Colorectal cancer is the second leading cause of death due to cancer in the United States [1
]. The incidence rate of colorectal cancer increases with age. Excessive alcohol consumption may lead to an increase in the colorectal cancer incidence rate by 60%; insufficient fibre intake and smoking may increase the colorectal cancer incidence rate by 20%. Previous research has found that the excessive intake of high-fat diets can also lead to colorectal cancer [2
]. At least 15%–20% of cancers are attributed to infection or inflammation, including malignant tumours such as colorectal cancer and inflammatory bowel disease (including ulcerative colitis and Crohn’s disease). Research has suggested that patients with the above infections are more likely to develop colorectal cancer [3
Probiotics are live microorganisms and can exert health benefits to their hosts when ingested in sufficient quantities [4
]. The fundamental criteria for probiotic selection include safety (must be a generally recognised as a safe strain), acid and bile salt tolerance, ability to adhere to the intestinal tract and colonise and exhibit health benefits to the host [5
]. The most common effects of lactobacilli are to reduce the activity of tumour-promoting enzymes, increase host immunity, produce metabolites that benefit the host and resist pathogens.
In vitro human intestinal cell models have been used to study the function of specific intestinal cells in humans, with Caco-2 and HT-29 the most widely used human intestinal cell lines [7
]. Caco-2 cell lines and clones display a spontaneous differentiation-dependent enterocyte-like phenotype of the small intestine [8
]. The parental colon cancer HT-29 cell line is composed mainly of undifferentiated cells, with a small minority of differentiated cells (~3% to 5% of total cells) [8
]. Lactobacillus helveticus
or a mix of Streptococcus thermophilus
and L. bulgaricus
significantly reduced the growth rate of HT-29 cells, resulting in a 10%–50% decrease in the total cell number. The most effective strains in lowering the HT-29 growth rate were L. helveticus
]. In Lactobacillus
-fermented soy milk, soy isoflavones such as genistein and daidzein and cell walls and extracellular components of the two probiotics engaged in the interaction with tumours and inhibited tumour growth [10
The inhibitory mechanisms of Lactobacillus
against colorectal cancer cells include reducing tumour-promoting enzymatic activity, binding to mutagens, increasing short-chain fatty acids, lowering pH and enhancing immunity [12
]. This study aimed to investigate the probiotic characteristics and their ability to inhibit the growth of the colorectal cancer cell line HT-29 with detection of Bax/Bcl-2, LDH and NO.
Conway et al. [16
] found that the viability of Lactobacillus
is lower in PBS than in gastric juice because certain components in gastric juice may confer protective effects on bacterial cells. Research has suggested that the acid tolerance of Lactobacillus
is attributed to the presence of a constant gradient between extracellular and cytoplasmic pH [17
]. Under acidic conditions in the absence of sugars, lactobacilli produce less ATP, revealing that sugar is an important factor affecting Lactobacillus
]. In the simulated intestinal fluid experiment to examine bile salt tolerance, the reason for the decrease in bacterial numbers may be damage to bacterial lipid membranes by bile salts, which thus influences the integrity of bacterial cell membranes [19
]. Additionally, bile salts can affect the expression of genes responsible for maintaining the cytoplasm and cell wall in lactobacilli [20
]. Huang et al. [21
] indicated that after culturing L. acidophilus
BCRC10695, L. paracasei
BCRC14023 and B. bifidum
BCRC14615 in 0.1% peptone water with added 0.1, 0.2 and 0.3% (w
) bile salts for 1.5 and 3 h, respectively, the viability of B. bifidum
BCRC14615 after the 3-h culture with 0.2 and 0.3% (w
) bile salts approached zero and the viability of the other two strains also decreased with elevated bile salt concentrations.
The adhesion ability of probiotic lactobacilli has been considered significant in the colonisation of bacteria within the gastrointestinal tract and in the beneficial benefits of bacteria on the hosts. Tuo et al. [22
] demonstrated that L. rhamnosus
IN1L had the highest adhesive capability (251 ± 14 bacterial cells/100 cells) to the HT-29 cells; L. rhamnosus
J5L had the second highest (32.3 ± 2.5 bacterial cells/100 cell) and L. rhamnosus
SB5L and L. rhamnosus
SB31L had the worst (2.5 ± 1.1, 2.6 ± 0.4 bacterial cells/100 cells) adhesive capabilities. In our study, except for BCRC14759 that had no adhesion capability, the other six strains of lactobacilli exhibited better adhesion capabilities to the HT-29 cells than those used in the above literature.
Lan et al. suggested that some probiotic activities result from the organic acids level and pH value of probiotics cultures [23
]. However, Motevaseli et al. demonstrated that the lactic acid level was a more important part of the lactobacilli inhibitory effect than pH alone [24
]. In the present study, HT-29 cells line growth inhibition was by the pH value, lactic acid level as well as culture supernatants. Sadeghi-Aliabadi et al. [25
] determined the influence of L. plantarum
A7 and L. rhamnosus
GG supernatants in three concentrations (2.5, 5 and 10 mg/mL) on HT-29 cell viability. Their results have indicated that the viabilities of HT-29 cells cultured with the L. plantarum
A7 supernatants were 46% ± 2.6%, 15% ± 7%, and 0%, respectively. Under the same condition, the viabilities of HT-29 cells cultured with L. rhamnosus
GG supernatants were 54% ± 14%, 19% ± 2.9%, and 0.4% ± 0.75%, respectively. Furthermore, a significant difference in HT-29 cell viability was found between the group with added 10 mg/mL Lactobacillus
supernatants and the control group, indicating that in the comparison of the original supernatant with the supernatant adjusted to pH 7, the lower pH has a greater impact on the growth inhibition of the HT-29 cells.
Wang et al. [26
] suggested that lactobacilli isolated from cheese induce cell apoptosis. Sun et al. [27
] showed that in the intestinal epithelium, the ratio of Bax and Bcl-2 protein expression can serve as an index of the mitochondria-mediated apoptotic pathway, with a ratio of more than 1 indicating that the tested molecules exert pro-apoptotic effects. Khoury et al. [28
] reported that kefir significantly upregulates the ratio of Bax and Bcl-2 in HT-29 and Caco-2 cells, indicating that kefir promotes cell apoptosis in the HT-29 and Caco-2 lines. Iyer et al. [29
] revealed that L. reuteri
may participate in the Lactobacillus
-induced extrinsic pathway of apoptosis to prevent the occurrence of colorectal cancer. However, although BCRC14625, a strain of L. reuteri
used in our study, and the strain in the aforementioned literature both belong to the same species, BCRC14625 does not exhibit the ability to induce cell apoptosis.
can produce NO by utilising nitrate and nitrite, thus exhibiting immunoregulatory and antibacterial effects [30
]. NO has a dual function: damaging DNA to cause cell death and inducing cell apoptosis [31
]. Literature has also indicated that NO inhibits Bcl-2 [32
]. Based on our findings, following the 24-h treatment of the HT-29 cells with the BCRC14625 supernatants at various concentrations, the corresponding amounts of Bcl-2 were significantly lower than that in the control group (Figure 3
A), providing evidence that NO can inhibit the anti-apoptotic protein Bcl-2.
4. Materials and Methods
4.1. Bacteria Strains, Culture Medium and Growth Conditions
A total of 7 strains of Lactobacillus, including L. johnsonii BCRC17010, L. delbrueckii subsp. bulgarius BCRC10696, L. salivarius BCRC14759, L. reuteri BCRC14625, L. brevis PM150, L. plantarum PM153 and L. brevis PM177, were obtained from isolated strains of Lactobacillus found in fermented plant products (PM150, PM153 and PM177) or from the Bioresource Collection and Research Center (BCRC), Hsin-Chu, Taiwan. To prepare Lactobacillus strains for use in this study, Lactobacillus stored at −80 °C was inoculated into Lactobacilli MRS broth (Difco) containing 0.05% l-cysteine and activated two times prior to incubation for 20 h at 37 °C before use. The l-lactic acid was detected by National Standards of the Republic of China (CNS) 12635 N6224 method.
4.2. LAB Resistance to Simulated Gastrointestinal Conditions
One milliliter of culture containing approximately 109 cfu/mL of LAB was centrifuged (1000× g, 10 min) and added into 10 mL phosphate-buffered saline (PBS) with 0.3% Pepsin (Sigma-Aldrich, St. Louis, MO, USA). The pH was adjusted to 2.0, 3.0 and 7.0 using 0.1 N HCl, and the solution was incubated at 37 °C for 0, 1.5 and 3 h. After incubation, viable bacterial counts were determined by serially diluting the culture in PBS (pH 7.2) and plating on MRS agar. Plates were incubated anaerobically at 37 °C for 48 h.
4.3. The Epithelial Cell Line Culture and Adhesion Assay
The HT-29 cell line was obtained from BCRC, Hsin-Chu, Taiwan. HT-29 cells were grown in Eagle’s minimal essential medium (EMEM) (GIBCO) supplemented with 10% (v
) fetal bovine serum, 1% nonessential amino acid (NEAA), and 50 unit·mL−1
Penicillin-Streptomycin (GIBCO). The HT-29 cell line was cultured in 75 cm2
plastic tissue culture flasks (GIBCO). The cells were washed twice with PBS and then transferred (4 × 105
cells/mL) with 0.05% trypsin into a 24-well multi-dish containing fresh tissue culture medium without Penicillin-Streptomycin. The mixtures were kept at 37 °C in 5% CO2
/95% air atmosphere until cell lines formed a monolayer in each well. Prior to the adhesion test all bacterial strains were washed twice with PBS and centrifuged for 10 min at 8000 × g
. One milliliter of broth inoculated with Lactobacillus
was centrifuged for 10 min, washed with 1 × PBS buffer and re-dissolved in cell culture medium without antibiotics. Then, 100 μL of the broth was added to a 24-well plate and incubated in a 37 °C CO2
incubator for 2 h. After incubation cells were washed twice with PBS, fixed with 10% formalin for 30 min, washed four times with PBS and then stained with crystal violet for 5 min. The numbers of LAB cells adhered to the cultured cell lines were counted according to the method of [33
4.4. Analysis of Cell Viability
This experiment focused on analysing the ability of Lactobacillus to inhibit colorectal cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a colourless, transparent tetrazolium salt, is reduced to yield a purple formazan crystal by mitochondrial dehydrogenase in living cells. In total, 500 μL (104 cells/mL) of cells was seeded into a 24-well plate, and the cells were subjected to overnight culture at 37 °C in a CO2 incubator, which made the cells attach, divide and grow in the 24 wells. The cells were gently washed twice with 1× PBS, and after discarding PBS, 1 mL of mixed solutions of Lactobacillus culture supernatants and cell culture media were added to the respective wells. The results were subsequently analysed after 24 h. For the analysis, first, the solution was aspirated from the 24-well plate, and the cells were gently washed with 1× PBS twice, followed by the removal of PBS by suction. Second, 300 μL of MTT solution was added to the cells. After a 1-h culture at 37 °C in a CO2 incubator, the supernatants were removed and 200 μL of dimethyl sulfoxide was added to the wells, which was followed by continuous shaking for 10 min to solubilise the purple formazan crystals. An ELISA reader (Model 680, BIO-RAD, Hercules, CA, USA) was used to read the absorbance at 570 nm; next, inhibitory rates were calculated to determine IC50 values. The formula to calculate the inhibitory rate is as follows: Inhibition ratio (%) = [(ODcontrol − ODtreated)/(ODcontrol)] × 100%; OD, optical density.
4.5. Quantitative Measurements of Bax and Bcl-2 Proteins Involved in the Apoptotic Pathway of Colorectal Cancer Cells
Apoptosis-associated protein expression in the HT-29 cells was determined by western blotting. The HT-29 cells were added to a 10-well plate and cultured overnight at 37 °C in the CO2 incubator to make the cells attach, divide and grow in the wells. The solutions of Lactobacillus supernatants in various concentrations mixed with the cell culture media were added to the wells, and the cells were cultured for 24 h at 37 °C in the CO2 incubator, which was followed by the addition of Lactobacillus cells (109 cfu/mL) mixed with the cell culture media. After a 6-h culture in the CO2 incubator at 37 °C, the cells were collected and then lysed in radioimmunoprecipitation assay buffer for 30 min on ice. Next, the cells were centrifuged at 12,000 rpm for 5 min at 4 °C. The resulting supernatants (extracts of cellular proteins) were subjected to quantitative protein analysis using the Invitrogen Qubit® fluorometer (Life Technologies, Waltham, MA, USA).
Cellular protein extracts were mixed with 5× loading dye, heated at 95 °C for 5 min and loaded in each well for SDS-PAGE. Following electrophoresis, proteins on the gel were transferred to a PVDF membrane for an hour at 37 °C with the addition of 5% skim milk as the blocking buffer. Subsequently, the membrane was washed in TBST [20 mM Tris–HCl, pH 8 and 137 mM NaCl containing 0.1% (v
) Tween-20] three times and was incubated with the primary antibody (monoclonal antibodies of Bax and Bcl-2) overnight at 4 °C. After three washes with TBST, the secondary antibody (HRP-conjugated goat anti-mouse IgG) was added and incubated with the membrane for 1 h, which was followed by TBST washes. The resulting gel images were captured using a luminometer, and bands were observed [34
4.6. Analysis of Lactate Dehydrogenase (LDH)
LDH stably exists in the cytoplasm and is released from cells with a damaged membrane; therefore, LDH activity in the cell culture medium is positively correlated with the number of necrotic cells. The CytoScanTM-LDH cytotoxicity assay kit (G-Biosciences, St. Louis, MO, USA) was used to measure LDH release: the HT-29 cells (104 cells/100 μL) were seeded in 96 wells and cultured overnight to ensure that the cells attached and grew in the wells. After the removal of the old culture medium, the cells were washed twice with PBS (including 1% BSA) and then cultured with a series of 100 μL mixed solutions of Lactobacillus supernatants in different concentrations and cell culture media for 24 h at 37 °C in the CO2 incubator. Alternatively, the HT-29 cells were cultured with mixed solutions of 100 μL of Lactobacillus cells (109, 108 and 107 cfu/mL), and following 4-, 8- and 12-h cultures at 37 °C in the CO2 incubator, the cells were centrifuged at 1100 rpm for 5 min. Next, 50 μL of supernatants were aspirated and transferred to a new 96-well plate. In addition, unprocessed cells in one of the 96 wells were used as the control group and cultured for 45 min at 37 °C in the CO2 incubator after the addition of 10 μL of 10× lysis buffer. Later, 50 μL of substrate mix was added to each well in the dark, left to stand for 20 min at 37 °C, followed by the addition of 50 μL of stop solution. The resulting absorbance in each well was measured at 490 nm.
4.7. Analysis of Nitric Oxide (NO) Concentration
The HT-29 cells were seeded in a 24-well plate and cultured overnight at 37 °C in the CO2 incubator to make the cells attach, divide and grow in the wells. The supernatants were collected, either after a 24-h co-culture at 37 °C in the CO2 incubator with the addition of a series of mixed solutions of Lactobacillus supernatants in various concentrations and cell culture media or after 4- and 8-h co-cultures at 37 °C in the CO2 incubator with the addition of mixed solutions of Lactobacillus cells (109, 108 and 107 cfu/mL) and cell culture media.
ParameterTM Total Nitric Oxide and Nitrate/Nitrite Assay (R&D Systems, Minneapolis, MN, USA) was used to determine the total amount of NO. First, the collected supernatants were diluted with a 1× reaction diluent. Next, in 96 wells, 50 μL of the reaction diluent (1×), 50 μL of the standard and sample, 25 μL of the NADH reagent, and 25 μL of nitrate reductase were added to each well and left to stand for 30 min at 37 °C. Then, 50 μL of Griess reagent Ι and 50 μL of Griess reagent ΙΙ were added to the wells, and the wells were left to stand for 10 min at room temperature. Lastly, the ELISA reader was used to measure the absorbance at 540 nm and 690 nm.
4.8. Statistical Analysis
Statistical analysis of the study data was performed using the SAS 9.4 statistical software (SAS Institute Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) or independent sample t-test was used to determine the statistical significance; p < 0.05 indicates significant difference.