摘要
乳腺癌是严重危害妇女健康的一种常见恶性肿瘤,如何有效防治乳腺癌,特别是通过膳食途径防治乳腺癌,目前已成为国内外营养学界一热门话题。大量流行病学调查和实验研究证实,膳食中的多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)与乳腺癌的关系极为密切,且不同种类的PUFA对乳腺癌的发生、发展具有不同甚至完全相反的作用。自然界中天然存在的PUFA可分为两大类,即n-3 PUFA和n-6 PUFA。研究发现,n-6 PUFA可以促进乳腺癌的发生发展,具有显著促癌效应,而n-3 PUFA则能够抑制乳腺癌的发生,具有显著抗癌效应,关于两者作用差异性的机制至今尚不清楚。近来有研究表明,膳食中的n-6/n-3 PUFA比例,而非这些脂肪酸的单一浓度,对乳腺癌的发生发展具有更为重要的意义,但目前也不清楚膳食中何种比例的n-6/n-3 PUFA摄入才能更好地预防和抑制乳腺癌的发生与发展。另有研究发现,大剂量n-3 PUFA的摄入会损害人体的免疫系统功能,且单纯n-3 PUFA对防治乳腺癌的效果也不理想。因此,深入研究膳食脂肪酸,特别是不同n-6/n-3 PUFA构成与乳腺癌发生发展的关系及其分子机制,探讨何种比例的n-6/n-3 PUFA摄入才能更有效地预防和抑制乳腺癌的发生、发展具有重要意义,同时这对从膳食途径防治乳腺癌也具有一定的现实意义。
PUFA是构成生物膜脂质结构极为重要的组成成分,对于生物膜结构与功能的维护和稳定至关重要。研究显示,n-6及n-3 PUFA对细胞膜脂均具有极高的亲和性,能够直接渗透嵌入细胞膜,参与并改变膜脂的构成,而膜脂组成的改变势必会对生物膜的物理特性和功能特性产生影响,进而影响到细胞的种种生命活动。因此,我们推测,不同比例的n-6/n-3 PUFA作用于乳腺癌细胞,能够对乳腺癌细胞膜脂组成、膜流动性以及关键跨膜信号蛋白的活性和表达等产生差异性影响,进而影响到乳腺癌细胞的增殖、凋亡和迁移等,最终表现出促癌或抗癌的不同生物学效应。
基于以上分析,并结合我国现阶段居民日常膳食特点,本课题以不同构成比的n-6/n-3 PUFA,即单纯n-6 PUFA、单纯n-3 PUFA、1:1 n-6/n-3 PUFA、5:1 n-6/n-3 PUFA、10:1 n-6/n-3 PUFA分别作用于雌激素受体阳性(positive estrogen receptor ,ER+)的MCF-7人乳腺癌细胞系和ER阴性(ER-)的MDA-MB-231人乳腺癌细胞系,采用MTT增殖检测、DAPI染色、TUNEL、RT-PCR、Western blotting、气相色谱(GC)、荧光漂白恢复(fluorescence recovery after photobleaching,FRAP)与激光共聚焦扫描显微镜等实验技术,观察和比较不同n-6/n-3 PUFA构成对两种乳腺癌细胞增殖和凋亡的影响,并从乳腺癌细胞膜磷脂脂肪酸组成、膜流动性和膜信号蛋白胰岛素样生长因子受体-1(insulin-like growth factor -1 receptor,IGF-1R)表达等方面,系统探讨不同n-6/n-3 PUFA构成对人乳腺癌细胞生物学效应及可能的作用机制。
本研究主要实验及结果如下
1.细胞生长曲线与MTT检测结果显示,单纯n-6 PUFA、5∶1 n-6/n-3 PUFA和10∶1 n-6/n-3 PUFA组MCF-7细胞增殖速度明显高于对照组,其中以n-6 PUFA组细胞增殖速度最快,10∶1 n-6/n-3 PUFA组次之,表明5∶1以上n-6/n-3 PUFA均能显著促进MCF-7细胞增殖,且此效应随n-6/n-3的比例增高而增强;与此相反,单纯n-3 PUFA和1∶1 n-6/n-3 PUFA处理细胞24 h可见MCF-7细胞大量死亡,活细胞量明显少于处理前和对照组,处理48 h后活细胞数有所增加,但增殖较缓,表明单纯n-3 PUFA和1∶1 n-6/n-3 PUFA均能显著抑制MCF-7细胞的增殖,且两者间抑制效应无显著差异。MDA-MB-231细胞状况与MCF-7细胞基本相似,提示至少高于5∶1比例的n-6/n-3 PUFA才能显著促进MDA-MB-231细胞增殖;此外1∶1 n-6/n-3 PUFA组细胞增殖速度略高于单纯n-3 PUFA组,表明单纯n-3 PUFA和1∶1 n-6/n-3 PUFA虽能显著抑制MDA-MB-231细胞的增殖,但前者抑制效应明显强于后者。
2.细胞形态观察可见,正常MCF-7细胞呈典型鹅卵石状,而MDA-MB-231细胞呈梭形,贴壁生长,聚集成甸;单纯n-6 PUFA、5∶1 n-6/n-3 PUFA和10∶1 n-6/n-3 PUFA组细胞形态与对照组比较均无明显变化;单纯n-3 PUFA组和1∶1 n-6/n-3 PUFA组细胞形态发生显著变化,细胞生长紊乱、形态异常,大量细胞皱缩变圆,胞质凝缩,并与其周边细胞分离,细胞贴壁性下降,表现出凋亡细胞的形态特征。
3. DAPI染色及TUNEL凋亡检测结果表明,与对照组比,单纯n-3 PUFA和1∶1 n-6/n-3 PUFA处理MCF-7和MDA-MB-231细胞24 h后,细胞凋亡率均显著升高(P<0.05),且两者间无统计学差异;而单纯n-6 PUFA组、5∶1 n-6/n-3 PUFA组和10∶1 n-6/n-3 PUFA组细胞无凋亡现象发生,表明n-3 PUFA和1∶1 n-6/n-3 PUFA能显著诱导乳腺癌MCF-7和MDA-MB-231细胞凋亡。
4.气相色谱分析表明,n-6/n-3 PUFA不同构成比对两种乳腺癌细胞膜的四种主要磷脂PE、PC、PS和PI的脂肪酸组成的影响基本一致,单纯n-3 PUFA和1:1 n-6/n-3PUFA处理组可以显著增加两种细胞膜四种磷脂的C20:5百分比含量,降低了各磷脂的n-6/n-3比例;单纯n-6 PUFA和10:1 n-6/n-3 PUFA处理组可以显著增加两种细胞四种磷脂的C18:2和C20:4的百分比含量,进而升高了各磷脂的n-6/n-3的比例,且四种磷脂中PE和PC的改变最为明显;在n-3 PUFA和1:1 n-6/n-3 PUFA处理组,还可以观察到PE和PC膜磷脂中的C22:6含量显著增加,提示了EPA向DHA的转化。以上表明不同n-6/n-3 PUFA构成能显著改变MCF-7和MDA-MB-231乳腺癌细胞膜磷脂脂肪酸的百分比组成。
5.以荧光探针NBD-C6标记细胞膜磷脂,利用荧光漂白恢复(FRAP)技术和激光共聚焦扫描显微镜检测膜流动性变化的结果表明,单纯n-6 PUFA、单纯n-3 PUFA和1∶1 n-6/n-3 PUFA处理MCF-7细胞和MDA-MB-231细胞2 h,细胞膜流动性均显著升高(P<0.05),表现为扩散系数显著升高;处理12 h,两种细胞膜流动性均较2 h时降低,单纯n-6组膜流动性仍显著高于对照组(P<0.05),而单纯n-3 PUFA和1∶1 n-6/n-3 PUFA组膜流动性则显著低于对照组(P<0.05);表明n-3 PUFA和1∶1 n-6/n-3 PUFA能使乳腺癌细胞膜流动性产生波动,先使其升高,后又降低。
6. RT-PCR和Western blotting的检测结果表明,不同n-6/n-3 PUFA构成比对MCF-7和MDA-MB-231乳腺癌细胞IGF-1R mRNA及蛋白的表达具有不同的影响,单纯n-6 PUFA、5:1 n-6/n-3 PUFA和10∶1 n-6/n-3 PUFA均能够显著上调两种乳腺癌细胞IGF-1R mRNA及蛋白水平的表达,与之相反的是,单纯n-3 PUFA和1∶1 n-6/n-3 PUFA显著下调细胞IGF-1R mRNA和蛋白的表达。
综上所述,本研究表明,不同n-6/n-3 PUFA构成对乳腺癌细胞的影响是不同的。n-6/n-3 PUFA构成比高于5:1时具有显著促进乳腺癌细胞增殖的效应,而n-6/n-3 PUFA构成比低于1:1时则具有抗乳腺癌细胞增殖、而诱导其凋亡的作用。这可能与两种多不饱和脂肪酸对乳腺癌细胞膜脂质构成的差异性改变,进而影响到膜流动性等膜生物物理特性和膜功能,以及某些关键信号蛋白的活性和表达等密切相关。
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.
引文
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