Analysis of anticancer and cytotoxic mechanism of an Aralia elata-derived antitumor protein, aralin.

龙牙楤木来源的抗肿瘤蛋白阿拉林的抗癌和细胞毒机制分析。

• 批准号: 17590098
• 负责人: TASHIRO Fumio
• 金额: $ 1.92万
• 依托单位: Tokyo University of Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590098/
• 关键词: Aralia elata; anticancer; lectin; RNA N-glycosidase; ribosome inactivating protein; callus

We found that aralin, a novel cytotoxic protein from Aralia elate, selectively induces apoptosis in transformed cells. Aralin is a lectin specific for galactose (Gal) and possesses RNA N-glycosidase activity. In this study, to elucidate the anticancer and cytotoxic mechanism evoked by aralin, we analyzed the tumorigencity when aralin administrate into the HeLa cells transplanted nude mice and intracellular localization of aralin using Tetramethylrhodamine (TAMRA)-conjugated aralin and its recognizing protein using far-Western blot analysis. The homogenate of Aralia elate including 2 μg of aralin were administer orally in the HeLa cells injected into nude mice for 3 weeks of 5 days on the week. The tumors formed by HeLa cells were remarkably decreased on the aralia elate administering group. TAMRA-aralin bound to cell membrane and then migrated into the cytosol, following to transport into endoplasmic reticulum in a time-dependent manner. The binding of TAMRA-aralin to cell membrane was significantly inhibited by the addition of Gal, which also repressed the cytotoxic effect of aralin. To analyze the aralin interacting proteins, we performed a far-Western blotting using aralin as a probe and anti-aralin antibody for the membrane fractions from HeLa cells, normal human lung fibroblast WI-38 cells and its SV40-transformed VA-13 cells. The results showed that 30 and 57 kDa proteins of VA-13 and HeLa cells were bound to aralin. These interactions were not influenced by ricin, whereas almost inhibited in presence of Gal. These data suggest that aralin is antitumor protein and incorporated into cells through its Gal-containing cell surface receptor, and induces cell death by the inhibition of protein synthesis.

我们发现，aralin，一种新的细胞毒性蛋白质从辽东木，选择性地诱导转化细胞的凋亡。Aralin是一种半乳糖凝集素，具有RNA N-糖苷酶活性。本研究以四甲基罗丹明（塔姆拉）标记的aralin及其识别蛋白为靶点，采用免疫印迹法分析aralin对裸鼠移植瘤HeLa细胞的致瘤性和细胞内定位，以阐明aralin的抗癌和细胞毒作用机制。用含2 μg阿拉灵的辽东木匀浆液灌胃给裸鼠接种HeLa细胞，每周5天，连续3周。土木香给药组HeLa细胞形成的肿瘤明显减少。TAMRA-aralin与细胞膜结合，然后进入胞质，随后以时间依赖性的方式运输到内质网。半乳糖的加入显著抑制了TAMRA-aralin与细胞膜的结合，也抑制了aralin的细胞毒作用。为了分析aralin相互作用的蛋白质，我们使用aralin作为探针和抗aralin抗体对来自HeLa细胞、正常人肺成纤维细胞WI-38细胞及其SV 40转化的VA-13细胞的膜组分进行远蛋白质印迹。结果表明，VA-13和HeLa细胞的30和57 kDa蛋白质与aralin结合。这些相互作用不受蓖麻毒素的影响，而几乎抑制存在的半乳糖。这些数据表明，aralin是抗肿瘤蛋白，并通过其含Gal的细胞表面受体掺入细胞，并通过抑制蛋白质合成诱导细胞死亡。