栝楼抗肿瘤蛋白的纯化及活性研究
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摘要
栝楼(Trichosanthes kirilowii Maxim)为葫芦科栝楼属植物,在我国主要分布于山东、河南、山西、陕西、江苏、浙江、安徽、四川、云南等省市。主要以果实和根部入药,均是历史悠久的常用中药材。瓜蒌,中国药典载为栝楼的果实,主要用来治疗心、肺方面的疾病。天花粉则来源于栝楼的块根,主要用于中期引产,主要成分为天花粉蛋白。天花粉蛋白曾在20世纪70年代在我国被广泛使用,但由于其在临床上容易引起发热、休克甚至死亡等过敏性反应,目前临床上很少使用。国外一些学者研究发现天花粉蛋白是一种单链核糖体失活蛋白,具有抗病毒、抗肿瘤等活性,尤其对HIV具有很好的抑制效果,因此天花粉蛋白又逐渐引起了人们的关注,但其毒副作用同样限制了其临床使用。目前很多医药工作者开始转向于寻找新的具有较低毒副作用的核糖体失活蛋白。本研究从所收集到的不同种质资源的栝楼中成功的分离、纯化到一种核糖体失活蛋白—Trichosanthrip,并对其性质和抗肿瘤活性进行了研究,取得了如下结果:
1.从14份不同居群栝楼的种子中,采用硫酸铵分级沉淀、阳离子交换色谱和葡聚糖凝胶过滤色谱成功的纯化到了Trichosanthrip,经测定其纯度高达96.6%,产率为1 mg/100g种仁。
2.Trichosanthrip经胰酶降解后使用LC-MS/MS分析,发现Trichosanthrip为AAI_LTSS超级蛋白质家族的一种新蛋白质。此类蛋白质广泛存在于植物的种子中,是植物种子中的一种自我防卫物质,具有抗真菌、病毒、细菌等病原物及抗昆虫的活性。
3.使用MALDI-TOF MS精确测定Trichosanthrip分子量为10964.717 D;Trichosanthrip具有很好的RNA N-糖苷酶活性和蛋白质生物合成抑制活性,对蛋白质生物合成的IC_(50)为1.6 ng/ml。
4.Trichosanthrip能体外抑制S_(180)肿瘤细胞增殖,使S_(180) G0/G1期细胞增加,S期细胞减少,导致其出现G0/G1期细胞阻滞现象,其对S_(180)的IC_(50)为19.91μg/mL。
5.Trichosanthrip能明显的抑制接种S_(180)昆明种小鼠体内肿瘤生长,在1.5mg/kg·d剂量下,抑瘤率可达91.36%;在最高剂量1.5 mg/kg·d下,Trichosanthrip对小鼠的体重、脾脏指数和胸腺指数几乎无影响,其体重和脏器指数与空白对照无明显变化;且Trichosanthrip对小鼠的急性毒性很小,LD_(50)为103 mg/kg,且当剂量达到75 mg/kg时才表现出急性毒性,远远高于实体瘤抑制实验中的最大剂量。
Trichosanthes kirilowii Maxim, a dioecious liana, is a member of the Cucurbitaceae. It grows in hillside, valley, brushwood, forest edge etc and can be planted all over the country. In the ancient East, it was widely used as a folk medicine because the tuber can reduce mid-term abortion and the fruit rind can help cure diseases of the heart and lungs. In 1989, McGrath purified a type 1 RIP, named trichosanthin (TCS), from the tuber of T. kirilowii. TCS displayed extreme inhibition of HIV. Hence, TCS and T. kirilowii have been studied by many researchers, and the antiviral and antitumor activities were founded. But its clinical usages were restricted by the associated fever, shock and even death. In comparison with the normal type 1 RIPs, several RIPs with a molecular mass of～10 kDa have high levels of bioactivity and exhibit low immune toxicity toward normal cells. In this study, Trichosanthrip, a novel RIP with a lower molecular mass, was purified from collected 14 populations of wild T. kirilowii, and its characterization and antitumor activity was assayed.
Trichosanthrip was purified from mature seeds of 14 populations of wild T. kirilowii Maxim by ammonium sulfate fractionation precipitation, CM-52 cation-exchange chromatography and Sephadex G50 gel-filtration chromatography. About 1.0 mg Trichosanthrips with purity of 96.6% were getted from 100 g seeds kernels. Trypsin-peptides of Trichosanthrip were identified by liquid chromatography- tandem mass spectrometry, and it was a novel protein belonged to AAI_LTSS super family. The proteins of the family were mainly presented in seeds of a variety of plants and played a role of defence with anti-insect and anti-pathogen activities.
Trichosanthrip migrated as a single band in SDS-PAGE, with an apparent molecular mass of～13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-fiight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC50 of 1.6 ng/ml.
Trichosanthrip could inhibit the growth of S_(180) with the IC_(50) of 19.91μg/ml by increased the cell of S_(180) in G0/G1 sterm, and meanwhile decreased the cell in S sterm.
Trichosanthrip could inhibit the growth of tumor in S_(180) transplanted Kunming strain mice, and the inhibitory rate was 91.36% when at the dose of 1.5 mg/kg·d. Meanwhile, there were not any effect on the weight and index of thymus and spleen in S180 transplanted Kunming strain mice at this dose. And the acute toxicity of Trichosanthrip on Kunming strain mice was extreme low with LD50 was 103 mg/kg.
So, Trichosanthrip has a good future in cancer care with higher antitumor activity and lower side effect.
1.从14份不同居群栝楼的种子中,采用硫酸铵分级沉淀、阳离子交换色谱和葡聚糖凝胶过滤色谱成功的纯化到了Trichosanthrip,经测定其纯度高达96.6%,产率为1 mg/100g种仁。
2.Trichosanthrip经胰酶降解后使用LC-MS/MS分析,发现Trichosanthrip为AAI_LTSS超级蛋白质家族的一种新蛋白质。此类蛋白质广泛存在于植物的种子中,是植物种子中的一种自我防卫物质,具有抗真菌、病毒、细菌等病原物及抗昆虫的活性。
3.使用MALDI-TOF MS精确测定Trichosanthrip分子量为10964.717 D;Trichosanthrip具有很好的RNA N-糖苷酶活性和蛋白质生物合成抑制活性,对蛋白质生物合成的IC_(50)为1.6 ng/ml。
4.Trichosanthrip能体外抑制S_(180)肿瘤细胞增殖,使S_(180) G0/G1期细胞增加,S期细胞减少,导致其出现G0/G1期细胞阻滞现象,其对S_(180)的IC_(50)为19.91μg/mL。
5.Trichosanthrip能明显的抑制接种S_(180)昆明种小鼠体内肿瘤生长,在1.5mg/kg·d剂量下,抑瘤率可达91.36%;在最高剂量1.5 mg/kg·d下,Trichosanthrip对小鼠的体重、脾脏指数和胸腺指数几乎无影响,其体重和脏器指数与空白对照无明显变化;且Trichosanthrip对小鼠的急性毒性很小,LD_(50)为103 mg/kg,且当剂量达到75 mg/kg时才表现出急性毒性,远远高于实体瘤抑制实验中的最大剂量。
Trichosanthes kirilowii Maxim, a dioecious liana, is a member of the Cucurbitaceae. It grows in hillside, valley, brushwood, forest edge etc and can be planted all over the country. In the ancient East, it was widely used as a folk medicine because the tuber can reduce mid-term abortion and the fruit rind can help cure diseases of the heart and lungs. In 1989, McGrath purified a type 1 RIP, named trichosanthin (TCS), from the tuber of T. kirilowii. TCS displayed extreme inhibition of HIV. Hence, TCS and T. kirilowii have been studied by many researchers, and the antiviral and antitumor activities were founded. But its clinical usages were restricted by the associated fever, shock and even death. In comparison with the normal type 1 RIPs, several RIPs with a molecular mass of～10 kDa have high levels of bioactivity and exhibit low immune toxicity toward normal cells. In this study, Trichosanthrip, a novel RIP with a lower molecular mass, was purified from collected 14 populations of wild T. kirilowii, and its characterization and antitumor activity was assayed.
Trichosanthrip was purified from mature seeds of 14 populations of wild T. kirilowii Maxim by ammonium sulfate fractionation precipitation, CM-52 cation-exchange chromatography and Sephadex G50 gel-filtration chromatography. About 1.0 mg Trichosanthrips with purity of 96.6% were getted from 100 g seeds kernels. Trypsin-peptides of Trichosanthrip were identified by liquid chromatography- tandem mass spectrometry, and it was a novel protein belonged to AAI_LTSS super family. The proteins of the family were mainly presented in seeds of a variety of plants and played a role of defence with anti-insect and anti-pathogen activities.
Trichosanthrip migrated as a single band in SDS-PAGE, with an apparent molecular mass of～13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-fiight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC50 of 1.6 ng/ml.
Trichosanthrip could inhibit the growth of S_(180) with the IC_(50) of 19.91μg/ml by increased the cell of S_(180) in G0/G1 sterm, and meanwhile decreased the cell in S sterm.
Trichosanthrip could inhibit the growth of tumor in S_(180) transplanted Kunming strain mice, and the inhibitory rate was 91.36% when at the dose of 1.5 mg/kg·d. Meanwhile, there were not any effect on the weight and index of thymus and spleen in S180 transplanted Kunming strain mice at this dose. And the acute toxicity of Trichosanthrip on Kunming strain mice was extreme low with LD50 was 103 mg/kg.
So, Trichosanthrip has a good future in cancer care with higher antitumor activity and lower side effect.
引文
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