Sugiyama Michiyo, Imai Atsushi, Furui Tatsuro, Tamaya Teruhiko
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu, Japan.
Am J Obstet Gynecol. 2004 Mar;190(3):680-5. doi: 10.1016/j.ajog.2003.09.069.
Lysophosphatidic acid stimulates the proliferation of ovarian cancer cell through specific members of the guanosine triphosphate-binding protein-coupled receptor family. We attempted to identify the guanosine triphosphate-binding protein subtypes that are linked to lysophosphatidic acid receptor-stimulated production of cytokine, which are involved potentially in ovarian cancer development.
Cytokine assay kits were used to determine interleukin-6, interleukin-8, and tumor necrotic factor-alpha that were produced from Caov-3 and SK-OV3 ovarian cancer cell lines. Thealpha-subunit of Gi was detected by pertussis toxin-catalyzed adenosine diphosphate ribosylation from nicotinamide adenine dinucleotide in isolated plasma membrane.
Pertussis toxin, but not cholera toxin, brought about adenosine diphosphate ribosylation of Galphai of 41 kd in the plasma membrane. Incubation with lysophosphatidic acid and nonhydrolyzable guanosine triphosphate analog decreased the adenosine diphosphate-ribosylation activity in a dose-dependent manner; a one half-maximal effect occurred with 10 micromol/L lysophosphatidic acid. The apparent inhibition by lysophosphatidic acid of the adenosine diphosphate ribosylation demonstrated that lysophosphatidic acid resolved the alpha-subunit of the Gi to guanosine triphosphate-bound form in the membranes. Pretreatment of the ovarian cancer cells with the pertussis toxin completely inhibited lysophosphatidic acid-stimulated production of interleukin-6, interleukin-8 and tumor necrosis factor-alpha. The lysophosphatidic acid-stimulated cytokine production was dose-dependent with a one half-maximal effect at 10 micromol/L. Phosphatidic acid and ceramide 1-phosphate had no effect on the lysophosphatidic acid action on cytokine expression.
These data demonstrate the coupling of lysophosphatidic acid receptor to Gi protein subfamily in ovarian cancer cell. The Gi that couples lysophosphatidic acid receptor to the effector may define the differences in the signaling pathways of lysophosphatidic acid-activated cytokine expression and proliferation.
溶血磷脂酸通过鸟苷三磷酸结合蛋白偶联受体家族的特定成员刺激卵巢癌细胞增殖。我们试图鉴定与溶血磷脂酸受体刺激的细胞因子产生相关的鸟苷三磷酸结合蛋白亚型，这些细胞因子可能参与卵巢癌的发展。
使用细胞因子检测试剂盒测定Caov-3和SK-OV3卵巢癌细胞系产生的白细胞介素-6、白细胞介素-8和肿瘤坏死因子-α。通过百日咳毒素催化的烟酰胺腺嘌呤二核苷酸在分离的质膜中的二磷酸腺苷核糖基化来检测Gi的α亚基。
百日咳毒素而非霍乱毒素导致质膜中41kd的Gαi发生二磷酸腺苷核糖基化。用溶血磷脂酸和不可水解的鸟苷三磷酸类似物孵育以剂量依赖性方式降低了二磷酸腺苷核糖基化活性；在10μmol/L溶血磷脂酸时出现半数最大效应。溶血磷脂酸对二磷酸腺苷核糖基化的明显抑制表明溶血磷脂酸使膜中Gi的α亚基转化为鸟苷三磷酸结合形式。用百日咳毒素预处理卵巢癌细胞可完全抑制溶血磷脂酸刺激的白细胞介素-6、白细胞介素-8和肿瘤坏死因子-α的产生。溶血磷脂酸刺激的细胞因子产生呈剂量依赖性，在10μmol/L时出现半数最大效应。磷脂酸和1-磷酸神经酰胺对溶血磷脂酸对细胞因子表达的作用无影响。
这些数据证明了卵巢癌细胞中溶血磷脂酸受体与Gi蛋白亚家族的偶联。将溶血磷脂酸受体与效应器偶联的Gi可能定义了溶血磷脂酸激活的细胞因子表达和增殖信号通路的差异。
