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

我们发现花生中的一种新的细胞毒蛋白aralin选择性地诱导转化细胞的凋亡。花生凝集素是半乳糖(Gal)的特异性凝集素，具有RNA N-糖苷酶活性。在本研究中，我们利用四甲基罗丹明(TAMRA)标记的阿拉林及其识别蛋白，通过Far-Western印迹分析，分析了阿拉林对HeLa裸鼠移植瘤的致瘤性以及阿拉林在细胞内的定位，以阐明阿拉林的抗癌和细胞毒作用机制。将龙牙菜匀浆2μg灌胃裸鼠HeLa细胞，连续给药3周，共5天。刺五加给药组HeLa细胞形成的肿瘤明显减少。Tamra-aralin结合到细胞膜上，然后迁移到胞浆中，然后以时间依赖的方式进入内质网。加入Gal可显著抑制Tamra-aralin与细胞膜的结合，从而抑制Tamra-aralin的细胞毒作用。为了分析花生蛋白相互作用的蛋白，我们以花生蛋白为探针，用抗花生蛋白抗体对HeLa细胞、正常人肺成纤维细胞WI-38细胞及其SV40转化的VA-13细胞的膜组分进行了Far-Western印迹。结果表明，VA-13和HeLa细胞中分别有30和57 kDa的蛋白与花生四烯酸结合。这些相互作用不受蓖麻毒素的影响，而在半乳糖的存在下几乎被抑制。这些数据表明，阿拉林是一种抗肿瘤蛋白，通过其含有Gal的细胞表面受体进入细胞内，并通过抑制蛋白质合成来诱导细胞死亡。

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.