Chai J G, Okamoto M, Bando T, Nagasawa H, Hisaeda H, Sakai T, Himeno K, Sato M, Ohkubo S
Department of Parasitology and Immunology, School of Medicine, University of Tokushima, Japan.
Immunopharmacology. 1995 Sep;30(3):209-15. doi: 10.1016/0162-3109(95)00024-n.
The extract from seeds of Aeginetia indica L. (AIL), a parasitic plant, induces potent antitumor immunity against Meth A fibrosarcomas in BALB/c mice. AIL also possesses a thymocyte co-stimulatory effect in vitro with suboptimal dose of Con A, a B cell mitogenic effect, and stimulates AIL-primed CD4+ T cells to produce Th1-type cytokines. In this study, we investigated the relationship between mitogenicity and antitumor activity with AIL. When AIL was analyzed by SDS-PAGE, there was strong and diffuse staining in the region between 14 kDa and the bottom of polyacrylamide gel and it was unaffected when AIL was digested with proteinase K (PK) before SDS-PAGE. Some bands with different molecular mass were also found in silver-stained gel and they disappeared completely by incubating AIL with PK before SDS-PAGE. The in vitro thymocyte co-stimulatory and B cell mitogenic effects were not influenced by digesting AIL with PK but were completely suppressed by the oxidation of AIL with sodium periodate before culture. In contrast, the in vivo antitumor activity was completely abolished by PK, but it was not affected by periodate oxidation. We generated mAbs specific for AIL and investigated the influence on the antitumor activity of AIL in vivo. Around 60-80% of tumor-bearing mice failed to recover from a challenge tumor when they were treated with supernatants isolated from mAb-induced precipitation reactions. Immunoblotting (Western blotting) revealed that all the mAbs reacted exclusively with a 50-60 kDa protein and that this reactivity was not influenced after oxidizing the blots with sodium periodate. We demonstrated that AIL contains polysaccharides and proteins. The polysaccharides induced B cell mitogenic and thymocyte co-stimulatory effects in vitro, while the proteins, especially a 50-60 kDa protein containing non-carbohydrate epitopes recognized by the mAbs, mediated antitumor activity in vivo.
印度野菰（Aeginetia indica L.，AIL）种子提取物，一种寄生植物提取物，可诱导BALB/c小鼠对Meth A纤维肉瘤产生强大的抗肿瘤免疫力。AIL在体外对亚最佳剂量的刀豆蛋白A（Con A）具有胸腺细胞共刺激作用、B细胞促有丝分裂作用，并刺激经AIL致敏的CD4 + T细胞产生Th1型细胞因子。在本研究中，我们调查了AIL的促有丝分裂活性与抗肿瘤活性之间的关系。当通过SDS-PAGE分析AIL时，在14 kDa至聚丙烯酰胺凝胶底部之间的区域出现强烈且弥散的条带，并且在SDS-PAGE之前用蛋白酶K（PK）消化AIL时该条带不受影响。在银染凝胶中也发现了一些不同分子量的条带，并且在SDS-PAGE之前将AIL与PK孵育后它们完全消失。用PK消化AIL不会影响体外胸腺细胞共刺激和B细胞促有丝分裂作用，但在培养前用过碘酸钠氧化AIL可完全抑制这些作用。相反，PK可完全消除体内抗肿瘤活性，但过碘酸钠氧化对其没有影响。我们制备了针对AIL的单克隆抗体（mAb），并研究了其对AIL体内抗肿瘤活性的影响。当用从mAb诱导的沉淀反应中分离的上清液处理时，约60 - 80%的荷瘤小鼠未能从肿瘤攻击中恢复。免疫印迹（Western印迹）显示，所有mAb仅与一种50 - 60 kDa的蛋白质反应，并且在用过碘酸钠氧化印迹后这种反应性不受影响。我们证明AIL含有多糖和蛋白质。多糖在体外诱导B细胞促有丝分裂和胸腺细胞共刺激作用，而蛋白质，尤其是一种含有被mAb识别的非碳水化合物表位的50 - 60 kDa蛋白质，在体内介导抗肿瘤活性。
