Study of Different n-6/n-3 Ratios on the Human Breast Cancer Cells and Relating Biomembrane Mechanism
|School||Third Military Medical University|
|Course||Nutrition and Food Hygiene|
|Keywords||Different n-6/n-3 PUFA constitute Human breast cancer cells MCF-7 MDA - MB- 231 Proliferation Apoptosis Morphology Cell membrane Phospholipid fatty acid composition Membrane fluidity Insulin-like growth factor receptor-1|
Breast cancer is serious harm to a common malignancy in women's health, and effective prevention and treatment of breast cancer, especially breast cancer prevention through dietary means, has become a hot topic in nutrition circles at home and abroad. A large number of epidemiological investigations and experimental studies have proven that dietary polyunsaturated fatty acids (polyunsaturated fatty acid, PUFA) very close relationship with breast cancer, and the different types of PUFA on the incidence of breast cancer development have different or even opposite the effect. PUFA naturally occurring in nature can be divided into two categories, namely n-3 PUFA and n-6 ??PUFA. N-6 PUFA can promote the development of breast cancer, with a significant cancer-promoting effects of n-3 PUFA can suppress the occurrence of breast cancer, has significant anti-cancer effects of differences in both the role of the mechanism has yet unclear. Recent studies have shown that n-6/n-3 PUFA ratio in the diet, rather than a single concentration of these fatty acids, has a greater significance on the development of breast cancer, but it is not clear what proportion of dietary n-6/n-3 PUFA intake in order to better prevent and suppress the occurrence and development of breast cancer. Another study found that large doses of n-3 PUFA intake can damage the body's immune system functions, and simple n-3 PUFA effect of prevention and treatment of breast cancer is not satisfactory. Therefore, in-depth study of dietary fatty acids, especially different n-6/n-3 PUFA composition with breast cancer occurrence and development of its molecular mechanism explore what proportion of n-6/n-3 PUFA intake can more effectively to prevent and suppress the occurrence of breast cancer, the development of great significance, prevention and treatment of breast cancer from dietary means has a certain practical significance. PUFA composition constituting the biofilm lipid structure is extremely important for the maintenance of the structure and function of the biofilm and vital to the stability. Studies have shown that n-6 and n-3 PUFA on membrane lipid has a very high affinity to direct penetration embedded in the cell membrane, participate in and change the composition of the membrane lipids, lipid composition change is bound to have a biofilm physical characteristics and functional properties of impact, thereby affecting the cells of various life activities. Therefore, we hypothesized that different ratios of n-6/n-3 PUFA role in breast cancer cells, breast cancer cell membrane lipid composition, membrane fluidity and key transmembrane signaling protein activity and expression of a difference, and thus impact on breast cancer cell proliferation, apoptosis and migration, and ultimately exhibit different biological effects of tumor promoters or anti-cancer. Based on the above analysis, combined with China's current residents' daily diet features subject to different constituent ratio of n-6/n-3 PUFA that simple n-6 PUFA, n-3 PUFA alone, 1:1 n-6 / n-3 PUFA, 5:1 n-6/n-3 PUFA, 10:1 n-6/n-3 PUFA role in estrogen receptor-positive (positive estrogen receptor, ER) MCF-7 human breast cancer cell lines and ER-negative (ER-) MDA-MB-231 human breast cancer cell lines by MTT proliferation assay, DAPI staining, TUNEL, RT-PCR, Western blotting, gas chromatography (GC), fluorescence recovery after photobleaching (fluorescence recovery after photobleaching, FRAP) and laser confocal scanning microscope experiments, observe and compare different the n-6/n-3 PUFA constitutes a two breast cancer cell proliferation and apoptosis, and breast cancer cell membrane phospholipid fatty acid composition, membrane fluidity and membrane signaling protein insulin-like growth factor receptor -1 (insulin-like growth factor -1 receptor, IGF-1R) expression system to explore different the n-6/n-3 PUFA constitutes a human breast cancer cells biological effects and possible mechanisms of action. In this study, experiments and results are as follows (1) Cell growth curve and MTT assay showed that pure n-6 PUFA, the 5:1 n-6/n-3 PUFAs and 10:1 n-6/n-3 the PUFA group of MCF- 7 cells proliferation rate was significantly higher than that in the control group, in which the N-6 PUFA group cell proliferation fastest, followed by a 10:1 n-6/n-3 PUFA group, indicating that the 5:1 n-6/n-3 PUFA can significantly promote the proliferation of MCF-7 cells, and this effect with the n-6/n-3 ratio was enhanced; contrast, pure n-3 PUFA and 1:1 n-6/n-3 PUFA Processing cells 24 h visible MCF-7 cells a large number of deaths, the amount of living cells was significantly less than before the treatment and control group for 48 h, the number of viable cells after an increase in but proliferation moderate, indicating that the simple n-3 PUFA and 1:1 n -6/n-3 PUFA could significantly inhibit the proliferation of MCF-7 cells, and the inhibitory effect was no significant difference between the two. Substantially similar situation of MDA-MB-231 cells and MCF-7 cells, suggesting that at least higher than the 5:1 ratio of n-6/n-3 PUFA can significantly promote the proliferation of MDA-MB-231 cells; Moreover 1:1 n-6 / N-3 PUFA group of cell proliferation rate slightly higher than the pure n-3 PUFA group, indicating that the simple n-3 PUFA and 1:1 n-6/n-3 PUFA could significantly inhibit the proliferation of MDA-MB-231 cells, but the former inhibitory effect was stronger than the latter. Cell morphology observed visible, normal MCF-7 cells were typical cobblestone-like, and MDA-MB-231 cells were spindle-shaped, adherent growth gathered into Austin; simple n-6 PUFA, 5:1 n-6 / n -3 PUFA and 10:1 n-6/n-3 PUFA group cell morphology compared with the control group did not change significantly; simple n-3 PUFA group and 1:1 n-6/n-3 the PUFA group of morphogenesis significant changes in cell growth disorders, abnormal shape, a large number of cell shrinkage round, cytoplasmic condensation, and its surrounding cell separation, cell adhesion decreased, showing the morphological characteristics of apoptotic cells. DAPI staining and TUNEL Apoptosis Detection results show that, compared with control group, the simple n-3 PUFA and 1:1 n-6/n-3 PUFA treatment of MCF-7 and MDA-MB-231 cells 24 h after cell apoptosis rate was significantly higher (P lt; 0.05), and no significant difference between the two; simple n-6 PUFA group, the 5:1 n-6/n-3 PUFA group and 10:1 n-6 / n-3 PUFA group cell apoptosis showed that n-3 PUFA and 1:1 n-6/n-3 PUFA can significantly induced breast cancer MCF-7 and MDA-MB-231 cell apoptosis. 4 gas chromatographic analysis showed that, basically consistent the n-6/n-3 PUFA different composition than the two breast cancer cell membrane four main phospholipids PE, PC, PS and PI fatty acid composition of, pure n-3 PUFA and 1:1 n-6/n-3PUFA treatment groups can be significantly increased two phospholipids of cell membranes four percentage content of C20: 5, to reduce the proportion of each phospholipid n-6/n-3; pure n-6 PUFA and 10: C18: 2 and C20: 4 percentage content of 1 n-6/n-3 PUFA treatment group two types of cells can be significantly increased four phospholipids, and thereby increase the n-6/n-3 ratio of the phospholipid, and four phospholipids, PE and PC, the most obvious change; can also be observed in the n-3 PUFA and 1:1 the n-6/n-3 PUFA treatment group, a significant increase to PE and PC in the membrane phospholipid content of C22: 6, suggests that the conversion of EPA to DHA. The above indicates that different n-6/n-3 PUFA can significantly change the MCF-7 and MDA-MB-231 breast cancer cell membrane phospholipid fatty acid composition as a percentage. The results showed that the fluorescent probe NBD-C6 tag membrane phospholipids, fluorescence recovery after photobleaching (FRAP) and laser confocal scanning microscope detection membrane fluidity, pure n-6 PUFA, n-3 PUFA and simple: 1 n-6/n-3 PUFA treatment of MCF-7 cells and MDA-MB-231 cells 2 h, membrane fluidity was significantly higher (P lt; 0.05), performance is the diffusion coefficient significantly increased; processing 12 h two membrane fluidity lower than those of 2 h, pure n-6 group of membrane fluidity was still significantly higher than that in control group (P lt; 0.05), and pure n-3 PUFA and 1:1 n-6/n-3 PUFA group of membrane fluidity was significantly lower than the control group (P lt; 0.05); show that n-3 PUFA and 1:1 n-6/n-3 PUFA breast cancer cell membrane fluidity can fluctuate, first make it rise , and then reduced. RT-PCR and Western blotting analysis showed that, of different n-6/n-3 PUFA ratio of MCF-7 and MDA-MB-231 breast cancer cells IGF-1R mRNA and protein expression have different effects, simple n-6 PUFA, 5:1 n-6/n-3 PUFA and 10:1 n-6/n-3 PUFA were significantly up-regulated the expression of two breast cancer cell IGF-1R mRNA and protein levels, contrast In contrast, the n-3 PUFA and 1:1 n-6/n-3 PUFA alone significantly down-regulated cells IGF-1R mRNA and protein expression. In summary, this study showed different n-6/n-3 PUFA form of breast cancer cells is different. the n-6/n-3 PUFA constituent ratio higher than 5:1 with the effect of significantly promote the proliferation of breast cancer cells, and the n-6/n-3 PUFA constitute less than 1:1 with anti-breast cancer cell proliferation induced apoptosis. This may be related to two polyunsaturated fatty acids differences in breast cancer cell membrane lipid composition changes, which affect membrane biophysical properties of the membrane fluidity and membrane function, as well as some of the key signaling protein activity and expression is closely related.