Awad A B, Ntanios F Y, Fink C S, Horvath P J
Nutrition Program, State University of New York at Buffalo 14214, USA.
Prostaglandins Leukot Essent Fatty Acids. 1996 Nov;55(5):293-302. doi: 10.1016/s0952-3278(96)90034-4.
The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12 x 10(3) cells/cm2 and supplemented with 30 microM of either 18:0, 18:2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18:0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18:2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18:0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18:2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 micrograms alamethicin and 42 microM free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation. PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.
本研究的目的是检测改变膜脂肪酸组成对HT - 29结肠癌细胞生长及磷脂酰肌醇特异性磷脂酶C（PLC）活性的影响。将细胞以12×10³个细胞/cm²的密度接种，并在DMEM培养基中添加30μM与牛血清白蛋白（BSA）络合的18:0、18:2（n6）或18:3（n3）。持续观察细胞生长12天。添加18:0的细胞（对照组）在第9天达到最大生长，其生长速度大于添加18:2（n6）或18:3（n3）的细胞。后两组细胞的生长无差异。为研究添加脂肪酸的脂肪酸掺入情况以及它们如何影响细胞膜组成，我们检测了每种磷脂（PL）种类的脂肪酸组成。磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）均受添加脂肪酸类型的显著影响。添加i8:0使HT - 29细胞膜的单不饱和脂肪酸含量高于在其他脂肪酸中生长的细胞。补充多不饱和脂肪酸（PUFA，18:2和18:3）导致PL组分中PUFA富集。与其他磷脂种类相比，添加18:3（n3）的细胞在膜PE中的不饱和指数最高。在存在15μg短杆菌肽和42μM游离钙的情况下，以PIP2为底物测量膜的PLC活性。使用GTPγ(S)评估G蛋白对该酶活性的贡献。在存在GTPγ(S)反应时，HT - 29细胞的PLC活性高16%。添加18:3（n3）的细胞中GTPγ(S)激活的PLC活性是添加18:0或18:2（n6）的细胞的81%。得出的结论是，添加18:3（n3）导致细胞增殖减少可能是通过其对PLC的抑制作用介导的，PLC为蛋白激酶C（PKC）激活提供第二信使。PLC可能受HT - 29肿瘤细胞膜PE组分不饱和指数增加的影响。
