Journal of Dairy Science
Volume 93, Issue 9 , Pages 3931-3939, September 2010

Self-assembled β-lactoglobulin–conjugated linoleic acid complex for colon cancer-targeted substance

Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin 150030, China

Received 10 January 2010; accepted 28 March 2010.

Article Outline

Abstract 

β-Lactoglobulin (β-LG) is a member of the lipocalin protein family and can bind a variety of hydrophobic molecules, such as fatty acids, in vitro. In this study, a potential colon-targeted antitumor drug was developed using bovine β-LG as a carrier loaded with cis-9, trans-11 conjugated linoleic acid (CLA). The intrinsic tryptophan fluorescence intensity of β-LG monitored by spectrofluorometer showed that 2.46mol of CLA can be bound per mole of β-LG. Dynamic light scattering showed the formation of a β-LG-CLA self-assembled complex with particle size of 170±0.08nm. After treatment with gastrointestinal pH and digestive enzymes, β-LG-CLA complex showed very good stability in gastrointestinal conditions in vitro, measured by zeta potential analyzer and sodium dodecyl sulfate PAGE, respectively. In an intestinal model in vitro, the concentration of CLA in Caco-2 cells was detected by reverse-phase HPLC, and the level of CLA in cells after treatment with β-LG-CLA complex was significantly greater than after treatment with CLA, which means β-LG served as a capsular vehicle of CLA for intracellular transport. According to cell proliferation assay, β-LG-CLA complex can inhibit the viability of Caco-2 cells, and the inhibition rate is significantly greater than with the same concentration of CLA (100 μM). The study revealed that bovine β-LG as a carrier binding with CLA can potentially be used for colon cancer therapy.

Key words: beta-lactoglobulin, conjugated linoleic acid, colon cancer, targeted substance

 

Back to Article Outline

Introduction 

Bovine β-LG, with an 18.4-kDa molecular mass for the monomer and 162 AA residues, is the major whey protein in cow's milk, and its properties have been studied extensively (Pervaiz and Brew, 1985). The secondary structure of β-LG has been reported: an 8-stranded antiparallel β-barrel forms a conical central calyx, with a 3-turn α-helix on the outer surface of the β-barrel and a ninth β-strand flanking the first strand (Papiz et al., 1986; Brownlow et al., 1997). The central cavity of β-LG provides a ligand-binding site for hydrophobic molecules, and this special property makes β-LG a core member of the lipocalin family, which shows a variety of biological functions related to the binding and transport of metabolites (Flower, 1994). It has been reported that β-LG has an observable affinity for retinol (Futterman and Heller, 1972) and many fatty acids, such as palmitic acid (Wu et al., 1999; Ragona et al., 2000), n-3 polyunsaturated fatty acids (Zimet and Livney, 2009), conjugated linoleic acid (CLA), and myristic acid (Considine et al., 2007), and in cow's milk, the main ligands that have been found to bind to β-LG are fatty acids (Pérez and Calvo, 1995). Therefore, it is likely possible to make a self-assembled complex by bovine β-LG binding cis-9, trans-11 CLA (c9,t11-CLA).

Conjugated linoleic acid was originally described as an anticarcinogen isolated from grilled ground beef (Ha et al., 1987). As positional and geometric isomers of linoleic acid that have conjugated double bonds, several isomers of CLA have been identified. It has been found that ruminant dairy products are good sources of CLA isomers, especially c9,t11-CLA (Kay et al., 2004). Many publications have reported CLA isomers altering carcinogenesis, with the main focus on 2 major forms: c9,t11-CLA and trans-10, cis-12 CLA (t10,c12-CLA). However, the growth inhibitory effects of CLA isomers varied with the model used, such as mammary, colon, and stomach cancer in vitro and in vivo, and different CLA isomers act through different mechanisms of inhibiting tumor growth (Kelley et al., 2007). There are very few studies of c9,t11-CLA in inhibiting cell growth of colon, colorectal cell lines, and more studies are needed to determine a preferable concentration of CLA during antitumor treatment.

Colorectal cancer is the third most common cause of cancer-related death worldwide (Parkin et al., 2005). Finding an effective therapeutic approach for colorectal cancer is very important to inhibit the rate of recurrence. A colon-specific drug-delivery system has the potential to cure bowel disease, including colorectal cancer (Zhao et al., 2008). Because the physiological conditions along the gastrointestinal tract include various enzymes and drastic changes in pH, which make the drugs likely to be absorbed or degraded before reaching colon, a special dosage form is required for the oral colon-specific drug-delivery system to the colon. It has been reported that β-LG can resist digestive enzyme hydrolysis in the gastrointestinal tract (Reddy et al., 1998) and remain intact while reaching the upper portion of the small intestine. In addition, β-LG is stable in low-pH conditions and could play a protective role for bound ligands in the acidic conditions of the stomach (Papiz et al., 1986). Thus, to some extent, β-LG has the ability to be a coating for CLA delivery to the colon.

In the present research, we synthesized a self-assembled β-LG-CLA complex beyond the hydrophobic ligand binding property of bovine β-LG, observed its stability in gastrointestinal conditions, detected the absorption level of CLA in the colon model after treatment with β-LG-CLA complex, and measured the inhibition effect of β-LG-CLA complex on colon cancer to evaluate whether this self-assembled complex has the potential to be a colon-specific drug for antitumor application in colon carcinoma.

Back to Article Outline

Materials and Methods 

Materials 

Bovine β-LG A was purchased from Sigma Chemical Company (St. Louis, MO), and the purity was >99%. The c9,t11-CLA was purchased from Cayman (Ann Arbor, MI), and the purity was >98%. Human colon tumor cancer Coca-2 cells were obtained from Cell Bank, Chinese Academy of Science (Shanghai, China). The 24-well and 96-well plates and a 6-well transwell plate were purchased from Corning Costar Corporation (Cambridge, MA). Pepsin and trypsin; Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum for cell culture; and acetonitrile, methanol, ethyl acetate, and acetic acid for reverse-phase HPLC (RP-HPLC) were all purchased from Sigma Chemical Company.

Preparation of β-LG-CLA Complex 

The β-LG-CLA was synthesized beyond the hydrophobic ligand binding property of bovine β-LG (Wu et al., 1999; Ragona et al., 2000; Kontopidis et al., 2004) with a titration experiment for c9,t11-CLA binding to bovine β-LG. Intrinsic fluorescence of the tryptophan residues of bovine β-LG was measured before and after the addition of different amounts of CLA, which was predissolved in ethanol (10 mg/100 μL), to 1 μM bovine β-LG solution. The CLA added ranged from 2 to 12.4 μL with 2 incremental aliquots, and bovine β-LG was dissolved in 2.5mL of 100mM Tris-HCl buffer solution with a pH of 7.0. After vigorous stirring, the β-LG-CLA mixtures (molar ratio range from 1:1 to 1:3) were incubated for 10min at room temperature before the measurements. The binding parameters were studied by measuring the binding-induced quenching of the intrinsic Trp 19 fluorescence of the protein, using a fluorescence spectrofluorometer (Hitachi, Tokyo, Japan), at excitation and emission wavelengths of 279 and 332nm, respectively (Christiaens et al., 2002). Measurements were performed in triplicate. The number of CLA molecules involved in binding per β-LG molecule was calculated by fluorescence intensity, expressed as the percentage of the initial fluorescence of CLA-free β-LG versus that with the added CLA concentration. The raw data were analyzed according to the model described by Christiaens et al. (2002). After the suitable binding molar ratio between β-LG and CLA was obtained, the β-LG-CLA complex was prepared by the titration experiment mentioned earlier, and the particle size at pH 7.0 was measured by dynamic light scattering using a Delsa Nano C particle size and zeta potential analyzer (Beckman Coulter, Brea, CA). Then, β-LG-CLA complex was synthesized and stored with 0.1% Tween 40 to keep a micelle state for the following research.

Stability of β-LG-CLA in the Gastrointestinal System 

The stability characteristics of β-LG-CLA were considered by electrophoretic mobility under a wide range of physical pH conditions, and the degree of digestion with various digestive enzymes in vitro was also studied. To investigate the stability of β-LG-CLA under stomach and intestinal pH-value conditions, the complex solutions contained were titrated at pH 1.0, 1.2, 1.4, 1.8, 2.4, 2.8, and 3.0 to imitate the acidic environment of the stomach and pH 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, and 7.4 to imitate the intestinal environment. A zeta potential analyzer (Delsa Nano C, Beckman Coulter) was used under a 3-V/cm electric field at 25°C to detect the electrophoretic mobility of the complex.

To investigate the stability of β-LG-CLA in the presence of digestive enzymes, pepsin and trypsin were used as digestive enzymes. The in vitro digestion experiments were performed at 37°C with an enzyme–substrate ratio of 1:100 (wt/wt) using 0.5 mg/mL enzyme and 5 mg/mL protein solutions. The pH for pepsin digestion was titrated at 1.2 using 1 mol/L HCl, and for trypsin digestion, the pH was titrated at 8.0 using 1 mol/L NaOH. Mixtures were stirred gently during digestion, and aliquots were withdrawn at 0, 5, 10, 15, 20, 25, 30, 60, 90, and 120min. The samples were immediately frozen at −80°C for 20% SDS-PAGE analysis. The time-dependent proteolysis of β-LG-CLA complex under digestive enzymes was expressed by the intensities of protein bands in gel.

Cell Culture and In Vitro Intestinal Model Establishment 

Caco-2 cells were grown on a 24-well plate with 1.0 × 104 cells/well in the presence of DMEM containing 20% fetal bovine serum, 2 mmol/L of l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. After 72h of incubation, cells were seeded in a 6-well transwell plate with 1.0 × 105 cells/well. After 6 d, the in vitro intestinal model had been established, and the permeability of each cell monolayer was tested following the method of During and Harrison (2005). Briefly, at the beginning of the test, the apical side received 2mL of serum-free DMEM containing 100 μM CLA or β-LG-CLA kept in 0.1% Tween 40, respectively, and the basolateral side received 2mL of serum-free medium. After 4h of incubation at 37°C, the cell monolayer was washed with PBS 3 times and collected for analysis of CLA by RP-HPLC.

Cell Proliferation Assay 

The influence of β-LG-CLA complex on the viability of Caco-2 cells was determined by methyl thiazolyl tetrazolium assay. Briefly, the cells were seeded in 96-well plates with 1.0 × 105 cells/well, which, after 24h of incubation, were transferred to another culture medium containing 100 μM β-LG, 100 μM CLA, and 100 μM β-LG-CLA. Because the CLA was dissolved with ethanol, the same amount of ethanol contained in DMEM was used as an ethanol control group. Cells cultured with DMEM with 20% fetal bovine serum were used as control. For each treated group, 3 replicates were employed. The cells were separately incubated for 0, 24, 48, and 72h. At different intervals, one plate was tested. Fifteen microliters of 5 mg/mL methyl thiazolyl tetrazolium solution in PBS was added to each well and incubated for 4h; 150 μL of dimethyl sulfoxide was added to each well after removing the medium. The plates were shaken for 10min and then read at 570nm in a Bio-Rad 680 ELISA reader (Bio-Rad Laboratories, Hercules, CA). The inhibitory rate was calculated using the following equation: Inhibitory rate (%)=(ODcontrol − ODtreatment) × 100%, where OD=optical density.

RP-HPLC for CLA in Caco-2 Cells 

Absorption of CLA in the intestinal model in vitro was detected by RP-HPLC. The methods for CLA extraction from the Caco-2 cell monolayer and determination of the CLA level in cells were described by Li et al. (2001). Briefly, the separation was performed on a C18 column (0.46cm × 25cm) with a particle size of 5 μm and pore size of 300 Å (Shiseido, Tokyo, Japan) kept at 25°C; the detection wavelength was 195nm. Eluant A was 25% HPLC-grade acetonitrile and 75% water, containing 0.12% acetic acid; eluant B was HPLC-grade acetonitrile containing 0.12% acetic acid. The elution gradient was set as follows: 0∼70min changes from 100 to ∼12.5% A, 70∼80min changes from 12.5 to ∼100% A. The flow rate was 1 mL/min.

Statistical Analysis 

All data detected were analyzed by Statistix 8.0 software; P-values <0.05 were considered significant. Data were reported as mean ± standard deviation. Each value is the mean of at least 3 repetitive experiments in each group.

Back to Article Outline

Results 

Synthesis of β-LG-CLA Complex 

The β-LG-CLA complex was prepared by the titration experiment. The number of CLA binding to bovine β-LG was measured by the binding-induced quenching of intrinsic fluorescence of tryptophanyl residue Trp 19 of β-LG. The binding constant Kb was analyzed according to the model derivation used by Christiaens et al. (2002). As the concentration of CLA added in bovine β-LG solution increased, the fluorescence intensity decreased gradually (Figure 1). The analysis suggested that 2.46±1.05mol of CLA were bound per mole of β-LG; Kb=3.7 × 106 M−1. Therefore, β-LG-CLA complex was synthesized in the solution containing CLA:β-LG with a molar ratio of 2.46:1 CLA:β-LG. Particle size of β-LG-CLA complex at pH 7.0 was measured by dynamic light scattering. As shown in Figure 2, β-LG-CLA complex exhibits a narrow size distribution with an average diameter of around 171.8nm (peak value 197.7nm, polydispersity index 0.181), which means that β-LG-CLA complex is a microsized compound. After 3 parallel tests of β-LG-CLA complex for particle size, we got the final diameter result of 170.7±0.08nm.

  • View full-size image.
  • Figure 1. 

    Spectrofluorometric analysis of the binding of cis-9, trans-11 conjugated linoleic acid (CLA) to bovine β-LG at pH 7.0 and 25°C. The symbols represent experimental points of triplicate titration runs.

Stability of β-LG-CLA Complex in Gastrointestinal pH Conditions 

The stability of β-LG-CLA complex in gastrointestinal pH conditions was monitored using a zeta potential analyzer. Freshly prepared β-LG-CLA complex at pH 7.0 had a zeta potential of −47.62±0.02mV (Figure 3). To investigate the stability of β-LG-CLA under stomach and intestinal pH value conditions, NaOH and HCl were added, respectively, to shift the pH drastically to imitate the stomach pH range from 1.0 to 3.0, the distal-end of the colon pH of 6.0 to 6.4, and the proximal end of the colon pH of 7.0 to 7.4. The zeta potential of β-LG-CLA complex reduced from 37.25±2.40mV to 18.48±2.46mV as the pH increased from 1 to 3, with the point of zero net charge at pH 5.12. As the pH increased from 6.0 to 7.4, the zeta potential of β-LG-CLA complex changed into negative values from −44.01±2.26mV to −47.47±2.45mV, and when the pH increased to 8.0, the zeta potential changed to −51.41±3.40mV.

In Vitro Digestibility of β-LG-CLA Complex by Digestive Enzymes 

The stability of β-LG-CLA complex in gastrointestinal conditions was also investigated under the function of digestive enzymes and shown by SDS-PAGE (Figure 3). In the β-LG-CLA complex–pepsin mixture, there were 3 major bands in the gel (Figure 4A). The intensity of the β-LG band (Figure 4A, band b, 18.4 kDa) reduced gradually with the simultaneous appearance of the other band (Figure 3A, band c, 10 to ∼17 kDa). The intensity of band a (Figure 4A, ∼36 kDa) appeared to decrease slowly during the 2-h incubation period, but the tendency was not significant. The intensity of band c seems to have been the same from 0 to 60min but decreased significantly from 60 to 120min of incubation. In the β-LG-CLA complex–trypsin mixture, there were also 3 major bands in the gel (Figure 4B). Band b, which represents the β-LG monomer, is similar to the pattern in the gel of the β-LG-CLA complex–pepsin mixture; however, the intensity of band a (Figure 4B, ∼36 kDa) appeared to increase up in 120min of incubation. Band c (Figure 4B, 10 to ∼17 kDa) appeared and separated from band b slowly from 0 to 30min, and the intensity decreased significantly from 60 to 120min of incubation.

  • View full-size image.
  • Figure 4. 

    The SDS-PAGE patterns obtained from β-LG–conjugated linoleic acid (CLA) complex solution as a function of digestive enzymes. A) The continuous phase of β-LG-CLA complex treated by pepsin in 2h; B) the continuous phase of β-LG-CLA complex treated by trypsin in 2h. Bands a, b, and c are 3 major bands that appeared in the SDS-PAGE during gastrointestinal digestion. The molecular weight of a is about 36.8 kDa, b is about 18.4 kDa, and c is about 10 to 16 kDa. M=molecular weight marker.

Intestinal Absorption of CLA from β-LG-CLA Complex 

The Caco-2 cells were cultured to mimic the bowl model in vitro for investigating intestinal absorption of CLA from β-LG-CLA complex. Cells were incubated with 100-μM c9,t11-CLA isomer or β-LG-CLA complex loading 100-μM c9,t11-CLA isomer for 4h. The extent of absorption of c9,t11-CLA through Caco-2 cells was analyzed by RP-HPLC. To quantify c9,t11-CLA level, the correlation between the peak area and the injection quantity for c9,t11-CLA standard was obtained by linear regression. In our experiment, the linearity of the standard curves was y=3.4E+07x+1.1E+5, with a correlation coefficient (R2) value of 0.998. Compared with the peak area of 100-μM c9,t11-CLA (Figure 5A), the data (Figure 5) showed that the level of c9,t11-CLA absorbed was 45.8 μM in the cells treated by c9,t11-CLA isomer (Figure 5B) and 85.9 μM in the cells treated by β-LG-CLA complex (Figure 5C).

  • View full-size image.
  • Figure 5. 

    Chromatographic profiles of cis-9, trans-11 conjugated linoleic acid (c9,t11-CLA) from Caco-2 cells. A) The 100-μM standard c9,t11-CLA; B) the level of c9,t11-CLA absorbed in the cells treated by c9,t11-CLA isomer; C) the level of c9,t11-CLA absorbed in the cells treated by β-LG binding CLA complex. AU=arbitrary units. Color version available in the online PDF.

Antitumor Effects of β-LG-CLA Complex on Colon Cancer Cells 

The antitumor effect of β-LG-CLA complex on colon cancer cells was expressed according to Caco-2 cell viability by methyl thiazolyl tetrazolium assay. Caco-2 cell viability was significantly inhibited in a time-dependent manner by β-LG-CLA complex (Figure 6). In the present experimental conditions, ethanol involved in the culture media influenced cell viability slightly. Treatment with 100 μM c9,t11-CLA isomer showed a very low inhibitory effect on colon cancer cells within 24, 48, and 72h, and the inhibitory rate varied little. Under the treatment of bovine β-LG loading 100 μM c9,t11-CLA, the inhibition rate was 26.02±0.31% at 24h, 49.09±0.01% at 48h, and 61.89±0.02% at 72h compared with the control.

  • View full-size image.
  • Figure 6. 

    The inhibition effect of β-LG binding cis-9, trans-11 conjugated linoleic acid (β-LG-CLA) complex on Caco-2 cells up to 72h. Color version available in the online PDF.

Back to Article Outline

Discussion 

Bovine β-LG has been studied extensively over the past 60 years. Since the discovery of the retinol-β-LG complex by Futterman and Heller (1972), many methods have been used to observe the properties and structure of β-LG for binding hydrophobic ligands (Pérez and Calvo, 1995; Wu et al., 1999; Kontopidis et al., 2004; Konuma et al., 2007). As a major member of the lipocalin family, β-LG has a main ligand-binding site formed by a calyx. At least 2 other distinct binding sites exist per monomer for a variety of ligands: one is in a crevice near the α-helix on the external surface of the β-barrel (Kontopidis et al., 2004), and another possible binding site was suggested to be located at the dimer interface (Harvey et al., 2007). In our research, we measured the binding-induced quenching of the intrinsic Trp 19 fluorescence of β-LG to estimate the binding constant. The result showed that 2.46mol of c9,t11-CLA were bound per mole of β-LG, which might be due to the 2 other binding sites just mentioned (Kontopidis et al., 2004; Harvey et al., 2007) being saturated simultaneously or before c9,t11-CLA binding with Trp 19. The binding constant of β-LG-CLA complex is 3.7 × 106 M−1, which is not as high as the binding of linoleic acid (5.26 × 106 M−1; Frapin et al., 1993) but higher than 6.75 × 105 M−1 for docosahexaenoic acid (DHA) (Zimet and Livney, 2009). This might be because the smaller size and straighter structure of CLA can fit well into protein binding sites.

The β-LG-CLA complex we successfully synthesized is a micro-grade compound, with a particle size of 170.7±0.08nm. To study the stability of β-LG-CLA complex in gastrointestinal conditions, we used a zeta potential test under pH ranging from 1.0 to 8.0 and a digestive enzymes hydrolysis experiment with pepsin and trypsin. Suspensions with zeta potential values more negative than −40mV generally have good stability (ASTM, 1985). In our study, zeta potential values of β-LG-CLA complex were more negative than −40mV in colon pH conditions (pH 6.0 to ∼7.4). In gastric pH conditions (pH 1.0 to 3.0), the potential value was decreased to less positive than 40mV. At physiological pH, β-LG mostly exists as dimers; at pH below 3.5 and above 7.5, the protein dissociates into monomers (McKenzie, 1971), at which point c9,t11-CLA located at the dimer interface of β-LG would be released and influence the stability of β-LG-CLA complex in gastric conditions. However, at pH 8.0, the zeta potential value became more negative than −40mV, which might be because the structure of β-LG is still in the dimeric form at pH 8.4 (Ragona et al., 2000). It has been said that β-LG can resist peptic hydrolysis and remain stable and intact until reaching the upper portion of the small intestine (Chobert et al., 1995; Kiabatake and Kinekawa, 1998).

In our research, β-LG-CLA complex was digested by pepsin (pH 3.0) and trypsin (pH 8.0) for 2h, and the samples were withdrawn equally and adjusted to pH 7.0 before adding buffer loading to avoid abnormal migration in the SDS-PAGE. We found that β-LG-CLA complex was hydrolyzed slightly by both pepsin and trypsin. Although β-LG is a monomer (18.4 kDa) at pH 3.0 and 8.0, the SDS-PAGE showed bands at ∼36 kDa, which might be associated with some β-LG changing to dimeric form at pH 7.0. It is worth mentioning that the intensity of the band at ∼36kDa of β-LG-CLA complex treated by trypsin was increased with extended time, which was also found in the antioxidant study of bovine milk β-LG by Liu et al. (2007). Considine et al. (2007) used β-LG type B to bind with c9,t11-CLA but merely studied the influence of heat and high pressure on the stability of the complex. Our research offers a new perspective by studying the stability of β-LG-CLA complex in gastric intestinal conditions. In nuclear magnetic resonance spectroscopy research on bovine β-LG binding with palmitic acid, it was reported that the release of palmitic acid starts at pH lower than 6.0 and is nearly complete at acidic pH, which suggests β-LG cannot be used as a carrier through the alimentary tract for purposes of nutrition (Ragona et al., 2000). In the present research, β-LG-CLA complex was kept in Tween 40 to maintain a micelle state; although the stability of the complex is not very good in low pH conditions, we cannot confirm the binding extent of c9,t11-CLA when Tween 40 is present. The release dynamics of CLA from β-LG-CLA complex will be observed in a future study.

For the study of intestinal absorption of c9,t11-CLA from β-LG-CLA complex, RP-HPLC was used to show c9,t11-CLA uptake in Caco-2 cells. We found that the concentration of c9,t11-CLA in Caco-2 cells treated by β-LG-CLA complex was significantly greater than in those treated by c9,t11-CLA isomer. This could have 2 explanations. One explanation is that bovine β-LG has the ability to cross the Caco-2 cell monolayer membrane (Bernasconi et al., 2006); the other is that, as a member of the lipocalin family, bovine β-LG has specific membrane receptors for transcellular transport, such as cytoplasmic celluar retino-binding protein and human STRA6 protein (Sundaram et al., 1999; Kawaguchi et al., 2007). Although vitamin-A-binding β-LG is a classic model for the character study of lipocalin family members, whether the membrane receptors can recognize other hydrophobic ligands still needs to be demonstrated.

High incidence rate of colorectal cancer is most likely due to high dietary intake of red or processed meat (Center et al., 2009). It is noteworthy that CLA, a mixture of positional and geometric isomers of LA found mainly in dairy and meats (Parodi, 1997), has been extensively studied both in vivo and in vitro as a possible anticarcinogen. In ruminant meat and dairy products, c9,t11-CLA is the major component of CLA isomers; thus, there have been recent assessments of whether c9,t11-CLA is more potent than other isomers for commercial CLA preparations. It has been reported that c9,t11-CLA is more potent than t10,c12-CLA in inhibiting the growth of colon cancer cell lines (Palombo et al., 2002). In our study, 100-μM CLA binding β-LG had the ability to inhibit Caco-2 cell growth in a time-dependent manner, but the 100-μM CLA isomer had very little inhibitory effect on cell proliferation. This can be explained by the fact that culture conditions including the concentration of CLA, duration of the treatment, and the cell lines used seem to determine whether CLA isomers will affect cell growth. Yamasaki et al. (2002) reported that CLA at 10 μM did not affect cell growth in the presence of 5 or 10% fetal bovine serum; fatty acids can form a complex with bovine albumin (Beppu et al., 2006). The present study demonstrated the greater inhibitory effect of β-LG-CLA complex compared with c9,t11-CLA isomer on the growth of human colon cancer cells.

Back to Article Outline

Conclusions 

In the present study, a microsized β-LG-CLA self-assembled complex, with mean particle size of 170.07±0.08nm, was synthesized. The binding constant of c9,t11-CLA to bovine β-LG was also presented as Kb=3.7 × 106 M−1. The complex stored in Tween 40 showed very good stability at intestinal pH conditions and was hydrolyzed very little by digestive enzymes. However, the stability of β-LG-CLA complex was not good in gastric pH conditions. The release dynamics of CLA from β-LG-CLA complex in gastrointestinal conditions needs to be studied and will be observed in a future study. With the in vitro intestinal model with Caco-2 monomer in this study, we found that the extent of absorption of c9,t11-CLA in Caco-2 cells was increased, which might be related to the lipocalin character of β-LG. The inhibition rate in colon cells is much greater with β-LG-CLA complex than with CLA isomers. More aspects still need to be observed to certify β-LG-CLA complex as a potential colon-targeted antitumor substance.

Back to Article Outline

Acknowledgment 

This work was supported by the National High Technology Research and Development Program of China (863 program), No. 2008AA10Z331.

Back to Article Outline

References 

  1. ASTM (American Society for Testing and Materials).. Standard test methods for zeta potential of colloids in water and waste water. ASTM Standard D 4182–82. Annual Book of ASTM Standards. West Conshohocken, PA: ASTM International; 1985;
  2. Beppu F, Hosokawa M, Tanaka L, Kohno H, Tanaka T, Miyashita K. Potent inhibitory effect of trans9, trans11 isomer of conjugated linoleic acid on the growth of human colon cancer cells. J. Nutr. Biochem. 2006;17:830–836
  3. Bernasconi E, Fritsché R, Corthésy B. Specific effects of denaturation, hydrolysis and exposure to Lactococcus lactis on bovine β-lactoglobulin transepithelial transport, antigenicity and allergenicity. Clin. Exp. Allergy. 2006;36:803–814
  4. Brownlow S, Cabral JHM, Cooper R, Flower DR, Yewdall SJ, Polikarpov I, et al. Bovine β-lactoglobulin at 1.8 Å resolution—Still an enigmatic lipocalin. Structure. 1997;5:481–495
  5. Center MM, Jemal A, Smith RA, Ward E. Worldwide variations in colorectal cancer. CA Cancer J. Clin. 2009;59:366–378
  6. Chobert JM, Briand L, Grinberg V, Haertlé T. Impact of esterification on the folding and the susceptibility to peptic proteolysis of β-lactoglobulin. Biochim. Biophys. Acta. 1995;1248:170–176
  7. Christiaens B, Symoens S, Vanderheyden S, Engelborghs Y, Joliot A, Prochiantz A. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. Eur. J. Biochem. 2002;269:2918–2926
  8. Considine T, Patel HA, Singh H, Creamer LK. Influence of binding conjugated linoleic acid and myristic acid on the heat- and high-pressure-induced unfolding and aggregation of β-lactoglobulin B. Food Chem. 2007;102:1270–1280
  9. During A, Harrison EH. An in vitro model to study the intestinal absorption of carotenoids. Food Res. Int. 2005;38:1001–1008
  10. Flower DR. The lipocalin protein family: A role in cell regulation. FEBS Lett. 1994;354:7–11
  11. Frapin D, Dufour E, Haertle T. Probing the fatty-acid-binding site of beta-lactoglobulins. J. Protein Chem. 1993;12:443–449
  12. Futterman S, Heller J. The enhancement of fluorescence and the decreased susceptibility to enzymatic oxidation of retinol complexed with bovine serum albumin, lactoglobulin and the retinol-binding protein of human plasma. J. Biol. Chem. 1972;247:5168–5172
  13. Ha YL, Grimm NK, Pariza MW. Anticarcinogens from fried ground beef: Heat-altered derivatives of linoleic acid. Carcinogenesis. 1987;8:1881–1887
  14. Harvey BJ, Bell E, Brancaleon L. A tryptophan rotamer located in a polar environment probes pH-dependent conformational changes in bovine β-lactoglobulin A. J. Phys. Chem. B. 2007;111:2610–2620
  15. Kawaguchi R, Yu J, Honda J, Hu J, Whitelegge J, Ping P, et al. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science. 2007;315:820–825
  16. Kay JK, Macle TR, Auldist MJ, Thomson NA, Bauman DE. Endogenous synthesis of cis-9, trans-11 conjugated linoleic acid in dairy cows fed fresh pasture. J. Dairy Sci. 2004;87:369–378
  17. Kelley NS, Hubbard NE, Erickson KL. Conjugated linoleic acid isomers and cancer. J. Nutr. 2007;137:2599–2607
  18. Kiabatake N, Kinekawa YI. Digestibility of bovine milk whey protein and β-lactoglobulin in vitro and in vivo. J. Agric. Food Chem. 1998;46:4917–4923
  19. Kontopidis G, Holt C, Sawyer L. β-Lactoglobulin: Binding properties, structure and function. J. Dairy Sci. 2004;87:785–796
  20. Konuma T, Sakurai K, Goto Y. Promiscuous binding of ligands by β-lactoglobulin involves hydrophobic interactions and plasticity. J. Mol. Biol. 2007;368:209–218
  21. Li Z, Gu T, Kelder B, Kopchick JJ. Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography. Chromatography A. 2001;54:463–467
  22. Liu HC, Chen WL, Mao SJT. Antioxidant nature of bovine milk β-lactoglobulin. J. Dairy Sci. 2007;90:547–555
  23. McKenzie HA. β-Lactoglobulins. In:  McKenzie HA editors. Milk Proteins. London, UK.: Academic Press Inc. Ltd.; 1971;p. 2257–2330
  24. Palombo JD, Ganguly A, Birstrian BR, Menard MP. The antiproliferative effects of biologically active isomers of conjugated linoleic acid on human colorectal and prostatic cancer cells. Cancer Lett. 2002;177:163–172
  25. Papiz MZ, Sawyer L, Eliopoulos EE, North ACT, Findlay JBC, Sivaprasadarao R. The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein. Nature. 1986;324:383–385
  26. Parkin DM, Bray FJ, Pisani P. Global cancer statistics. 2002. CA Cancer J. Clin. 2005;55:74–108
  27. Parodi PW. Cow's milk fat components as potential anticarcinogenic agents. J. Nutr. 1997;127:1055–1060
  28. Pérez MD, Calvo M. Interaction of β-lactoglobulin with retinol and fatty acids and its role as a possible biological function for this protein: A review. J. Dairy Sci. 1995;78:987–988
  29. Pervaiz S, Brew K. Homology of β-lactoglobulin, serum retinol-binding protein and protein HC. Science. 1985;228:335–337
  30. Ragona L, Fogolari F, Zetta L, Pérez DM, Puyol P, Kruif KD, et al. Bovine β-lactoglobulin: Interaction studies with palmitic acid. Protein Sci. 2000;9:1347–1356
  31. Reddy M, Kella NKD, Kinsella JE. Structural and conformational basis of the resistance of β-lactoglobulin to peptic and chymotryptic digestion. J. Agric. Food Chem. 1998;36:737–741
  32. Sundaram M, Sivaprasadarao A, DeSousa MM, Findlay JBC. The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. J. Biol. Chem. 1999;273:3336–3342
  33. Wu SY, Pérez MD, Puyol P, Sawyer L. β-lactoglobulin binds palmitate within its central cavity. J. Biol. Chem. 1999;274:170–174
  34. Yamasaki M, Chujo H, Koga Y, Oishi A, Rikimaru T, Shimada M, et al. Potent cytotoxic effect of the trans10, cis12 isomer of conjugated linoleic acid on rat hepatoma dRLh-84 cells. Cancer Lett. 2002;188:171–180
  35. Zhao XL, Li KX, Zhao XF, Chen DW. Study on colon-specific 5-Fu pH-enzyme Di-dependent chitosan microspheres. Chem. Pharm. Bull. (Tokyo). 2008;56:963–968
  36. Zimet P, Livney YD. Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for ω-3 polyunsaturated fatty acids. Food Hydrocoll. 2009;23:1120–1126

PII: S0022-0302(10)00417-0

doi:10.3168/jds.2010-3071

Journal of Dairy Science
Volume 93, Issue 9 , Pages 3931-3939, September 2010

Article Tools

* Image Usage & Resolution