Contributed equally
Kefir, a fermented milk product, exhibits anti-tumoral activity
Kefir is a well-known fermented milk product obtained by the fermentation of milk with kefir grains. It is highly consumed in many countries, mostly in Eastern Europe, but also in Asia and America (
Kefir grains are a white, soft, gelatin-like mass composed of bacteria and yeast existing in a matrix of proteins, fat, and polysaccharides, with kefiran being the most important water-soluble polysaccharide (
It was always assumed among Bulgarian farmers that kefir held special healing powers, and they believed that their longevity was attributed to their ingestion of kefir (
The first publication to show antitumoral activity of a water-soluble polysaccharide (KGF-C) separated from kefir grains was published in 1982 by Shiomi
Recently,
In 2013, the American Cancer Society ranked colorectal cancer (CRC) as the third leading cause of cancer-related mortalities in the US in both men and women. It was estimated that by the end of 2013, >50,000 death cases will arise due to CRC (
The current study aims at examining the effect of cell-free fractions of kefir on the viability, proliferation, apoptosis and motility of CRC cells
Human colorectal adenocarcinoma cell lines (Caco-2 and HT-29) and human breast cancer cell lines (MCF-7 and MDA-MB231), obtained from ATCC (American Type Culture Collection), were cultured in DMEM medium supplemented with 10% FBS and 100 U penicillin/streptomycin at 37°C and 5% CO2 in a humidified chamber.
Mouse monoclonal IgG anti-β-actin, anti-Bax, anti-Bcl2, anti-p53, anti MMP-2, and anti-MMP-9 antibodies and rabbit polyclonal anti-p21 were obtained from Santa Cruz Biotechnology, Inc. Anti-mouse and anti-rabbit IgG HRP-conjugated secondary antibodies were obtained from Promega.
Pasteurized skimmed milk (150 ml) was inoculated with kefir grains (50 g). Inoculated milk samples were incubated at 20°C for 24 h in a sealed-glass container. At the end of fermentation, the milk was strained to remove the kefir grains. The yeast and bacteria in the filtrate were removed by centrifugation (35,000 rpm for 10 min at 4°C). The supernatant was stored at −20°C until needed for treatment of cells. On the day of treatment, the kefir supernatant was thawed and then passed through a 0.22-μm filter (Millipore). This cell-free fraction of fermented milk, also termed kefir, was applied directly to the cells in different volumes to establish the different concentrations required.
Non-inoculated milk samples were similarly prepared but passed consecutively through a 0.45-μm then 0.22-μm filter (Millipore).
HT-29 and Caco-2 cells were grown in 24-well plates (growth area: 2 cm2) at a density of 2×106 cells/ml. Cells were treated with milk and kefir at the following concentrations (v/v): 0, 5, 10, 15, and 20%. After 24, 48, and 72 h, the supernatant from each well was collected, cells were washed with phosphate-buffered saline (PBS), and the washes were added to the supernatant of each well. Cells were then trypsinised and collected separately from the well contents and PBS. From each collection tube 20 μl were mixed with 20 μl of trypan blue (Sigma-Aldrich). This mixture was placed in a counting chamber under the microscope and the percentage of viable cells was reported.
HT-29 and Caco-2 cells were seeded in 96-well plates (growth area: 0.6 cm2) at a concentration of 1×106 cells/ml. After 24 h of seeding, cells were treated with 0, 5, 10, 15, and 20% milk and kefir (v/v). For every milk and kefir concentration, a blank well was prepared, containing only media and the corresponding volume of kefir or milk. After 24, 48, and 72 h, 10 μl of cell proliferation reagent (WST-1; Roche) was added to each well. The plates were put in a humidified incubator (37°C) for 1.5 h. Absorbance was then read at 450 nm. The absorbance of the each blank well was subtracted from the corresponding sample well. The results were normalized to the untreated controls, and the percent proliferation was reported.
Caco-2 and HT-29 cells were seeded in 6-well plates (growth area: 9.5 cm2). Treatment was done for 24 h, with 10% milk and kefir. After treatment, cells were trypsinized and detached, then centrifuged at 1,200 rpm at 5°C for 5 min. The pellet was washed in 1 ml of ice-cold PBS, centrifuged, and resuspended again in 1 ml of ice-cold PBS. Ethanol was then added to a final concentration of 70%. The fixed cells were left overnight at 40°C. The following day, cells were centrifuged and washed with PBS. The pellet was resuspended in 500 μl of binding buffer, and then 10 μl of propidium iodide (PI) was added to each sample. The samples were incubated in the dark for 10 min.
Cells were analyzed using an Accuri C6 flow cytometer (Accuri Cytometers Inc.), which indicated the distribution of the cells into their respective cell cycle phases based on their DNA content. Cell DNA content was determined by CFlow® software. An increase in cells in the pre-G phase is indicative of an increase in cell death. The percentage of cells in the sub-G0/G1 phase was compared to that of the control.
HT-29 and Caco-2 cells were grown in 96-well plates (growth area: 0.6 cm2) at 1×105 cells/ml. After 24 h, cells were treated with relative concentrations (v/v): 0, 5, 10, and 15%. After 24 or 48 h, cells were lysed with lysis buffer, and incubated for 30 min at room temperature. The plates were then centrifuged for 10 min at 200 g. The supernatant (20 μl) was placed in streptavidin-coated microtiter plates, followed by the addition of biotin-labeled anti-histone and peroxidase-conjugated anti-DNA antibodies. The anti-histone antibody, bound to the plate via biotin-streptavidin, also bound histones from released nucleosomes. The plate was then incubated at room temperature for 2 h, after which 2,2′-azino-di[3-ethylbenzthiazolin-sulfonate] (ABTS) was added as a substrate for peroxidase enzyme. Enrichment factor (EF) was calculated as the recorded absorbance of each sample, divided by that of the untreated cells, according to manufacturer’s instructions (Roche).
Cell lysates were prepared by scraping the cells in a sample buffer consisting of 4% SDS, 10% β-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl at pH 6.8. The resulting lysates were boiled for 5 min. Protein samples were separated by SDS-PAGE on 10% (for β-actin, p53, Mmp-2, and Mmp-9) or 12% (for Bax, Bcl-2, and p21) gels and transferred to PVDF membranes overnight at 30 V. The membranes were then blocked with 5% BSA in PBS containing 0.1% Tween-20 for 1 h at room temperature and incubated with primary antibody at a concentration of 1:1,000 for 2 h at room temperature. After the incubation with the primary antibody, the membranes were washed and incubated with secondary antibody at a concentration of 1:1,000 for 1 h at room temperature. The membranes were then washed, and the bands visualized by treating the membranes with western blotting enhanced chemiluminescent reagent ECL (GE Healthcare). The results were obtained on X-ray film (Agfa Healthcare). The levels of protein expression were compared by densitometry using the ImageJ software.
Cells were grown in 6-well plate at density of 1×106 cells/ml. After 24 h, cells were treated with 0, 5, and 10% cell-free fractions of kefir for 24 h, after which total RNA was extracted using RNeasy extraction kit (Qiagen) according to manufacturer’s instruction. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify RNA of β-actin (
Cells were grown to confluence on culture plates and a wound was made in the monolayer with a sterile pipette tip. After wounding, the cells were washed twice with PBS to remove debris and fresh medium was added. Phase-contrast images of the wounded area were taken at 0 and 21 or 24 h after wounding. Wound widths were measured at 11 different points for each wound, and the average rate of wound closure was calculated in μm/h using ImageJ software.
For motility analysis, images of cells moving randomly in serum were collected every 60 sec for 2 h using a 20× objective. During imaging, the temperature was controlled using a Nikon heating stage which was set at 37°C. The medium was buffered using HEPES and overlayed with mineral oil. The speed of cell movement was quantified using the ROI tracker plugin in ImageJ software, which was used to calculate the total distance travelled by individual cells. The speed was then calculated by dividing this distance by the time (120 min) and reported in μm/min. The speed of at least 10 cells for each condition was calculated.
Cells were grown in 6-well plates. After 24 h, cells were treated with 0, 5, and 10% of kefir for another 24 h. Invasion assay was performed following treatment period using the collagen-based invasion assay (Millipore) according to manufacturer’s instructions. Briefly, 24 h prior to assay, cells were starved with serum free medium. Cells were harvested, centrifuged and then resuspended in quenching medium (serum free). Cells were then brought to a concentration of 1×106 cells/ml. Inserts were rehydrated with 300 μl of serum free medium for 30 min at room temperature, 250 μl of media was then removed from inserts and 250 μl of cell suspension was added. Inserts were then placed in a 24-well plate, and 500 μl of complete media was added to the lower wells. Plates were incubated for 24 h at 37°C in a CO2 incubator. Later, inserts were stained for 20 min at room temperature with 400 μl of cell stain provided with the kit. Stain was then extracted with extraction buffer. The extracted stain (100 μl) was then used for colorimetric measurement using a plate reader. Optical density was measured at 560 μm.
All reported results represent average values from three independent experiments. The error estimates are given as mean ± SEM. The p-values were calculated by t-tests or χ2 tests depending on the experiment, using the VassarStats: Website for Statistical Computation (
To assess kefir’s cytotoxicity on the two cell lines, we began by determining the percentage viability after treating the cells with increasing kefir concentrations. Upon kefir treatment, the viability of the cells decreased in a time- and dose-dependent manner.
Results demonstrated that kefir’s inhibitory concentration 50 (IC50) for Caco-2 cells ranges between 10, 12, and 18% (v/v) at 72, 48, and 24 h, respectively (
The viability of both cell lines was not reduced 6 h post-treatment (data not shown), suggesting that the cells were dying through apoptosis rather than necrosis.
The effect of kefir on proliferation of CRC cells was determined by the activity of mitochondrial dehydrogenases. Results have shown that kefir significantly inhibited the proliferation of HT-29 and Caco-2 cells (p<0.05) (
HT-29 cells as well showed significant inhibition of proliferation upon kefir treatment, even though the effect was slightly less than that exhibited by the Caco-2 cells (
All cells treated with milk showed a significant increase in proliferation, compared to the untreated and kefir-treated cells (p<0.05). Hence, kefir treatment significantly reduces proliferation of CRC cell lines in a time- and dose-dependent manner.
After verifying that kefir inhibited cell proliferation in CRC cells, we aimed to evaluate whether this effect was through an induction of cell cycle arrest, using flow cytometry. After analyzing the cell’s DNA content, cells were assigned to their respective phases: sub-G 0/G1 cells were <2n, G0/G1 cells were 2n, and S/M phase cells were >2n.
Consistent with the results of the proliferation assay, the sub-G0/G1 population of Caco-2 cells increased from 14 to 5.2% as a result of 10% kefir treatment, while the S/M phase cells decreased from 15.5 to 3.6% (
It is thus implied that kefir causes a cell cycle arrest at the G1 transition checkpoint which explains its anti-proliferative effect.
To verify if kefir reduce the viability of the CRC cells through an induction of apoptosis, we used the cell death detection ELISA assay, where the absorbance obtained at 405 nm reflects the quantity of released nucleosomes, a hallmark of apoptosis. The EF, which is the ratio of the absorbance measured for each concentration to that of the untreated controls, was calculated. In Caco-2 kefir-treated cells, the EF increased around 2.3-, 2.6-, and 6-fold, 24 h after treatment with 5, 10, and 15% kefir, respectively (
In order to determine a possible mechanism for the anti-proliferative effect of kefir observed in the CRC cells with the WST-1 assay, the expression of
The effect of kefir on the metastatic ability of cancer cells was assessed in a single study,
After looking at the motility of colorectal and breast cancer cell lines in two-dimensions and observing no effect upon kefir treatment, we decided to look at whether kefir has any effect on the invasive ability of the CRC HT-29 cells. Using the collagen-based invasion assay, we observed no significant difference in the invasive ability of HT-29 cell lines between control and 10% kefir-treated cells
In the present study, we aimed to investigate whether kefir’s anti-cancerous effect, previously proven on several types of cancers, both
We first determined that the IC50 is reached with 18, 12, and 10% v/v (kefir cell-free fractions) at 24, 48, and 72 h respectively, for Caco-2 cells, compared to 12 and 10% at 48 and 72 h, respectively, for HT-29 cells. For both cell lines, treatment with kefir reduced viability in a time- and dose-dependent manner. Since 6 h post-treatment, the viability of the cell lines was not reduced (data not shown), it was assumed that the cell numbers were reduced due to cell death. The observation that the milk-treated cells showed no decrease in viability suggests that kefir’s effect is due to products released by the microorganisms during fermentation.
A notable decrease in proliferation upon kefir treatment was detected in the cells using the WST-1 reagent. Caco-2 cells exhibited a 91, 74, and 96% decrease in proliferative activity upon treatment with 18, 12, and 10% at 24, 48, and 72 h, respectively (IC50 concentrations). For HT-29 cells, treatment with IC50 concentrations (12 and 10% at 48 and 72 h, respectively), caused 64 and 40% decrease. As expected, milk-treated cells results showed an increase in proliferation. Through flow cytometry, it was verified that kefir, but not milk, causes a shift in the cell cycle of the treated cells towards the sub-G0/G1 phase, by inducing cell cycle arrest at the G1 checkpoint. Kefir did not only increase the percentage of cells in the sub-G0/G1 phase, but also caused a reduction in the S/M cell population as well. The increase in sub-G0/G1 implies that kefir induces death in CRC cell lines. To verify that the cells were dying through apoptosis and not necrosis, as assumed, the cell death ELISA kit assay was performed. The results of this assay confirmed that kefir was indeed inducing apoptosis in Caco-2 and HT-29 cells.
TGF-α is a known mitogen whose expression is upregulated in many types of tumors, especially CRC, where its expression exceeds four times that of normal colorectal tissues (
A downregulation in the expression level of
To determine a possible explanation for the pro-apoptotic effect of kefir, the expression of Bax and Bcl-2 at the protein level was assessed. Our findings show upregulation in Bax:Bcl-2 expression in HT-29 cells, consistent with the observed increase in apoptosis using the cell death ELISA assay. For the milk-treated HT-29 cells, a decrease in Bax:Bcl-2 ratio was observed which is also in accordance with the decrease in apoptosis and increase in proliferation observed for milk-treated cells.
To further elucidate the mechanism of action of kefir, the expression levels of p53 and p21 proteins were assessed. Kefir treatment at 5 and 10% (v/v) caused no difference in p53 levels but a noticeable increase in p21 levels when treated with 10% kefir. This increase in p21 levels might explain the cell cycle arrest observed at the G1 phase through cell cycle analysis by flow cytometry. Our data suggest that p21 induction is p53-independent.
Since metastasis remains the main cause of death in cancer patients, we assessed the effect of cell-free fractions of kefir on the motility of CRC cells
In conclusion, kefir has become globally known as a complex probiotic, to which many health benefits have been attributed. These include anti-microbial, anti-inflammatory, immunomodulatory, and metabolic benefits. This study focused on assessing kefir’s anti-cancerous potential. Through several experiments, we have established that kefir exhibits pro-apoptotic and anti-proliferative properties on colorectal adenocarcinoma cells, namely Caco-2 and HT-29,
ROI tracker software was supplied by David Entenberg and John Condeelis as supported by CA100324 and GM064346. This study was partially funded by the National Council for Scientific Research-Lebanon, and the Univeristy Research Council-Lebanese American University.
colorectal cancer
reverse transcriptase-polymerase chain reaction
human T-lymphotropic virus type I
epidermal growth factor receptor
transforming growth factor-α
transforming growth factor-β1
inhibitory concentration 50
enrichment factor
Effect of kefir on viability of colorectal cancer (CRC) cell lines using trypan blue assay. Viability of Caco-2 cells after (A) 24 h, (B) 48 h, and (C) 72 h of treatment with different concentrations of milk and kefir cell-free fractions. Viability of HT-29 cells after (D) 24 h, (E) 48 h, and (F) 72 h of treatment with different concentrations of milk and kefir cell-free fractions. Results are reported as the percent of viable cells out of the total number of cells (dead and alive). Data represent mean ± SEM from three independent experiments.
Effect of kefir on the proliferation of colorectal cancer (CRC) cell lines. Proliferation of Caco-2 cells after (A) 24 h, (B) 48 h, and (C) 72 h of treatment with different concentrations of milk and kefir cell-free fractions. Proliferation of HT-29 cells after (D) 24 h, (E) 48 h, and (F) 72 h of treatment with different concentrations of milk and kefir cell-free fractions. Results were normalized to the untreated cells. Data represent mean ± SEM from three independent experiments.
Cell cycle analysis by flow cytometry of kefir-treated colorectal cancer (CRC) cells. Cell cycle analysis for Caco-2 cells after 24 h of treatment with (A) 0% kefir, (B) 10% milk, and (C) 10% kefir. Cell cycle analysis for HT-29 cells after 24 h of treatment with (D) 0% kefir, (E) 10% milk, and (F) 10% kefir.
Effect of kefir on apoptosis of colorectal cancer (CRC) cell lines. Induction of apoptosis in Caco-2 cells (A) 24 h and (B) 48 h after kefir and milk treatment. Induction of apoptosis in HT-29 cells (C) 24 h and (D) 48 h after kefir and milk treatment. Results shown represent the enrichment factor (EF) calculated as absorbance of sample/absorbance of untreated control. Data represent mean ± SEM from three independent experiments.
Expression of genes involved in proliferation and apoptosis in kefir-treated HT-29 cells. The expression of transforming growth factor α (TGF-α) and transforming growth factor-β1 (TGF-β1) in 0, 5, and 10% (v/v) (A) kefir- and (B) milk-treated HT-29 cells. Representative western blotting images for Bax and Bcl-2 in (C) kefir- and (D) milk-treated cells. Representative western blot images for p21 and p53 in (E) kefir- and (F) milk-treated cells. Representative western blot images for MMP-2 and MMP-9 in (G) kefir and (H) milk-treated cells.
Effect of kefir on the migration ability of treated cells
Effect of kefir on the invasive ability of treated HT-29 cells