We examined the apoptotic effect of a novel Probiotics Fermentation Technology (PFT) kefir grain product; PFT is a natural mixture composed primarily of
Lactic acid bacteria (LAB) have been used throughout history to produce fermented food and milk products. In the early 1900s, biologist Eli Metchinkoff suggested that the intake of LAB could increase the life span of humans (
The idea of treating cancers with LAB is not as recent as one might think. Over a century ago, Metchnikoff suggested that LAB had a protective effect against colorectal cancer (
Conventional treatments for cancer such as chemotherapy aim to initiate apoptosis. However, these drugs can be toxic and can decrease a cancer patient’s quality of life. Therefore, there has been an effort to investigate alternative treatments that have fewer side effects and improve the health of the patient. A recent study has shown that
The product we use in this study is a symbiotic microbe of lactic acid bacteria and yeasts known as Probiotics Fermentation Technology (PFT) kefir grain product. PFT is separated from kefir, a popular drink across Eastern and Northern Europe and Russia. Kefir has been shown to have various health benefits, for example, it protects the intestine against disease-causing bacteria (
The results of this study show that PFT possesses the ability to pierce holes in MDR human myeloid leukemic (HL60/AR) cells, which induces apoptosis in the cancer cells by intrinsic (mitochondrial) pathway of apoptosis. This study suggests that PFT may exert a therapeutic effect in treating MDR cancers.
Human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells were used in the present study. Cells were kindly provided by Dr S. Gollapudi at the University of California, Irvine, CA, USA. Tumor cells were maintained in our laboratory in a complete medium (CM) that consisted of RPMI-1640, 10% fetal calf serum (FCS), 2 mM glutamine and 100
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), was employed.
PFT is a mixture that mainly (∼90%) contains a freeze-dried form of heat-killed
HL60/AR cells were cultured in the presence or absence of PFT at different concentrations (0, 0.6, 1.25, 2.5 and 5 mg/ml) for 3 days and the percentage of dead cancer cells was examined by the propidium iodide (PI) technique using a FACScan flow cytometery. Briefly, PI was added to the cells (1×106/ml) to give a final PI concentration of 50
For detection of Bcl-2, cells were first fixed and permeabilized with ice-cold 70% methanol. Cells were then stained with FITC-labeled anti-Bcl-2 or isotype control (Dako Corp., Carpinteria, CA, USA), washed and analyzed by FACScan. The percentage of cells expressing Bcl-2 and mean fluorescent intensity (an indicator of density of the molecules/cell) was determined.
The method for measuring intracellular activity of caspase 3 is based on carboxyfluorescein labeled fluromethyl ketone (FMK)-peptide inhibitors of caspases. These inhibitors are cell permeable and non-toxic. Once inside the cells, these inhibitors bind covalently to the active caspase. Caspase-positive (+) cells are distinguished from caspase-negative (−) cells with the aid of flow cytometry. Briefly, cells undergoing apoptosis were loaded with fluorescein labeled FAM-DEVD-FMK for caspase 3 (Intergen Co., NY, USA). After 1-h incubation, the cells were washed to remove unbound caspase and cells that contained bound inhibitor were quantified using a FACScan flow cytometer.
Variations of the mitochondrial transmembrane potential ΔΨm during apoptosis were studied using tetramethylrhodamine ethylester (TMRE, Molecular Probes, Eugene, OR, USA). Briefly, after treatment with PFT for 3 days, cancer cells (5×105 cells/ml) were incubated with 50 nM TMRE for 30 min at 37°C. The cells were washed with PBS and analyzed with FACS Forward, the side scatters were used to gate and exclude cellular debris using a FACScan. The cells were excited at 488 nm and the emission was collected on the FL2 channel. Five thousand cells were analyzed. The data were acquired and analyzed using CellQuest software (Becton-Dickinson).
HL60/AR cells (1×106 cells/ml) were cultured with PFT (5 mg/ml) for 2 min and 24 h. Results were compared to those of cells without treatment. Cytospin preparations (Shandon Southern Inst., Sewickley, PA, USA) of cells were air-dried, fixed in 100% MeOH for 5 min and prepared for AFM studies. AFM studies were carried out to examine the morphological changes associated with PFT treatment of HL60/AR cells such as hole induction and membrane blebbing. Dimension 5000 AFM (Veeco) under contact mode was used to image the HL60/AR cells with Bruker’s Sharp Nitride Lever (SNL) silicon probes (Veeco). Topographic height images were recorded at 512×512 pixels at a scan rate of 0.8 Hz. Image processing was performed using SPIPTM Software. Usually an MLCT-AFM tip (with a ‘k’ value of 0.03N/m) contributes to the broadening effect because of its specific geometry (
Statistical significance for cell apoptosis in
We examined the effect of PFT on tumor cell survival using propidium iodide and FACScan flow cytometry. The data in
To verify activation of the apoptotic pathway, the level of caspase 3 activation was investigated by treating HL60/AR cells with PFT at varying concentrations for 24 h and analyzed using flow cytometry. Data depicted in
Experiments were carried out to examine the ability of PFT to disrupt MMP. HL60/AR cells were treated in the presence or absence of PFT at varying concentrations and MMP was determined by flow cytometry using membrane potential sensitive TMRE dye. The data in
The expression of Bcl-2 was examined to determine the anti-apoptotic activity post-treatment of HL60/AR cells by PFT. Results depicted in
AFM studies were carried out to examine the morphological changes associated with PFT treatment of HL60/AR cells. Results showed a low percentage of the control untreated HL60/AR cells with holes, as well as a small number of holes per cell (
In this study, we examined the apoptotic activity of PFT, a mixture with the main constituent of
Several studies have shown that LAB exerts antitumor activity via several mechanisms. These include the ability of LAB for binding mutagens and removing carcinogens from colon (
Hole piercing has been observed with nanoparticles such as nickel (
Vacuoles appear in the cancer cells apparently due to the cells ability to phagocytize microorganisms, such as bacteria and yeast (
The unique properties of the
In conclusion,
The authors would like to thank Dr Sastry Gollapudi, Professor of Immunology, Division of Basic and Clinical Immunology, University of California, Irvine, CA, USA for assistance in revision of the manuscript. The authors would also like to thank Paitos Co., Ltd., Yokohama, Kanagawa, Japan; grant no. T0099108. The authors thank Dr Peggy Vorwald for her contributions. The authors acknowledge the use of resources provided by Dr James Gimzewski and for the use of the SPM facility and atomic force microscopy, at the Nano and Pico Characterization Lab at the California NanoSystems Institute.
PFT induces apoptosis of HL60/AR cells. Tumor cells were cultured with PFT at different concentrations (0, 0.6, 1.25, 2.5 and 5 mg/ml) and the percentage of dead cancer cells was examined by the propidium iodide (PI) technique using a FACScan flow cytometry. Data represent the mean ± SD of 4 experiments. Statistical analysis compared the concentrations to control (0 mg/ml). *p<0.05, **p<0.0005, ***p<0.0001.
PFT induces apoptosis through activation of caspase 3. The activation of caspase 3 was determined in cancer cells post-culture with PFT at different concentrations (0, 0.6, 1.25 and 2.5 mg/ml). The lines represent cancer cells in the absence of PFT (control, purple) and cancer cells treated with PFT at concentration of 0.6 mg/ml (green), 1.25 mg/ml (pink), or 2.5 mg/ml (blue). The activation of caspase 3 in cells was detected using carboxyfluorescein-labeled cell permeable peptide substrate and was analyzed using flow cytometry.
PFT reduces MMP. Tumor cells were cultured with PFT at different concentrations (0, 0.6, 1.25 and 2.5 mg/ml) and the MMP was determined by flow cytometry using TMRE dye. Decrease in TMRE fluorescence indicates loss of membrane potential. PFT depolarized membrane potential in a dose-dependent manner. Statistical analysis was performed by the Kolmogorov-Smirnov test using CellQuest Software System. A D-value >0.2 is considered statistically significant.
PFT reduces the expression of Bcl-2. HL60/AR cells (1×106 cells/ml) were cultured with PFT at a concentration of 1.25 mg/ml for 24 h. Expression of Bcl-2 was determined by staining the cells with anti-human Bcl-2 antibody and by flow cytometry. It is shown as mean fluorescent intensity (MCF).
PFT induces holes and membrane blebbing in cancer cells. HL60/AR cells (1×106 cells/ml) were cultured with PFT and the changes were examined by AFM. (A) Control untreated cells. (B) Cells treated with 5 mg/ml of PFT for 2 min; note the attachment of cancer cells to
Hole detection using AFM (peak force imaging). Darker color indicates greater hole depth. Note the variation in hole shape, size and depth. (A and B) Several smaller, distinctly circular holes. (C and D) A broad, irregularly shaped, deep indentation. (E and F) A large, circular hole.
Determining the depth of PFT-induced hole formation. The red and blue lines indicate the surface contour of an HL60/AR cell treated with PFT. The arrow indicates a large hole detected by the SNL tip and arrowheads indicate smaller holes. This image is representative of many HL60/AR cells during PFT treatment.
Test | P-IF characteristic |
---|---|
99.6% sequence homology | |
16S ribosome identification | |
Cell shape | Rod |
Gram staining | + |
Motility | Non-motile |
Colony growth | 3D growth |
Carbon utilization | Glucose |
Fructose | |
Galactose | |
L-arabinose | |
Ribose | |
Maltose | |
Lactose | |
Melibiose | |
Gluconate | |
Acid/gas production | Carbonic acid gas |
pH tolerance | >4.3 pH |
Characteristics not common in other kefiri strains.