Study Of Different N-6/n-3 Ratios On The Human Breast Cancer Cells And Relating Biomembrane Mechanism

Breast cancer is one of the most common malignancy which severely threatens the health of females and how to prevent breast cancer, especially through the dietary intake, has been a matter of concern in global nutrition academia. Epidemiological investigations and empirical studies have revealed the extremely close interrelationship between the dietary intake of polyunsaturated fatty acid (PUFA) and breast cancer that different types of PUFA played varied roles in the tumorigenesis and progression of breast cancer. There are two types of natural PUFA, namely, n-6 PUFA and n-3 PUFA. Previous studies demonstrated that pure n-6 PUFA could remarkably promote the tumorigenesis and progression of breast cancer, whereas pure n-3 PUFA presented anti-cancer effects. However, the mechanism of different effects of n-3 and n-6 PUFAs still remains unestablished. Recent studies indicate that the ratio of n-6 PUFA to n-3 PUFA rather than absolute concentration is more important to the development of breast cancer, nevertheless, it is still unclear to determine an rational ratio of n-6/n-3 PUFA in daily diet. Moreover, several studies indicate that large dose intake of n-3 PUFA can cause damage to human immune system , furthermore, n-3 PUFA intake can not exert effective anti-cancer effects merely. Therefore, it is of theoretical and practical significance to investigate the effects of fatty acids especially the optimal proportion of n-6 to n-3 PUFA on breast cancer for more effective prevention through the dietary intervention.PUFA is a vital component of lipid conformation and is critical for structural and functional maintenance and stabilization of cellular biomembrane. It has been proved that both n-6 PUFA and n-3 PUFA have specific high-affinity with biomembrane lipid, so they can infiltrate into the cellular membrane directly and consequently alter the composition of membrane lipid leading to different impacts on physical and biological characteristics and various biological behaviour of cells eventually. Therefore, it is rational to speculate that different ratios of n-6/n-3 PUFA may bring about variant effects on membrane lipid composition, membrane fluidity and the expression of critical transmembrane signal proteins. Furthermore, it may affect the proliferation, apoptosis and migration of breast cancer cells and ultimately show varied biological effects of cancer promotion or cancer inhibition.Based on the analysis above, combined with the characteristics of daily prandium of Chinese inhabitants, we took human breast cancer MCF-7 (positive estrogen receptor, ER+) and MDA-MB-231 (negative estrogen receptor, ER-) cells as experimental objects in our study, applied several experimental techniques such as MTT assay, DAPI staining, TUNEL, RT-PCR, Western blotting, gas chromatography (GC), fluorescence recovery after photobleaching (FRAP) and confocal laser scanning microscope to observe and compare the effects of different constituent ratios of n-6 and n-3 PUFA on proliferation and apoptosis of these two cell lines, including pure n-6 PUFA, pure n-3 PUFA, 1:1 n-6/n-3 PUFA, 5:1 n-6/n-3 PUFA, 10:1 n-6/n-3 PUFA. We also focused on exploring the biological effects and possible mechanism by detecting the effects of different ratios of n-6/n-3 PUFA on composition of breast cancer cell membrane lipid, membrane fluidity and the expression of insulin-like growth factor 1 receptor (IGF-1R).1. Cellular growth curve and MTT assay showed that cell proliferation of MCF-7 cells treated with pure n-6 PUFA, 5:1 n-6/n-3 PUFA and 10:1 n-6/n-3 PUFA were significantly higher than the control group in which pure n-6 PUFA group was the highest, followed by 10:1 n-6/n-3 PUFA group, suggesting that n-6/n-3 PUFA at or above 5:1 can significantly promote the proliferation of MCF-7 cells and the promotive effect was enhanced with the increase of n-6/n-3 PUFA ratio. On the contrary, the viability of MCF-7 cells were obviously suppressed 24 h after pure n-3 or 1:1 n-6/n-3 PUFA treatment, indicating that both pure n-3 and 1:1 n-6/n-3 PUFA can inhibit the proliferation of MCF-7 cells, but there was no significant difference between these two groups. The situation in MDA-MB-231 cells was similar to that of MCF-7 cells, yet 5:1 n-6/n-3 PUFA treatment can not promote the proliferation of MDA-MB-231 cells significantly, suggesting that at least more than 5:1 n-6/n-3 PUFA was required for cell proliferation promoting. Furthermore, the proliferation of cells treated with 1:1 n-6/n-3 PUFA was significantly higher than that of pure n-3 group, indicating that the inhibitory effect of pure n-3 on MDA-MB-231 cell viability was stronger than that of 1:1 n-6/n-3 PUFA. 2. Morphological observation showed that normal MCF-7 cells formed typical cobblestone appearance, compared to MDA-MB-231 cells with a typical fibroblastic, flattened, spindly appearance. There were not apparent differences between the controls and the pure n-6 PUFA, 5:1 n-6/n-3 PUFA and 10:1 n-6/n-3 PUFA treatment groups in either cell line. However, dramatic morphological changes including cell growth disorder and paramorphia were observed in pure n-3 and 1:1 n-6/n-3 PUFA treatment groups. Morphological characteristic of apoptosis cells including cell shrinkage, cytoplasmic condensation, cell detachment and suspension were observed in MCF-7 cells.3. The results of DAPI staining and TUNEL assay showed that the apoptosis of MCF-7 and MDA-MB-231 cells were significantly increased after pure n-3 PUFA or 1:1 n-6/n-3 PUFA treatment for 24h compared with control, and there was not significant difference between these two groups. On the other hand, cell apoptosis were not distinctly changed in pure n-6 PUFA, 5:1 n-6/n-3 and 10:1 n-6/n-3 groups in these two cell lines. These results suggested that both pure n-3 PUFA and 1:1 n-6/n-3 PUFA could effectively induce apoptosis of breast cancer cells.4. Gas chromatographic analysis showed that different n-6/n-3 PUFA ratio treatments markedly altered the fatty acid compositions of phospholipids (PE, PC, PS, PI) in breast cancer cells. An increased concentration of C20∶5 along with a decreased n-6/n-3 proportion were found in pure n-3 PUFA and 1:1 n-6/n-3 PUFA treatment groups. The higher levels of C18∶2 and C20∶4 along with an increased n-6/n-3 proportion were found in n-6 PUFA and 10:1 n-6/n-3 PUFA treatment groups. Our results also revealed that the concentration of C22∶6 in PE and PC of the breast cancer cells was significantly increased in n-3 PUFA and 1:1 n-6/n-3 PUFA treatment groups, indicating a transformation from EPA to DHA.5. Results of the laser confocal scanning microscopy and fluorescence recovery after photobleaching using NBD-C6 as membrane phospholipid fluorescent probe showed that the membrane fluidity of breast cancer cells was significantly increased after pure n-6, pure n-3 or 1:1 n-6/n-3 PUFA treatment for 2 h, and then declined 12 h later. However, the membrane fluidity mobility in pure n-6 PUFA group was still higher compared with control group whereas pure n-3 and 1:1 n-6/n-3 PUFA showed the opposite effects. The results indicated that pure n-3 and 1:1 n-6/n-3 PUFA treatments could cause significant fluctuation of the membrane phospholipids mobility in breast cancer cells .6. The results of RT-PCR and western blotting showed that different n-6/n-3 PUFA treatments had different effects on the expression of IGF-1R in breast cancer cells. Pure n-6 , 5∶1 and 10∶1 n-6/n-3 PUFA obviously up-regulated the expression of IGF-1R at both mRNA and protein level, while pure n-3 and 1:1 n-6/n-3 PUFA greatly inhibited the IGF-1R expression.To conclude, our findings suggested that different ratios of n-6/n-3 PUFA had different effects on breast cancer cells. The n-6/n-3 PUFA ratio above 5:1 can significantly promote the tumorigenesis and development of breast cancer, while n-6/n-3 PUFA ratio below 1:1 n-6/n-3 PUFA can significantly inhibit the oncogenesis of breast cancer, including proliferation inhibition and apoptosis induced effect. The mechanism could be elucidated by the changes of membrane biophysical properties, membrane function and cellular signal transduction due to the different ratios of n-6 PUFA to n-3 PUFA in breast cancer cells.