Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells

Breast cancer is the most commonly diagnosed cancer in women. Resistance to therapy is the major reason for failure of cancer treatment (Mesner et al., 1997). There is a critical need to identify new chemotherapeutic agents that can increase susceptibility to anti-breast cancer drugs or overcome resistance mechanisms, which would improve patient outcomes, prevent relapse, and prolong patient survival. Kefir is an acidic and mildly alcoholic fermented milk that originated in the Caucasian mountains of former Soviet Union, and enjoys a rich tradition of health benefits. Consumption of fermented dairy foods has been associated with lower incidence of breast cancer (Ronco et al., 2002). Research on the putative health benefits of fermented milks suggested that by-products of bacterial fermentation of proteins, lipids and carbohydrates present in fermented milks exert health benefits beyond basic nutrition including anti-tumor action, immune system enhancement and antioxidant effects (Parvez et al., 2006). Conjugated linoleic acid (Schonberg et al., 1995), sphingolipids (Dillehay et al., 1994), polysaccharides (Shiomi et al., 1982), organic acids (Garrote et al., 2000) and some proteins and peptides (Svensson et al., 1999) have been shown to have antimutagenic and antitumor effects. Promising results regarding anti-tumor activity of yogurt extracts in cell culture (Biffi et al., 1997) and kefir extracts in animal feeding studies (Cervikbas et al., 1994; Furukawa et al., 2000; Shiomi et al., 1982) have been reported. However, the bioactive components in kefir and the mechanisms involved in the various biofunctional effects of kefir are still not well characterized, particularly in terms of anti-cancer activities. In our recent studies, kefir extracts exerted potent anti-proliferative effects on cultured human mammary tumor cells as compared to extracts of yogurt or milk (Chen et al., 2007). The aim of the present study was to fractionate and characterize bioactive components in kefir mother culture that exert antiproliferative effects in MCF-7 cells.


Introduction
Breast cancer is the most commonly diagnosed cancer in women.Resistance to therapy is the major reason for failure of cancer treatment (Mesner et al., 1997).There is a critical need to identify new chemotherapeutic agents that can increase susceptibility to anti-breast cancer drugs or overcome resistance mechanisms, which would improve patient outcomes, prevent relapse, and prolong patient survival.Kefir is an acidic and mildly alcoholic fermented milk that originated in the Caucasian mountains of former Soviet Union, and enjoys a rich tradition of health benefits.Consumption of fermented dairy foods has been associated with lower incidence of breast cancer (Ronco et al., 2002).Research on the putative health benefits of fermented milks suggested that by-products of bacterial fermentation of proteins, lipids and carbohydrates present in fermented milks exert health benefits beyond basic nutrition including anti-tumor action, immune system enhancement and antioxidant effects (Parvez et al., 2006).Conjugated linoleic acid (Schonberg et al., 1995), sphingolipids (Dillehay et al., 1994), polysaccharides (Shiomi et al., 1982), organic acids (Garrote et al., 2000) and some proteins and peptides (Svensson et al., 1999) have been shown to have antimutagenic and antitumor effects.Promising results regarding anti-tumor activity of yogurt extracts in cell culture (Biffi et al., 1997) and kefir extracts in animal feeding studies (Cervikbas et al., 1994;Furukawa et al., 2000;Shiomi et al., 1982) have been reported.However, the bioactive components in kefir and the mechanisms involved in the various biofunctional effects of kefir are still not well characterized, particularly in terms of anti-cancer activities.In our recent studies, kefir extracts exerted potent anti-proliferative effects on cultured human mammary tumor cells as compared to extracts of yogurt or milk (Chen et al., 2007).The aim of the present study was to fractionate and characterize bioactive components in kefir mother culture that exert antiproliferative effects in MCF-7 cells.
Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells 151 assembled device was centrifuged at 4°C, 3000 x g for 290 min.The filtrates were collected for further analysis.

Size exclusion HPLC (SEC) separation
One hundred microliters of kefir mother culture extract was injected into a TSK G2000SWXL column (78 mm x 30 mm, SUPELCO, Bellefonte, USA) and separated with a Shimadzu LC-6AD Liquid Chromatograph system (Shimadzu Scientific Instruments, Inc. Columbia, USA) with UV detection at 210 nm.The separation buffer used was a mixture of 45% acetonitrile in 0.1% trifluoroacetic acid (TFA) with a flow rate of 0.4 ml/min for 40 min.Nine fractions were collected for each HPLC run, and fractions from five to ten runs were pooled.The above nine fractions were evaporated with N 2 and then freeze dried, stored at −80°C for cell culture incubations and for further analytical analyses.

Reverse phase HPLC (RP-HPLC) fractionation
The fraction(s) collected with SEC HPLC that showed antiproliferative effects on MCF-7 cells were further analyzed with a Prosphere 300 C4 column (5µm, 250 mm x 4.6 mm) (Alltech Associate, Inc. Deerfield, USA) using a Beckman Gold HPLC System (Beckman Coulter, Fullerton, USA).After the column was equilibrated with buffer A (0.1% TFA in water) at a flow rate of 1 ml per min, the fractions were eluted with a linear gradient of buffer A (0.1% TFA in water) and buffer B (60% of acetonitrile in 0.1% TFA: 40% of 0.1% TFA in water) as follows: 0 to 60 min, 0 to 90% B; and 61 to 65 min, 90 to 0% B. Dual channel absorbance was monitored at 210 nm (channel A) and 280 nm (channel B).

Preparative HPLC fractionation
Three batches of extracts of kefir mother culture were pooled and fractionated using the Centriplus centrifugal filter devices to obtain fractions of compounds with molecular weights less than 3000 Da.The fractions of MWCO less than 3000 Da were freeze-dried using a FLEXI-DRY MP Freeze Dryer (FTS Systems, Inc. Stone Ridge, U SA). Five g of the lyophilized MWCO fractions were dissolved in 20 ml of water.Ten milliliters of reconstituted solution were loaded on a C4 preparative column (300 Å, 5 µm, 300 mm x 50 mm) (Vydac Company, Herperia, CA) and separated with a Water Delta Prep 4000 HPLC system (Waters Corporation, Milford, USA).After the column was equilibrated with buffer A (0.6% acetic acid in water) at a flow rate of 13 ml and the fractions were eluted with a linear gradient of buffer A and buffer B (0.6% acetic acid in acetonitrile) as follows: 0 to 70 min, 0 to 60% B; 70 to 80 min, 60 to 70% B; 80 to 100 min, 70 to 80% B; 100 to 105 min, 80 to 0% B. A total of 100 fractions were collected between 0 and 100 min, with samples taken at 1-min intervals.The fractions were then lyophilized and kept at −80°C for further cell culture and analysis.The fractions were reconstituted by adding water and peptide and protein concentrations were determined before cell culture assays.

Capillary electrophoresis
Capillary zone electrophoresis (CE) was performed using a P/ACE TM 2200 HPAC instrument controlled by System Gold software (Beckman, Fullerton, CA, USA) coupled to an IBM PC 486 computer (IBM Corp., Portmouth, England) for data acquisition and analysis.A neutral uncoated fused silica capillary column (57 cm × 50 µm, the length from intake to detector was 50 cm) was assembled in the P/ACE cartridge (Polymicro www.intechopen.comBreast Cancer -Current and Alternative Therapeutic Modalities 152 Technologies, Phoenix, Arizona USA) for capillary separations.Injection volume, buffer concentration and running voltage were optimized to achieve the best resolution with the shortest running time.The capillary was flushed with 1 M sodium hydroxide, followed by nanopure water, 0.1 M sodium hydroxide, nanopure water, 1 M hydrochloric acid, and again water, each for 5 min at a pressure of 40 psi.A solution of 5% polybrene and 2% ethylene glycol was then passed for 10 min at 40 psi.Excessive coating was then removed by flushing with water for 2 min.Further capillary flushing was then performed for additional 10 min using 200 mM formic acid buffer.Before each sample application, the capillary was rinsed with 1 min water, 1 min 0.1 M sodium hydroxide, 1 min water and 3 min separation buffer (200 mM formic acid in water, pH 2.0).After the completion of each run, the capillary was rinsed with nanopure water for 1 min, 0.1 M sodium hydroxide for 1 min and nanopure water for 1 min.Peptide standard and sample injections were carried out at the anode end of the capillary using N 2 pressure (0.5 psi) for 5 sec and was separated at a constant temperature of 20°C with a 200 mM formic acid (pH 2.0) as separation buffer.On-line detection was performed using an UV detector at 200 nm.Twenty kilovolts was applied across the run buffers for best separation.The capillary was re-coated after every 5 runs.

MALDI-TOF for estimation of molecular weight
Reconstituted SEC-HPLC and RP-HPLC fractions were analyzed using a MALDI-TOF mass spectrometer (Voyager DE-STR; Applied Biosystems, Palo-Alto, CA, USA) with a laser at 337 nm and an acceleration voltage of 20.000 V.

Mass spectrometry
Mass spectrometric analysis in the positive ion mode was performed on a triple quadupole mass spectrometer (SCIEX API III Biomolecular mass analyzer, Thornhill, Ontario, Canada).Lyophilized reversed phase HPLC fractions having antiproliferative effect on MCF-7 cells were reconstituted in 0.5 mM ammonium acetate in methanol or in 10% acetic acid in 20% aqueous methanol.The resulting solution was then infused into the electrospray ion-source by a syringe pump (Harvard Apparatus Model 22, South Natick, MA) at a flow rate of 1.5 ml/min.The ion-spray voltage was set at 5.5 kV and the orifice potential was set at 50 V.Argon was used as the collision gas at a collision gas thickness (CGT) of 1.5 x 10 14 for collision-induced fragmentation MS-MS analysis.
2.10 Cell Culture screening for antiproliferative effects MCF-7 cells were purchased from ATCC (ATCC, Manassas, USA).Cells were routinely propagated as a monolayer culture in Dulbcco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), in a 75-cm 2 plastic dish at 37°C in a humidified atmosphere with 5% CO 2 , and passaged every 3-4 days.Normal human mammary epithelial cell lines (HMEC) were graciously provided by Dr. M.R. Stampfer (Lawrence Berkeley National Laboratory, Berkeley, USA).Cells were routinely propagated as a monolayer culture in Mammary Epithelial Growth Media (MEGM, Clonetics, San Diego, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in 75-cm 2 plastic dish at 37°C in a humidified atmosphere with 5% CO 2 , and passaged every week.For the experiments, both MCF-7 and HMEC cells were harvest from the dish using 0.25% trypsin-EDTA solution (Sigma, St Louis, MO, USA).

Cell proliferation experiments in 24-well plates
Cells previously harvested were seeded in 24-well plates, i.e., 10,000 cells for MCF-7 per well in DMEM supplemented with 10% FBS and 5,000 cells for HMEC per well in MEGM supplemented with 10% FBS.The cells were allowed to attach for 24 h.After that period, old media were removed and fresh media and extracts were added to each well.To study the dose response, a serial dilution of each the extract using the culture media was made to achieve final concentrations of extracts at 5%, 2.5%, 1.3%, 0.6%, and 0.3% (vol./vol.),respectively.Because the kefir extracts were acidic (approximately pH 4.5), Dulbecco's Phosphate Buffered Saline (PBS) buffer (Gibco BRL, Grand Island, NY) was added to the culture media to adjust the pH between 7.0 and 7.4.Cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 for 6 d.Cell nuclei were counted in order to eliminate the difficulty in counting whole cells due to clumping.Furthermore, a more uniform distribution of nuclei over the grids of a hemacytometer was seen compared with whole cells.Media was aspirated from wells and cells were rinsed with 500 µl of PBS.250 µl of hypotonic buffer (0.01 M HEPES, 1.5 M MgCl 2 , pH 7.5) was added to each well.After 2 min, 250 µl of cell lysing solution (10% ethyl hexadecyl dimethyl ammonium bromide, 3% glacial acetic acid, in water) was added.The plate was shaken lightly every minute for 5 min.Cell lysis was confirmed microscopically as indicated by a suspension of clean nuclei.The suspension was mixed and the nuclei were counted using a Coulter Counter (Coulter Counter Corporation, Fullerton USA).Each sample was run in quadruplicate.Control cells were incubated with the culture medium with the dosing vehicle (PBS).To standardize results, each plate had its own control wells with cells treated with PBS and cell proliferation of different treatment was expressed as a percent of control.

Cell proliferation experiments in 96-well plates
Cells previously harvested were seeded in 96-well plates at 1,000 cells per well for MCF-7 in DMEM supplemented with 10% FBS and for HMEC in MEGM supplemented with 10% FBS.The cells were allowed to attach for 24 h.After that period, old media were removed and fresh media and extracts were added to each well.A serial dilution of each test fraction was made to study the dose-response.PBS buffer was added to the culture media to keep the final pH between 7.0 and 7.4.Cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 for 6 d and the cell numbers in each well were determined by using a CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, USA).Each sample was run in quadruplicate.Control cells were incubated with the culture medium with the dosing vehicle (PBS).To standardize results, each plate had its own control wells with cells treated with PBS and cell proliferation of different treatments was expressed as a percent of control.

Statistics
All statistics test were performed using SAS 8.2 for PC (SAS, Cary, NC USA).Means were compared with Student's t test.Two-way ANOVA was used to analyze the effects of treatments and doses for the cell culture experiments.The differences among doses and treatments were determined by the Student-Newman-Keuls (SNK) multiple comparison test.Statistical significance was considered at P< 0.05.

Results
Moisture, macronutrients and some minerals in kefir mother culture and final kefir described in Table 1.No significant differences among nutrients and organic acids were found, except that lactic acid in the kefir commercial product was higher than that of kefir mother culture (Table 2).Capillary electrophoresis analyses showed that the profiles of extracts of mother culture and kefir were similar (data not shown).In our earlier study, the extract of kefir mother culture showed a tendency of having stronger antiproliferative effect on MCF-7 cells than other extracts (Chen et al., 2007).Hence, mother culture was chosen for further fractionation and cell culture tests to identify the bioactive component(s).Extracts of kefir mother culture that were fractionated via the 3000 Da MWCO were analyzed for protein and peptide content (Table 3).Both protein and peptide concentrations were significantly lower in the fraction of MWCO less than 3000 Da (P < 0.05) relative to the whole kefir mother culture extract.Both the whole extract and the fraction with the MWCO less than 3000 Da were screened by MCF-7 cell culture using 24-well plates.As shown in Figure 1, the filtrate of MWCO less than 3000 Da had a dose dependent antiproliferative effect on MCF-7 cells comparable to that of the whole mother culture extract.
Fig Fractions 7 and 8 were the only fractions to demonstrate a dose-dependent antiproliferative effects on MCF-7 cells with the most potent effect exhibited with fraction 7 (Figure 2).Fraction 7 was also tested with HMEC cells, and no antiproliferative effects were observed (Figure 3).Fraction 7 was further analyzed with RP-HPLC, which showed the presence of about 11 peaks in Fraction 7 (Figure 4).SEC-HPLC Fraction 7 was also analyzed via MALDI-TOF, which indicated that the peak masses ranged from 659 to 2074 Da. (Figure 5).A preparative C4 column was utilized to obtain sufficient amounts of mother culture kefir fractions of MWCO less than 3000 Da for more extensive structure analysis and cell culture studies.The average nitrogen content of the lyophilized filtrate of MWCO less than 3000 Da was 0.58%.Five g of the lyophilized MWCO less than 3000 Da fractions was dissolved in 20 ml of water.Ten milliliters of reconstituted solution were loaded on the column, and 100 fractions were collected in 1 min intervals (Figure 6).Fig. 6.Elution profile of MWCO less than 3000 Da fraction of kefir mother culture extract analyzed by preparative RP-HPLC.Appropriate amount of samples were loaded on a C4 preparative column (300 Å, 5 µm, 300 mm x 50 mm) (Vydac Company, Herperia, CA) and separated with Waters Delta Prep 4000 HPLC system.After the column was equilibrated with buffer A at a flow rate of 13 ml, the fractions were eluted with a linear gradient of Buffer A (0.6% acetic acid in water) and Buffer B (0.6% acetic acid in acetonitrile) as follows: 0 to 70 min, 0 to 60% B; 70 to 80 min, 60 to 70% B; 80 to 100 min, 70 to 80% B; 100 to 105 min, 80 to 0% B. The absorbance was measured at 210 nm.A total of 100 fractions were collected between 0 and 100 min, with samples taken at 1-min intervals.
The peptide concentration of each preparative HPLC fraction is shown in Figure 7, Fractions 28 to 58, 62 to 64, and 72 to 74 were screened for antiproliferative effects on MCF-7 cells with three concentrations of 0.4%, 2% and 10% (Figure 8).Fractions 29 to 41, 62 were found to have antiproliferative effects on MCF-7 cells.These fractions were further screened for antiproliferative effects with serial dilution.A dose dependent antiproliferative effect was observed when MCF-7 cells were treated with fractions 29, 30, 34, and 37, while not on HMEC cells (Figure 9).B C D Fig. 9. Dose effect of preparative RP-HPLC Fraction 29 (A), 30 (B), 34 (C), and 37 (D) on MCF-7 and HMEC cells.Cell proliferation was analyzed in 96-well plate using a CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay kit.Values were graphed as Mean±SD, n=4.
Tamoxifen is a commonly used medication for breast cancer patients.In this study, only the dosage above 0.2 µM/l showed antiproliferative effects on MCF-7 cells.The effective dose reported here was higher than the value reported by Doisneau-Sixou et al. (2003).RP-HPLC Fraction 30 of kefir mother culture extract significantly increased MCF-7 cell susceptibility to tamoxifen (Figure 10).A dose dependent antiproliferative effect was also observed.Electrospray mass spectrometry was used to determine the molecular weight profile of the active Fractions 29, 30, 34, and 37. Figure 11 is the ESI-mass spectrum of HPLC fraction 30.
The spectrum represents a typical molecular ion profile of the active HPLC fractions (29, 30, 34 and 37).The similarity in the molecular ion profile of the active fractions indicated that the active component(s) is spread over HPLC Fractions 30 to 37. The spectrum showed mainly singly charged molecular ions with m/z ratios ranging from 300 to 900.The peaks below m/z 300 were found to fragments of the peaks with m/z ranging from 300 to 900.The mainly singly charged molecular ions with m/z ratios range from 300 to 900.The peaks appearing below m/z 300 were found to be fragments of the peaks with m/z ranging from 300 to 900.
To characterize the composition and chemical structure of the active components in the HPLC fractions, molecular ions ranging from m/z 300 to 900 were subjected to collision ion dissociation (CID) tandem mass spectrometry.Figure 12A, showed the CID spectrum of the molecular ion at m/z 821.Fragmentation of the molecular ion at m/z 821 produced a neutral loss of 342 mass units, which corresponds to the loss of a lactose moiety.It also produced a series of neutral loses of 60 mass units, which to the loss of acetic acid moieties.The neutral loss of 60 atomic mass units may also be derived from sugar cleavage products (Figure 13).The CID spectrum of the molecular ion at m/z 813 (Figure 12B) also produced a neutral loss of 342 (lactose) and neutral losses of 120 atomic mass units, which may be derived from fragmentation of lactose (Figure 13).A neutral loss of 162, which corresponds to the loss of a hexose moiety (glucose or galactose), is also observed.This was also observed at m/z 759.The fragmentation patterns of the molecular ions at m/z 821 and 813 indicate that they have similar structural features.The molecular ions with m/z 707 (Figure 12E) and 723 (Figure 12D) represent sodium and potassium ion dimmers of lactose, respectively.The peaks at m/z at 365 and 381 represent the sodium and potassium ions of lactose respectively (Figure 14).
Another interesting feature about the fragmentation pattern of with m/z ranging from 707 to 823 is that they fragmented into common fragment ions at m/z 261 and 98.Further isolation and fragmentation (MS-MS) of the ion at m/z 261 showed that its major product ion is the ion at m/z 98.Further isolation and fragmentation of the ion at m/z 98 produced two ions at m/z 54 and 39.The tandem mass spectrometric analysis of the molecular ion of the active HPLC fraction show that most of the peaks produced a stable product ion at m/z 98, but the ion at 98 could not be ascribed to known structure peptide.Analysis of the fragmentation pattern of the molecular ion at m/z 759 (Figure 12C), however, revealed that the molecular ion could be derived from the interaction between serine and lactose.As indicated in Figure A, fragmentation of the molecular ion at m/z 821 produced a neutral loss of 342 and 60 mass units, which corresponds to the loss of a lactose moiety and acetic acid moiety respectively.There is also a neutral loss of 98, which could not be ascribed to a known structure.Ion at m/z =261 was also observed.Fragmentation of the molecular ion at m/z 813 produced a neutral loss of 342 and 120 mass units, which might be derived from lactose and hexose moieties (glucose or galactose) respectively.Fragmentation of the molecular ion at m/z 759 produced a neutral loss of 342 and serial loss of 60 mass units.Fragmentation of the molecular ion at m/z=723 produced neutral losses of 342 and 120 atomic mass unit.The main fragment is the ion at m/z 365 with the neutral loss of 342 (lactose) from m/z 707.Fig. 13.Scheme 1: Generation of neutral loss of 60 and 120 atomic mass unit from fragmentation of lactose.Fragmentation at "a" and "b" will generate a neutral loss of 60 and fragmentation at "a" and "c" will generate a neutral loss of 120 atomic mass unit.They can be derived from fragmentation of acetic acid and lactose.A possible scheme for the generation of the ion at m/z 759 is presented in Scheme 2 (Figure 15).The proposed structure agrees with the fragmentation pattern.This interaction may be due to chemical reactions.The interaction between the amino group of serine and the reducing end of lactose is likely a chemical reaction similar to the Maillard reaction, and the interaction between the hydroxyl groups of lactose and the carboxyl group of serine is a simple esterification reaction.The reactions, particularly the esterification reaction may also be driven biochemically by esterases.The active fraction likely still contains a mixture of chemicals as suggested by the results of RP-HPLC (Figure 16).The exact molecular structure of these compounds is not known.However, results of the extensive MS analysis suggested that the compounds are possibly complex polymers that have lactose as the backbone structure.The proposed structure shown in Scheme 2 is a product with two lactose units linked by a serine.A more complex molecule with linkage with acetic acid is also possible.Fig. 15.Scheme 2: A possible interaction for the generation of the ion at m/z 759.As shown in the scheme, the interaction between the amino group of serine and the reducing end of lactose is likely a chemical reaction similar to the Maillard reaction, and the interaction between the hydroxyl groups of lactose and the carboxyl group of serine is a simple esterification reaction.The reactions, particularly the esterification reaction may also be driven biochemically by esterases.
The isolated bioactive fraction showed antiproliferative effects on cancer cells while not on normal cells.As indicated in the RP-HPLC chromatograph, the isolated fractions such as Fraction 30 are likely composed of multiple components that may be acting either singly or synergistically to exert the antiproliferative effects.Although the presence of peptides was suggested in the fractions as determined by the OPA methodology, the MS data did not support the presence of known peptide structures.The failure to identify any known peptides is likely due to the limitation of the OPA method, which can only detect the presence of primary amines and thus the primary amines in peptides cannot be differentiated from other amine containing compounds.

Conclusion
Due to the complex mixture of components in the specific fractions and the whole kefir extracts, the antiproliferative role of specific components such as proteins, peptides, organic acids and some small molecules, such as free amino acids, oligosaccharides and their interactions has been unclear.One of the main components of the bioactive antiproliferative kefir fractions is composed of lactose and acetic acid, possibly an end product of fermentation of the milk protein and sugar by the kefir bacteria and yeast.The lactose polymer suggests that the active component may be fragments of previously isolated polysaccharides termed as kefirans (Kooiman, 1968).Kefiran is a water-soluble polysaccharide consisted of approximately equal proportions of D-glucose and D-galactose (Kooiman, 1968;Micheli et al., 1999).Kefiran has been reported to have antitumor activity (Shiomi et al., 1982;Murofushi et al., 1983).It was also shown to be one of the substances having few or no side effect(s) in functioning to retard tumor growth in vivo.
It is noteworthy that one of the proposed novel bioactive structures contains serine and glucose or galactose; hence, it is conceivable that this molecule could be a sphingolipid compound.The ceramide component of sphingolipids is derived from serine and sphingolipids such as cerebrosides, which contain either a glucose or galactose.Sphingolipids such as gangliosides are sphingosine compounds that contain several glucose or galactose units.Also, lower molecular weight ceramide moieties have been detected in dairy gangliosides (Colarow et al., 2003) and cultured dairy products have been shown to be a rich source of gangliosides (Kathleen et al., 2000).Ceramide is derived from sphingomyelin (SpM) and can act as an intracellular second messenger for tumor necrosis factor-alpha, IL-1beta, and other cytokines.Ceramide has also been implicated in the acquired drug resistance that often characterizes breast cancer cells (Liu et al., 1999).C2 and C6 ceramides are cell permeable ceramide analogs that have been shown to induce cell apoptosis (Fillet et al., 2003).Osada et al. (1993) demonstrated that kefir contains an active substance, which enhances IFN-beta secretion of a human osteosarcoma line MG-63 treated with a chemical inducer, poly I: poly C. The active substance in the fermented milk was identified to be SpM.SpM from fermented milk (F-SpM) was a mixture of four molecular species of SpMs having C21-, C22-, C23-and C24-fatty acids.F-SpM enhanced the IFN secretion 14 times; SpMs from other food sources also enhanced IFN secretion but more moderately (2-3 times).We have observed a synergistic effect when MCF-7 cells were treated with ceramide analogs in the presence of extract of mother culture (data not shown).The putative sphingolipid component(s) in kefir that we have detected may have acted synergistically with the ceramide analogs leading to apoptosis of MCF-7 cells.In support of this concept, SpM has been shown to enhance the ceramide formation and ceramide-induced apoptosis in concert with the chemotherapeutic agent, gemcitabine, in human pancreatic cancer cells (Modrak et al., 2004).Modification of ceramide metabolism also increases MCF-7 cells sensitivity to cytotoxics (Lucci et al., 1999).
In summary, we have isolated the bioactive components from kefir mother culture that show antitumor cell proliferative effects.The MS data indicate that the isolated compounds are likely SpM complexes such as gangliosides that might have anti-tumor activities that are similar to that of ceramide.The biological functions of these ceramide-like compounds warrant further studies.The appearance of multiple components in the isolated bioactive antitumor fractions suggests the possibility that such components may be acting both independently and synergistically to exert the antiproliferative effects.Instead of searching for single compound in kefir, a mixture or a combination of components may be the best approach to have the highest potency for antitumor properties.The potential of using the kefir extract as co-drug for chemotherapy should be explored.In that regard, RP-HPLC Fraction 30 increased significantly the susceptibility of MCF-7 cells to tamoxifen, a commonly used anti-breast cancer drug.These results thus provide the rationale for future research to explore the potential of using kefir extracts as co-drugs for breast cancer chemotherapy and/or a functional food used for prevention of breast cancer.

Acknowledgments
This study was supported in part by grants from Liberty Brand Products, Inc. and a Natural Science and Engineering Research of Canada grant to S.K. and H.M.C.The authors thank

Fig. 2 .
Fig. 2. Antiproliferative effects of 9 SEC-HPLC fractions on MCF-7 cells.Cell proliferation was analyzed in 96-well plate using a CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit.Values were graphed as Mean±SD, n=4.

Fig. 7 .
Fig. 7. Peptide concentrations in preparative RP-HPLC fractions.One hundred fractions obtained from preparative RP-HPLC were freeze-dried and then reconstituted with nanopure water to certain concentration.Peptide concentrations were determined in triplicates by OPA method.Mean values were plotted.

Fig
Fig. 8. Antiproliferative effects of fractions of preparative RP-HPLC on MCF-7 cells.Cell proliferation was analyzed in 96-well plate using a CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay kit.Each fraction was diluted at three concentrations and was run in quadruplicate.Mean values were plotted.
Fig. 10.Antiproliferative effect of RP-HPLC Fraction 30 of kefir alone or in combination with tamoxifen on MCF-7 cells.Means±SD (n=4) with the same letter are not significantly different at P < 0.05 for comparison among treatments at the same dose level.

Fig. 11 .
Fig. 11.Mass spectrum of HPLC Fraction 30 done by electrospray mass spectrometry.The mainly singly charged molecular ions with m/z ratios range from 300 to 900.The peaks appearing below m/z 300 were found to be fragments of the peaks with m/z ranging from 300 to 900.
.intechopen.comFractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells 165

Fig. 14 .
Fig. 14.Spectrum of molecular ion at m/z 365 and 381 done by collision ion dissociation (CID) tandem mass spectrometry.The peaks at m/z at 365 and 381 represent the sodium and potassium ions of lactose respectively.
.com Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells 169

Table 3 .
Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells Protein and peptide concentrations in different MWCO fractions of extracts of kefir mother culture ( 1 Determined by using Nitrogen Determinator; 2 Determined by Bradford method (1976); 3 Determined by OPA method of Church et al. (1983); 4 Mean±SD, n=3; 5 ND: No data is available; *Significant different at P < 0.05 when compared to protein or peptide concentration in whole extract).

Table 4 .
. 1. Antiproliferative effects on MCF-7 cells induced by extracts of: (A) whole kefir mother culture; and (B) fraction of kefir mother culture obtained with MWCO less than 3000 Da.Cell proliferation was analyzed in 24-well plate.Values were graphed as Mean±SD, n=4.Estimated molecular weight, protein and peptide concentration of the nine SEC HPLC fractions, Mean±SD, n=3.