Goat and buffalo milk fat globule membranes exhibit better effects at inducing apoptosis and reduction the viability of HT-29 cells

Abstract

Bovine milk fat globule membrane (MFGM) has shown many health benefits, however, there has not been much study on non-cattle MFGMs. The purpose of this study was to compare the anti-proliferation effects and investigate the mechanisms of MFGMs from bovine, goat, buffalo, yak and camel milk in HT-29 cells. Results showed that protein content in MFGM of yak milk is the highest among five MFGM. All MFGMs reduced cellular viability which was in agreement with cell morphology and apoptosis. However, the number of cells in S-phase from 24 h to 72 h was increased significantly by treatment with goat, buffalo and bovine MFGMs (100 μg/mL), but not yak and camel. All MFGMs treatment significantly reduced the mitochondrial membrane potential (with an order of goat > buffalo > bovine > camel > yak) and Bcl-2 expression, but increased the expression of both Bax and Caspase-3. Taken together, the results indicate that all MFGMs, especially goat and buffalo MFGMs, showed better effects at inducing apoptosis and reduction the viability of HT-29 cells. The mechanism might be arresting the cell cycle at S phase, depolarization of mitochondrial membrane potential, down-regulation of Bcl-2 expression and increase of Bax and Caspase-3 expression.

Figure 1

SDS-PAGE of MFGM proteins from bovine, goat, buffalo, yak and camel milk. The main protein bands identified were matched with previous report.

Figure 2

Effect of five MFGMs on HT-29 cell viability by MTT assay. HT-29 cells were treated with each of five MFGMs (1–100 μg/mL), positive control (10 μg/mL, camptothecin) for 24, 48 and 72 h respectively. Data values are expressed as mean ± SD of triplicate determinations. In the same time point and the same concentration, there are significant differences between any two bars labelled with different letters (a–e) (p < 0.05).

Figure 3

The effect of MFGMs on cell cycle phase distribution of HT-29 cell. Cells were treated with MFGMs (100 μg/mL) for 24, 48, and 72 h respectively. Then the cells were fixed and stained with Propidium Iodide (PI), and the cell cycle was analysed by flow cytometry. Data values are expressed as mean ± SD of triplicate determinations. In the same cellular cycle phase, there are significant differences between any two data sets labelled with different letters (a–d) (p < 0.05).

Figure 4

Cytomorphology of HT-29 cells treated with five MFGMs (100 μg/mL) for 72 h. The magnification is ×200. HT-29 cells not treated were used as control cells. Cells with shrinkage () and lost contact between adjacent cells ().

Figure 5

Cytomorphology of HT-29 cells treated with five MFGMs (100 μg/mL) for 72 h. Dead cells including shrinkage and lost contact cells were counted in five view fields. The magnification is ×200. The death rate was calculated by the death/all cells × 100%. There are significant differences between any two bar groups labelled with different letters (a–d) (p < 0.01).

Figure 6

Fluorescence microscopic images of staining annexin V-FITC/PI for apoptotic morphology of HT-29 cells treated with five MFGMs (100 μg/mL) for 72 h. Normal HT-29 cells were used as control cells. Cells stained with V-FITC turn green and indicate they are in the earlier apoptotic phase. While cells stained with both V-FITC (which stains the cell membrane green) and PI (which stains the nuclei red) turn orange, this indicates the cells are at later stages of apoptotic phase. Cells stained with red are only labelled with PI and indicate the dead cells. The magnification is ×200.

Figure 7

Cell number of apoptotic or dead cells of HT-29 cells treated with five MFGMs (100 μg/mL) for 72 h. There are significant differences between any two bars labelled with different letters (a–f) (p < 0.05).

Figure 8

Nuclear changes of apoptotic HT-29 cells treated with MFGM (100 µg/mL) for 72 h. HT-29 cells without given treatment were used as control cells. Images of TEM, The magnifications is ×2500. Cell membrane (); nuclei (); cytoplasm ().

Figure 9

Effect of MFGMs on apoptosis by flow cytometry. HT-29 cells without given treatment (Control group) or treated with each of five MFGMs (100 µg/mL) were labelled with annexin-V- FITC (V-FITC) and PI.

Figure 10

The early apoptotic rate of HT-29 cells induced by MFGMs was analysed with the flow cytometer. Data values are expressed as mean ± SD of triplicate determinations. There are significant differences between any two bars labelled with different letters (a–c) (p < 0.05).

Figure 11

The mitochondrial content was labelled with a mitochondrial green fluorescent probe-Mito-Tracker Green in HT-29 cells treated with MFGMs (100 μg/mL) for 72 h and visualized with a fluorescence microscope.

Figure 12

Cells labelled with green are analysed. Data values are expressed as mean ± SD. Between any two bars labelled with different letters, p < 0.05.

Figure 13

Effect of each of five MFGMs on the expressions of Caspase-3 (19 kDa and 17 kD), Bax (21 kDa), and Bcl-2 (26 kDa) were analysed through Western blot assay.

Figure 14

Effect of each of five MFGMs on the expressions of Caspase-3, Bax, and Bcl-2 was calculated by the comparison to β-Actin (42 kDa) (Left). The ratio of Bax/Bcl-2 was calculated as apoptosis index (Right). Data values are expressed as mean ± SD of triplicate determinations. *p < 0.01 as compared with control group.