appl Biochem Biotechnol (2013) 169:511–525
DOI 10.1007/s12010-012-9995-y
M. Thirabunyanon (*) : P. Hongwittayakorn
Division of Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
e-mail: mthirabun@yahoo.co.th
against diseases and other gastrointestinal pathogenic infection [3–5]. Microbial metabo-
lisms produce several harmful enzymes such as β-glucuronidase, nitroreductase, azoreduc-
tase, and 7-α-dehydroxylase which mediate the formation of the carcinogenic process, and
later release them inside the intestinal tract leading colon cancer initiation [6, 7]. Similarly, a
normal intestinal resident such as pathogenic Escherichia coli has been associated with the
initiation of colon carcinoma and which secretes a harmful toxin especially called cytotoxic
necrotizing factor I [8]. Conversely, one biotherapeutic source of potential probiotic bacteria
is of human origin, referred to as human microbiota.
Colon cancer is considered a leading cause of numerous deaths and has been nowadays
implicated with dietary behavior such as consumption of high-fat, low-fiber diet which is a
major risk for colon cancer [7, 9]. An alternative protection and/or therapy against this
cancer as recently studied is the use of potential probiotic bacteria [10, 11]. In fact, it is not
the general probiotic bacteria that have this biotherapeutic action but those that have a
particular anticancer activity that is more than the usual probiotic bacteria criteria. The
original sources of probiotic bacteria are also viewed with importance based on their
efficiencies and therapeutic applications. Our previous study [12] indicated that potential
lactic acid probiotic bacteria, Enterococcus faecium RM11 and Lactobacillus fermentum
RM28, showed anticancer action such as antiproliferation of colon cancer cells which is an
important process of colon cancer formation. These potential bacteria are found in naturally
fermented dairy milks which are finally applied as functional food.
The human microbiota comprise diverse complex species in relative proportions; in the
environment of the human intestines where colon cancer originates, these bacteria are
beneficial and normally have self-adaptive functions, maybe having potential for colon
cancer protection and/or therapy. These microbiota are an important source of probiotic
bacteria of human origin, and the isolation of these probiotic bacteria has previously been
performed [13–16]. The alternative biotherapy from the use of probiotic bacteria showed
successful actions for protection against gastrointestinal pathogenic infection as has also
been suggested [4]. Likewise, a biotherapeutic alternative method of potential probiotic
bacteria in colon cancer prevention and/or treatment has recently been investigated [10–12].
Moreover, some relevant reports of much-needed investigation have emerged especially for
probiotic bacteria of human origin since the biotherapeutic mode of action of probiotic
bacteria that function in the antiproliferation of colon cancer cells is also still unclear.
In this study, the probiotic criteria of lactic acid bacteria (LAB) which were screened from
infant feces for use as potential probiotics and their efficiency towards antiproliferation of
colon cancer cells were investigated. The biotherapeutic mode of action of these potential
probiotic bacteria including the inhibition of the proliferation of colon cancer cells partic-
ularly with probiotic bacteria directly adhered to the cells and which produce SCFAs was
also investigated.
Materials and Methods
Bacterial Isolation
The original bacterial specimen was collected from 17 healthy infant feces samples in the
hospital of Chiang Mai, Thailand, at 1–14 days after birth. One gram of fecal sample was
diluted with 0.85 % sodium chloride (NaCl) in sterilized distilled water. Suitable dilutions of
100 μl were spread onto plated de Man, Rogosa and Sharpe (MRS) agar (Criterion, Santa
Maria, USA) and incubated at 37 °C using anaerobic conditions for 48 h. All differentially
512 Appl Biochem Biotechnol (2013) 169:511–525
morphological colonies were collected in order to obtain pure isolation. Gram staining and
microscopic examinations were later performed. Only isolates which were Gram positive and
rod or cocci shaped were collected. A total of 81 bacterial isolates were obtained. Each isolate
was kept at −80 °C in MRS broth supplemented with 20 % glycerol until further analysis.
Antagonistic Activity
The antimicrobial activity of 81 bacterial isolates was evaluated against Helicobacter pylori
DMST 20165, E. coli TISTR 780, Salmonella enteritidis DMST 15676, Salmonella typhi-
murium TISTR 292, Staphylococcus aureus TISTR 118, Bacillus cereus TISTR 687,
Listeria monocytogenes DMST 1783, and Vibrio cholerae DMST 2873. These pathogens
were prepared by culturing in nutrient broth (NB; Merck, Darmstadt, Germany). The tested
strains in MRS broth were incubated at 37 °C for 18 h. The MRS agar plates were overlaid
with 10 ml of molten MRS broth containing 0.7 % agar at 45 °C and inoculated with
pathogenic strains to obtain a final concentration of about 105CFU/ml. Upon solidification
of both agar layers, a sterile cork borer was applied to create wells of 8 mm in diameter.
Subsequently, the cell-free supernatant (100 μl) from the broth containing the tested bacteria
which was cultured was transferred into the wells and incubated at 37 °C for 24 h. A sterile
MRS broth and 0.3 % (v/v) hydrogen peroxide(Sigma, St. Louis, MO, USA) were used as
control. The inhibition of a clear zone around the well showing no growth of indicator
pathogens was recorded. Triplicates of each sample were done.
Hemolytic Activity and Antibiotic Resistance Assay
The bacterial strains were tested in order to determine their harmless property by using
hemolysis analysis. The hemolytic property of red blood cell was evaluated using Columbia
blood agar (Sigma) plates supplemented with 5 % (v/v) human blood and incubated at 37 °C
for 48 h. Recorded characteristics of hemolysis on blood agar were shown in different
phenomena as clear zones around colonies and classified as hemolytic (β-hemolysis); green-
hued zones around colonies (α-hemolysis) and no zone around colonies (γ-hemolysis) were
considered non-hemolytic. The assay was performed in triplicates.
The antibiotic resistance was evaluated by disk diffusion method. Tested bacteria were
grown in MRS broth at 37 °C for 18 h. Bacterial suspensions were swabbed on MRS agar
plates with sterile cotton. An antibiotic dish (Oxoid, Basingstoke, Hampshire, England)
consisting of chloramphenicol (30 μg), ampicillin (10 μg), erythromycin (15 μg), tetramycin
(30 μg), and kanamycin (30 μg) was placed on MRS agar plates and incubated at 37 °C for
48 h. The inhibition zone around the antibiotic disk was recorded. The assay was done in
triplicates.
Acid and Bile Salt Tolerance Assay
To evaluate the ability of tested bacterial strains to survive in the gastrointestinal tract, they
were subjected to a tolerance test in gastrointestinal tract models of acid and bile salt
conditions as discussed below. For the acid tolerance assay, the modified method of [17]
was performed. Tested bacterial strains were incubated in MRS broth at 37 °C for 18 h. One
milliliter of culture was transferred into 9 ml of sterile phosphate buffer saline solution (PBS)
adjusted to a pH value of 2.5 with 5 N HCl (Merck) and further incubated at 37 °C. The
number of viable bacteria was determined at 0 and 3 h of incubation on an MRS agar plate
after incubation. Triplicates of each isolate were performed.
Appl Biochem Biotechnol (2013) 169:511–525 513
For the bile salt tolerance assay, the method of [17] was used. Tested bacterial strains were
incubated inMRS broth at 37 °C for 18 h, and 1 ml of culture was transferred into 9 ml of sterile
MRS broth containing a final concentration of 0.3 % (w/v) bile salt (Sigma). The suspension
was further incubated at 37 °C. The number of viable bacteria was determined at 0 and 24 h of
incubation on an MRS agar plate after incubation. Triplicates of each sample were done.
Cell Culture
Cells of Caco-2 (colon cancer cell line) were routinely cultured in Dulbecco's modified
Eagle's minimal essential medium (DMEM, Sigma) supplemented with 10 % (v/v) fetal calf
serum (Hyclone, Logan, UT, USA) inactivated at 56 °C for 30 min, with 1 % (v/v) non-
essential amino acid (Hyclone) and 1 % (v/v) penicillin–streptomycin (10,000 IU/ml and
10,000 μg/ml; Invitrogen, Carlsbad, CA, USA). Cells were grown in a culture flask until
becoming confluent at 37 °C under 5 % CO2 environment, and cells were then seeded into
24- or 96-well tissue culture plates according to the experimental method.
Bacterial Forms of Live Whole Cells and a Cultured Medium
The bacteria of each strain were grown in MRS broth at 37 °C for 18 h. Two forms of
bacteria were applied, live whole cells (LWC) and cultured medium (CM), and both forms
were collected after centrifugation at 5,000×g for 10 min of bacterial culture. The LWC were
taken from the resuspended pellet in MRS broth while the CM was obtained after further
filtration of bacterial culture through a sterilized syringe filter of 0.2 μm (Sartorius,
Goettingen, Germany).
Effect of Probiotic Bacteria on Antiproliferation of Colon Cancer Cells
The activity of probiotic bacteria on the antiproliferation of colon cancer cells was evaluated by
using two methods, namely MTTand Trypan blue exclusion assays, as previously described by
[12, 18]. The MTT assay used in this experiment contained 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium (MTT, Sigma). Colon cancer cells were prepared using 100 μl at a density
of 106 cells/well in a 96-well plate (TPP, Trasadingen, Switzerland). The cell suspensions were
incubated in a CO2 incubator at 37 °C for 24 h. After incubation, 100 μl of CMwas added into
each well, and incubation continued further for another 24 h in the same conditions.
Cells were then washed twice with PBS; 10 μl of MTT solution with 0.5 mg/ml in
dimethyl sulfoxide (DMSO, Sigma) was added in each well and incubated further for
another 4 h. The precipitations of formazan were then solubilized by adding 100 μl of
DMSO and continually incubated for 5 min. Absorbance was measured at 595 nm using
a microplate reader for the cell viability calculation while the absorbance of the control
group which used MRS broth was set as 100 % of cell viability. This assay was done in
three independent experiments to secure a triplicate observation [% cell viability0(sample
O.D./control O.D.)×100].
For the Trypan blue exclusion assay, colon cancer cells were seeded at a concentration of
106 cells/ml/well into 24-well plates (TPP). The cells were incubated in a CO2 environment
incubator at 37 °C in a 5 % CO2 in air for 24 h. After incubation, 1 ml of LWC or CM was
added into each well and incubated further for another 24 h in the same conditions. Cell
exposures were examined using Trypan blue (Sigma) exclusion stained with a Neubauer
hemocytometer. This analysis was performed in three independent studies of each triplicate
observation [% cell viability0(live cell count/total cell count)×100].
514 Appl Biochem Biotechnol (2013) 169:511–525
Probiotic Bacteria Identification by 16S rRNA Sequences
Four isolates of probiotic bacteria were identified for their genera by using the method
indicated by [12]. In brief, total genomic DNAwas extracted using an UltraClean microbial
DNA isolation kit (MoBio Laboratories, Inc., Carlsbad, CA, USA) following the manufac-
turer's instructions. Two universal primers of 27F (5′-AGAGTTTGATCMTGGCTCAG-3′)
and 520R (5′-ACCGCGGCKGCTGGC-3′) (Operon, Cologne, Germany) were used in order
to amplify 16S rRNA gene sequences by using a polymerase chain reaction (PCR). For each
reaction, a 50-μl reaction mixture containing 25 μl of MasterMix (Eppendorf, New York,
USA), 2 μl of 27 F primer, 2 μl of 520R primer, 17 μl of sterile H2O, and 4 μl of 20 ng/μl
genomic DNA sample was used. The amplification program was performed using a dena-
turing step at 94 °C for 5 min, followed by 25 cycles at 94 °C for 1 min, 55 °C for 1 min, and
72 °C for 1 min, with a final 5-min extension at 72 °C. A total of 5 μl of the PCR product
was mixed with 2 μl of loading dye (Fermentas, MD, USA) and loaded onto a 1.5 % agarose
gel with ethidium bromide staining and visualized by UV illumination. The sizes of DNA
fragments were compared using a 100-bp DNA ladder (Fermentas). The PCR products were
purified using TaKaRa SUPREC-PCR, following the manufacturer's instructions and were
then sequenced. Data of the 16S rRNA gene sequences were compared with the GenBank
database by using BLAST (http://www.ncbi.nlm.nih.gov).
Probiotic Bacteria Adhesion to Colon Cancer Cells
For bacterial adhesion assay, the modified methods of [12, 19] were used. The colon cancer
cells were seeded with 1 ml of culture medium containing 106 viable cells/well in 24-well
tissue culture plates. The culture medium was changed every 48 h. These cells were used at
15 days post-confluence after becoming fully differentiated. The medium of non-
supplemented DMEM was replaced at least 1 h before assay. Tested bacteria from
18-h cultures in MRS broth were harvested and washed twice with PBS and later resus-
pended in non-supplemented DMEM to achieve a concentration of 108CFU/ml. Afterwashing the cells twice with PBS, 0.5 ml of bacterial suspension was added to each well
and incubated at 37 °C for 1 h in a 5 % CO2 environment. Unattached bacteria were removed
by washing with PBS three times. Cells were lysed with 0.1 % Triton X-100 (Merck) for
5 min. The concentration of adhering bacteria cells was enumerated by plate counting in
triplicate with an MRS agar and then incubated at 37 °C for 48 h. The adherence of probiotic
bacteria to colon cancer cells was compared between the initial and viable bacteria using
plate counting. The adhesion assays were done in five replicates.
Bioproduction as SCFAs of Probiotic Bacteria
Analysis of SCFA bioproduction was done using a modified method of [20]. The probiotic
bacteria were cultured anaerobically for 18 h in MRS broth, and samples were later
centrifuged at 4,500×g for 5 min. Ten milliliters of each supernatant sample was added with
4 g of NaCl and 2 ml of H2SO4 (50 %) (Fluka) and was then extracted with 5 ml of diethyl
ether (Merck) for 30 min. The samples were subsequently centrifuged at 1,500×g for 5 min.
One milliliter of the extracted compounds was collected for SCFA determination. This
analysis of SCFA was performed using a gas chromatograph–mass spectrometer (GC-MS)
of an Agilent 6890 series GC (Agilent Technologies, Palo, Alto, CA, USA) coupled to an
Agilent 5973 series MS and operated in electron impact mode (70 eV). An AT-Wax capillary
column of 30 m×0.25 mm×0.25 μm film thickness (Alltech Associates, Deerfield, IL,
Appl Biochem Biotechnol (2013) 169:511–525 515
USA) was used. Each extracted sample of 1 μl was injected with the injector operating in
split mode ratio of 20:1. The oven temperature of the gas chromatograph was programmed
as follows: at 60 °C for 2 min, increased at 15 °C/min to 220 °C, and then held constant for
15 min. Helium was used as carrier gas with flow velocity of 36 cm/s. Mass selective
detector scanning ions of 15–300 atomic mass units were applied. Temperatures of ion
source and quadrupole mass analyzer were kept at 230 and 150 °C, respectively. Analyzing
time for each sample was 27.67 min. Compounds were identified by MSD Chemstation
software (Agilent Technologies) using standards of propionic (Merck) and butyric acids
(Merck) and matching with mass spectra of the commercial library (NIST98.L and
WILEY275.L). This experiment was performed in triplicate.
Effect of Butyric and Propionic Acids on Antiproliferation of Colon Cancer Cells
The activity of butyric and propionic acids on antiproliferation was evaluated by MTT assay
using modified methods of [12, 21]. Colon cancer cells at 100 μl of the DMEM were used
and containing a density of 106 cells/well added into 96-well plates. Cells were incubated in
a CO2 incubator at 37 °C for 24 h. One-hundred microliters of PBS containing butyric or
propionic acids at different concentrations (0, 1, 2, 4, 8, 16 ppm) was then added into each
well and further incubated for 24 h in similar conditions. After co-incubation, cells were then
washed twice with PBS. Ten microliters of MTT solution with 0.5 mg/ml in DMSO was later
added in each well and incubated further for 4 h. Precipitations of formazan were solubilized
by adding 100 μl of DMSO and further incubated for 5 min. Absorbance of 595 nm was
used with a microplate reader for antiproliferative calculation while absorbance of the
control group was set as 100 % of cell viability. This assay was done in five replications
[% cell viability0(sample O.D./control O.D.)×100].
Statistical Analysis
A one-way analysis of variance was employed with the use of SPSS (version 17.0) to
evaluate the experimental data. Significant differences were accepted at P<0.05 by Duncan's
multiple range test.
Results
Bacterial Isolates
The bacterial isolates from healthy infant feces samples in this study were obtained in
diverse bacterial morphological colonies of an individual fecal sample. A total of 81
bacterial isolates were obtained from all of 17 samples. Summary of isolates after Gram
staining indicated only Gram-positive rod or cocci form was obtained, after which all
isolates were further analyzed.
Antagonistic Activity Against Enteric Pathogens
The antimicrobial activity of 81 bacterial isolates was found in different actions of each
isolate. Most of them showed no competence on growth inhibition of enteric pathogens.
Only 15 isolates indicated the ability to inhibit the growth of all enteric pathogens (Table 1),
which were then marked for further analyses. These zones of inhibition were different in
516 Appl Biochem Biotechnol (2013) 169:511–525
each isolate action and were also dependent on individual enteric harmful strain as well. In
general comparison, two isolates of FP25 and FP35 were shown to rather exhibit potential
antagonistic action against enteric pathogens (Table 1).
Hemolytic and Antibiotic Resistance Activity
Non-harmful isolates from the human red blood cell hemolysis were evaluated for bacterial
probiotic criteria and selection. Results showed that none of all the 15 isolates were
displayed to be β and α-hemolysis but instead were found to be only γ-hemolysis (no
hemolysis). On antibiotic resistance, all tested bacteria in this study were found to exhibit
sensitivity to ampicillin, chloramphenicol, erythromycin, and tetramycin. Besides, some of
them showed to have either a route of sensitive or resistance activity. Isolates of FP14, FP47,
FP52, FP66, FP70, and FP78 were also found sensitive to kanamycin. There were differ-
ences in FP3, FP12, FP15, FP25, FP35, FP51, and FP75 isolates regarding their resistance to
kanamycin.
Acid and Bile Salt Tolerance Activity
The tolerance of the 15 isolates within the gastrointestinal tract conditional model is
shown in Figs. 1 and 2. Evaluation by acid condition of pH2.5 (Fig. 1) exhibited that
almost isolates had good survival and tolerance in this acidic condition. In addition,
most of them showed normal growth in this model based on the observed growth rate
at the final stage more than that at the initial period (Fig. 1). Similarly, all isolates
were also shown to have good survival and tolerance in 0.3 % bile salt in the
intestinal conditional model (Fig. 2).
Antiproliferation Activity
The potential action of CM or LWC was correlated on the antiproliferation activity of
colon cancer cells. Of all the 15 isolates, the CM activity to inhibit proliferative rates
of colon cancer cells was found in significant difference in each active isolate,
ranking from 0 to 35.3 % by using MTT assays. High percentages of more than
15 % (35.3, 28.9, 25.3, and 17.7 %) were found in isolates of FP35, FP3, FP25, and
FP51, respectively (Fig. 3). In addition, the CM efficiency on antiproliferation rate
was also exhibited to be rather constant using another Trypan blue exclusion assay.
These antiproliferation rates of all four isolates were significantly increased (P<0.01)
as compared to the control group (Fig. 4).
The efficiency of LWC on these four isolates with a Trypan blue exclusion assay was also
significantly increased (P<0.01) on antiproliferation when compared with the control group
(20.5 to 33.7 %) (Fig. 4).
Probiotic Bacteria Identification by 16S rRNA Sequences
Using the 16S rRNA, the probiotic bacterial genera were identified and compared to those in
the GenBank data. The FP3 isolate exhibited a 100 % identity to database entries, and this
genus was identified to be P. pentosaceus (accession no: EU082192.1) while FP25 and FP35
isolates were L. salivarius at 99.8 and 100 % identity, respectively (accession no:
EU559602.1). Meanwhile, isolate FP51 was identified as E. faecium at 99.8 % identity
(accession no: AB362603.1).
Appl Biochem Biotechnol (2013) 169:511–525 517
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.3
±
2.
4
F
P
70
13
.0
±
0.
6
14
.0
±
1.
0
11
.0
±
0.
6
12
.0
±
0.
0
20
.7
±
1.
3
14
.0
±
0.
6
12
.0
±
0.
0
20
.3
±
3.
7
F
P
75
12
.3
±
0.
3
20
.7
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2.
3
14
.7
±
0.
3
16
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21
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9
19
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6
16
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5
23
.3
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7
F
P
78
12
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±
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9
16
.3
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11
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3
11
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3
21
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3
11
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0
518 Appl Biochem Biotechnol (2013) 169:511–525
Probiotic Bacteria Adhered to Colon Cancer Cells
The adhesion efficiency of potential probiotics on the colon cancer cells is shown in Fig. 5,
indicating an ability to adhere to colon cancer cells and showing percentages of adhesive
actions ranging from 5 to 15.7 %. Higher percentage rates were exhibited by P. pentosaceus
FP3, L. salivarius FP35, L. salivarius FP25, and E. faecium FP51, respectively.
Bioproduction of SCFAs from Potential Probiotic Bacteria
The ability to produce SCFAs by the beneficial probiotic bacteria is shown in Table 2. The
probiotic strain of L. salivarius FP35 produced SCFAs such as butyric acid at 1.67 ppm,
0
1
2
3
4
5
6
7
8
3 12 14 15 25 35 47 51 52 60 64 66 70 75 78
Lo
g 
CF
U
/m
l
Fig. 1 Acid tolerance activity of LAB isolates. Survival of LAB isolates was compared by plate counting
after challenge with low pH2.5 in PBS for 0 (black bars) and 3 h (gray bars). Values are mean with SE of
three replications
0
1
2
3
4
5
6
7
8
3 12 14 15 25 35 47 51 52 60 64 66 70 75 78
Lo
g 
CF
U
/m
l
Fig. 2 Bile salt tolerance activity of LAB isolates. Survival of LAB isolates was compared after challenge
with bile salt 0.3 % in MRS for 0 (black bars) and 24 h (gray bars). Values are mean with SE of three
replications
Appl Biochem Biotechnol (2013) 169:511–525 519
Fig. 3 Antiproliferation activity of probiotic LAB isolates by MTT assay. The effective CM of LAB isolates
had different rates of antiproliferation of colon cancer cells; only four isolates, FP3, FP25, FP35, and FP 51,
were higher than 15 %. Values are the mean with SE of three independent experiments of each triplications
0
5
10
15
20
25
30
35
40
Control FP3 FP25 FP35 FP51
A
nt
ip
ro
lif
er
at
io
n 
(%
)
a
b
a
b
a a
a
a
a
a
Fig. 4 Antiproliferation activity of probiotic LAB isolates by Trypan blue exclusion assay. Both effective CM
(black bars) and LWC (gray bars) of LAB isolates had significantly induced antiproliferation of colon cancer
cells compared to the control group. Values are mean with SE of three independent experiments of each
triplication. Means within each grouping with different letter designations (a, b) differ (P<0.01)
520 Appl Biochem Biotechnol (2013) 169:511–525
which was much higher than that of other probiotic strains. Higher production of propionic
acid (2.0 ppm) was found in a probiotic strain L. salivarius FP25. On the other hand, a
probiotic strain of E. faecium FP51 produced SCFAs but could not be determined as either
butyric or propionic acid.
Effect of Butyric and Propionic Acids on Antiproliferation of Colon Cancer Cells
The induction activities of butyric and propionic acids in colon cancer cell death are shown
in Figs. 6 and 7, respectively. In comparison with the control group, butyric acid was found
to trigger colon cancer cell death at the rates of 6.2–23.2 %, with the higher rate in the group
using the dose of 16 ppm (Fig. 6). These results were shown to be similar to the use of
propionic acid which indicated the induction of death of colon cancer cells by the dose
responding at the rates of 1.6–13.8 % (Fig. 7).
Fig. 5 Adhesion activity of probiotic LAB adhered to colon cancer cells. Apart from the higher antiprolif-
eration of colon cancer cells, the high adhesion potential to colon cancer cells was also found in the probiotic
LAB of FP3, FP35, and FP25. Values are mean with SE of five replications
Table 2 Bioproduction of SCFAs
through the activity of probiotic
LAB. Data are presented as mean
with SE of three replications
ND not detected
Probiotic LAB Short-chain fatty acids (ppm)
Butyric Propionic
Pediococcus pentosaceus FP3 1.40±0.6 1.82±0.7
Lactobacillus salivarius FP25 1.03±0.5 2.00±0.4
Lactobacillus salivarius FP35 1.67±0.1 1.54±0.2
Enterococcus faecium FP51 ND ND
Appl Biochem Biotechnol (2013) 169:511–525 521
Discussion
The prevention and/or biotherapy of colon cancer via probiotic bacteria is considered a
significant alternative method recently. As human microbiota are known to be associated
Fig. 6 Antiproliferation activity of butyric acid. Trigger inhibiting proliferation of colon cancer cells was
observed by dose response after exposure with butyric acidat 1, 2, 4, 8, and 16 ppm. Values are mean with SE
of five replications
Fig. 7 Antiproliferation activity of propionic acid. Trigger inhibiting proliferation of colon cancer cells was
observed by dose response after exposure with propionic acid at 1, 2, 4, 8, and 16 ppm. Values are mean with
SE of five replications
522 Appl Biochem Biotechnol (2013) 169:511–525
with human diseases and health, probiotic bacteria of human origin are an important
potential source of which several researches have been conducted [13–16]. However, there
is unclear evidence for antiproliferative activity by probiotic bacteria of human origin against
colon cancer cells, which is key for colon cancer prevention; this has formed the important
purpose of this study. As well, the biotherapeutic mode of action of these potential probiotic
bacteria was also investigated. A total of 81 isolates of LAB were obtained from infant feces.
All these isolates might have suitable functional modes of action in the intestine for
protection and/or biotherapy since they are normally resident in this area and where both
indicate probiotic bacterial action and colon cancer formation.
Potential application criteria of probiotic bacteria and colon cancer biotherapy were
investigated from these 81 isolates out of which only 15 isolates showed inhibiting activities
on the growth of eight enteric pathogens. It has been indicated that some of these harmful
strains are known to produce several harmful enzymes, such as β-glucuronidase and nitro-
reductase, which are being implicated in the carcinogenic process by notably releasing
carcinogens in the intestinal tract [6, 7]. In fact, the pathogenic strains of E. coli and H.
pylori are also known as initial causes of colon cancer [8, 22]. The inhibiting action of these
15 isolates might be effective towards the production of antimicrobial substances such as
bacteriocins, reuterin, hydrogen peroxide, organic acids, and biosurfactant [4, 13, 14]. This
growth inhibition of enteric pathogens suggests that these LAB isolates might have initial
activity of having a protective role against initiation of colon cancer.
One safe probiotic bacterial criterion is a non-pathogenic strain that was found in all of
these 15 isolates as shown by the absence of hemolysis of red blood cells, thus indicating
that these isolates were not virulent and were also suitable for further consideration as
beneficial probiotic bacteria [23]. In prior active areas where most of the intestinal tract and
colon cancer originate, probiotic bacteria must survive transit through the stomach and
intestines. One finding on the viability of all 15 isolates was the challenges in the gastro-
intestinal tract model by conditions such as acidity (pH2.5) and bile salt condition (0.3 %
bile salt). These LAB isolates showed that they could survive well in the model of the human
gastrointestinal tract. Similar to the results of this study, high tolerance to acidic pH and bile
salts was also found in potential probiotic bacteria of human origin such as L. fermentum
ACA-DC 179 [24], L. rhamnosus IMC 501, L. paracasei 502 [15], and L. plantarum CS23
[16]. The tolerance to this criterion was correlated with the fact that these LAB isolates
normally reside and adapt well in the environment of the human gastrointestinal tract which
is considered the functional active area of the potential probiotic bacteria that might defend
against colon cancer formation.
Although the alternative prevention and/or biotherapy of colon cancer by probiotic
bacteria have been investigated in recent years, only certain probiotic bacteria exhibited
these potential actions. Our finding showed that not all the 15 probiotic LAB isolates
exhibited characteristics of antiproliferative activity of colon cancer cells. Only four of these
probiotic LAB strains (P. pentosaceus FP3, L. salivarius FP25 and FP35, and E. faecium
FP51) had the ability to effectively inhibit proliferation of colon cancer cells both under CM
and LWC conditions. Similarly, the conditioned medium of probiotic LAB (VSL3) reduced
viability and induced apoptosis of colon cancer cells [18]. In a previous study [12], it was
indicated that two probiotic LAB strains of E. faecium RM11 and L. fermentum RM28
which originated from fermented dairy milks triggered the inhibition of proliferation of
colon cancer cells. These findings indicated that these probiotic LAB strains might be
applicable for colon cancer prevention and treatment.
The modes of action for these potential probiotic LAB in colon cancer prevention and/or
therapy have not been so clearly understood; thus, it was also included for investigation in
Appl Biochem Biotechnol (2013) 169:511–525 523
this study. Although the postulate mechanisms were not very clear, they gave hints that they
could result in several modes of action [7, 9, 25]. The hypothesis of probiotic bacteria that
consisted of their adhesion to colon cancer cells and later inducing death of these colon
cancer cells was included. Comparison of each probiotic strain that could produce SCFAs
was also investigated. In this study, the correlation among percentages of antiproliferation,
adhesion, and SCFA production from probiotic bacteria pointed to the same direction.
Among them, the probiotic bacteria P. pentosaceus FP3, L. salivarius FP25, and L. salivar-
ius FP35 had higher rates of antiproliferation of colon cancer cells, adhesion to colon cancer
cells, and production of SCFAs, than E. faecium FP51 which was low in these activities. In
particular, it was specifically very low in the adhesion to colon cancer cells and was not
detected to produce SCFAs, resulting in low percentage of proliferative inhibition of
the colon cancer cells. In similar studies, the anticarcinogenic activity of Bacillus
polyfermenticus SCD [19] and probiotic LAB of E. faecium RM11 and L. fermentum
RM28 [12] had high adherence to colon cancer cells and were also found to inhibit
proliferation of the colon cancer cells. These results suggested that adhesion of
probiotic bacteria to colon cancer cells as shown in this study may serve as an
important mechanism in directly inducing deaths of these cells and/or inducing other
synergic mechanisms such as bioproduction of SCFAs, which result in induced
apoptosis of the colon cancer cells.
SCFA production from probiotic bacteria of this study, which included butyric and
propionic acids, was found in higher concentration in probiotic strains of P. pentosa-
ceus FP3, L. salivarius FP25, and L. salivarius FP35, but was absent in E. faecium
FP51. These SCFAs may trigger antiproliferation of colon cancer cells in dose, as
clarified in this investigation through evaluation of P. pentosaceus FP3, L. salivarius
FP25, and L. salivarius FP35, which showed parallel potential actions of higher
production rate of SCFAs and also high induction of death of colon cancer cells.
Our study showed that these potential probiotic bacteria clearly induced colon cancer
cell deaths through the cause of apoptosis induction via caspase 3 activity and
morphological change when observed under a fluorescence microscope after staining
with 4′6-diamidino-2-phenylindole (unpublished data). It was also found that the dose
certainly affected the response in the case of using individual butyric and propionic
acids as investigated in this study. A probiotic LAB strain of L. plantarum NCIMB
8826 was indicated to produce these SCFAs by cereal-based substrates [26]. Similarly
to this, [21] proposed that propionibacteria that can produce SCFAs, mainly propio-
nate, Propionibacterium acidipropionici CNRZ80, and P. freudenreichii subsp. freu-
denreichii ITG18 induced death of colon cancer cells by apoptosis. These proliferative
inhibition activities have also been found in the case of using propionate as the dose
response. A probiotic bacterium of Butyrivibrio fibrisolvens MDT-1, a butyrate-
producingstrain, in a mouse model of colon cancer, showed decreased formation of
aberrant crypt foci (ACF) and inhibition of tumor growth progression [10]. These
results suggested that new probiotic LAB-producing SCFAs of human origin in this
study may induce growth inhibition of colon cancer cells via also the secretion of
butyric and propionic acids.
The alternative biotherapeutic potential for colon cancer was exhibited by selected
probiotic bacteria of human origin such as P. pentosaceus FP3, L. salivarius FP25,
and L. salivarius FP35. It is found that mechanisms of proliferative inhibition of
colon cancer cells may induce the probiotic LAB to directly adhere to these colon
cancer cells and/or thus trigger synergically the mode of action of bioproduction of
SCFAs.
524 Appl Biochem Biotechnol (2013) 169:511–525
Acknowledgments This research was financially supported by the National Research Council of
Thailand.
References
1. Bäckhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A., & Gordon, J. I. (2005). Science, 307, 1915–
1920.
2. McFall-Ngai, M. (2007). Nature, 445, 153.
3. O’Flaherty, S., & Klaenhammer, T. R. (2010). International Dairy Journal, 20, 262–268.
4. Thirabunyanon, M. (2011). Maejo International Journal of Science and Technology, 5, 108–128.
5. Thirabunyanon, M., & Thongwittaya, Ν. (2012). Research in Veterinary Science, 93, 74–81.
6. McBain, A. J., & Macfarlane, G. T. (1998). Journal of Medical Microbiology, 47, 407–416.
7. Rafter, J. (2003). Best Practice & Research Clinical Gastroenterology, 17, 849–859.
8. Travaglione, S., Fabbri, A., & Fiorentini, C. (2008). Infectious Agents and Cancer., 3, 4.
9. Commane, D., Hughes, R., Shortt, C., & Rowland, I. (2005). Mutation Research, 591, 276–289.
10. Ohkawara, S., Furuya, H., Nagashima, K., Asanuma, N., & Hino, T. (2005). The Journal of Nutrition,
135, 2878–8283.
11. Ma, E. L., Choi, Y. J., Choi, J., Pothoulakis, C., Rhee, S. H., & Im, E. (2010). International Journal of
Cancer, 127, 780–790.
12. Thirabunyanon, M., Boonprasom, P., & Niamsup, P. (2009). Biotechnology Letters, 31, 571–576.
13. Millette, M., Dupont, C., Shareck, F., Ruiz, M. T., Archambault, D., & Lacroix, M. (2008). Journal of
Applied Microbiology, 104, 269–275.
14. Pridmore, R. D., Pittet, A. C., Praplan, F., & Cavadini, C. (2008). FEMS Microbiology Letters, 283, 210–
215.
15. Verdenelli, M. C., Ghelfi, F., Silvi, S., Orpianesi, C., Cecchini, C., & Cresci, A. (2009). European Journal
of Nutrition, 48, 355–363.
16. Gaudana, S. B., Dhanani, A. S., & Bagchi, T. (2010). British Journal of Nutrition, 103, 1620–1628.
17. Pennacchia, C., Ercolini, D., Blaiotta, G., Pepe, O., Mauriello, G., & Villani, F. (2004).Meat Science, 67,
309–317.
18. Ewaschuk, J. B., Walker, J. W., Diaz, H., & Madsen, K. L. (2006). Journal of Nutrition, 136, 1483–1487.
19. Lee, N. K., Park, J. S., Park, E., & Paik, H. D. (2007). Letters in Applied Microbiology, 44, 274–278.
20. Hussain, M. A., Rouch, D. A., & Britz, M. L. (2009). International Dairy Journal, 19, 12–21.
21. Jan, G., Belzacq, A. S., Haouzi, D., Rouault, A., Métivier, D., Kroemer, G., & Brenner, C. (2002). Cell
Death and Differentiation, 9, 179–188.
22. Shmuely, H., Passaro, D., Figer, A., Niv, Y., Pitlik, S., Samra, Z., Koren, R., & Yahav, J. (2001). The
American Journal of Gastroenterology, 96, 3406–3410.
23. Maragkoudakis, P. A., Mountzouris, K. C., Psyrras, D., Cremonese, S., Fischer, J., Cantor, M. D., &
Tsakalidou, E. (2009). International Journal of Food Microbiology, 130, 219–226.
24. Zoumpopoulou, G., Foligne, B., Christodoulou, K., Grangette, C., Pot, B., & Tsakalidou, E. (2008).
International Journal of Food Microbiology, 121, 18–26.
25. Fotiadis, C. I., Stoidis, C. N., Spyropoulos, B. G., & Zografos, E. D. (2008). World Journal of
Gastroenterology, 14, 6453–6457.
26. Salmeron, I., Fuciños, P., Charalampopoulos, D., & Pandiella, S. S. (2009). Food Chemistry, 117, 265–
271.
Appl Biochem Biotechnol (2013) 169:511–525 525