WO2009015602A1 - Polypeptides having anticancer activity - Google Patents

Polypeptides having anticancer activity Download PDF

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Publication number
WO2009015602A1
WO2009015602A1 PCT/CN2008/071804 CN2008071804W WO2009015602A1 WO 2009015602 A1 WO2009015602 A1 WO 2009015602A1 CN 2008071804 W CN2008071804 W CN 2008071804W WO 2009015602 A1 WO2009015602 A1 WO 2009015602A1
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Prior art keywords
polypeptide
amino acid
present
seq
sequence
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PCT/CN2008/071804
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English (en)
French (fr)
Inventor
Songping Liang
Zhonghua Liu
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Xiamen Bioway Biotech Co., Limited
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Application filed by Xiamen Bioway Biotech Co., Limited filed Critical Xiamen Bioway Biotech Co., Limited
Priority to EP20080783797 priority Critical patent/EP2184295B1/en
Priority to JP2010518485A priority patent/JP5707594B2/ja
Publication of WO2009015602A1 publication Critical patent/WO2009015602A1/zh
Priority to US12/697,320 priority patent/US8207122B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to an anticancer active peptide, in particular to a polypeptide having anticancer activity isolated from the crude venom of the burrowing spiderweed in Xinjiang.
  • Polypeptide anticancer drugs are hotspots in the development of anticancer drugs in recent years. However, due to the low activity and high toxicity, few peptide anticancer drugs have been used in clinical applications so far, which are far from meeting the growing demand of the anticancer drug market. Therefore, how to obtain an active and highly toxic small anti-cancer polypeptide material to meet the demand for a large number of anticancer drugs is an urgent problem to be solved.
  • the present invention aims to provide a highly effective and low toxicity anticancer active peptide.
  • the anticancer active peptide provided by the invention has the amino acid sequence:
  • the anticancer active peptide of the present invention is a 24 peptide, which is isolated and purified from the crude venom of the burrowing burrowing spider in Xinjiang, and can also be synthesized by artificial chemistry.
  • the polypeptide can induce apoptosis to kill a variety of cancer cells, and can inhibit the proliferation of cancer cells, but is less toxic to normal cells and animals; and can also inhibit the transcriptional activity of hypoxia-inducible factor HIF a, thereby inhibiting tumor tissue blood vessels.
  • Regeneration, effective inhibition of solid tumor growth; its anti-cancer activity has the characteristics of high efficiency and low toxicity, and has a good application prospect for the development of drugs for the treatment of solid tumors such as lung cancer, liver cancer and cervical cancer. Due to the large-scale preparation by artificial chemical synthesis technology, it lays a foundation for the preparation of low-cost and high-yield preparations for future drug development.
  • the present invention relates to an active peptide which, in addition to encoding the sequence of SEQ NO. 1 involved, also includes a functional analog which encodes one or more amino acid substitutions, insertions or deletions of an amino acid of an active peptide molecule of the present invention.
  • the effect of the inventive active peptide also encompasses encoding at least 90% homology to the amino acid sequence set forth in SEQ NO.
  • the following amino acid sequences SEQ NO. 2 sequence
  • Lys G ly Trp Phe Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys G lu Lys Met Lys G lu H is Leu - NH 2
  • Lys G ly Trp Phe Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys G lu Lys Leu Lys G lu H is Leu - NH 2
  • SEQ NO. 2 is an analog isolated from the crude venom of the burrowing burrowing spider, Xinjiang, which differs from the sequence by only two amino acid residues, that is, the sequence 1N-terminal residue Arg is deleted, and the Leul 9 mutation is Met l 9 . But the two have the same activity.
  • sequence SEQ NO. 3 - 5 is derived from the sequence SEQ NO. 1 , a variant obtained by performing molecular design and anticancer activity studies on the sequence SEQ NO. 1, wherein the sequences SEQ NO. 3 and SEQ NO. 4 are active
  • sequence SEQ NO. 1 is identical, the sequence SEQ NO. 5 activity is increased by about 10-fold, but at the same time has a stronger hemolytic activity than the sequence SEQ NO.
  • the arrow marked in the figure is the target peak.
  • FIG. 6 The left panel shows volume changes before and after the last dose.
  • the graph shows the mean volume of all nude mice in the drug-administered and control groups. Black is before administration and gray is after administration. The tumor weight of the control group and the drug-administered group was compared, and the average weight of the tumor in the control group was about 5 times that of the drug-administered group;
  • the anticancer active peptide of the present invention is first isolated and purified from the venom of the burrowing spider venom of Xinjiang by high-sensitivity analytical RP-HPLC, and the amino acid sequence of the polypeptide of the present invention is determined by the Edman degradation technique, indicating that it exists with the existing The anticancer active peptide has a low amino acid sequence similarity and is a novel polypeptide molecule.
  • the specific operations are as follows:
  • the crude toxic powder of Xinjiang burrowing spider was dissolved in double distilled water to prepare a 5 mg/ml toxin solution, centrifuged at 8000 rpm for 5 minutes, and the supernatant was filtered with a Millipore disposable filter (0.22 ⁇ mol). , placed at 4 ° C to save.
  • the lysate is 0.1% trifluoroacetic acid / /.
  • the peak component was collected at a wavelength of 215 nm, and the molecular weight was determined by matrix-assisted dissociation time-of-flight mass spectrometry, followed by freeze-drying to obtain a dry powder of the anticancer active peptide of the present invention.
  • the anticancer active peptide of the present invention can be synthesized by artificial chemical, and the specific operation is as follows:
  • the polypeptide of the present invention is synthesized by a solid phase polypeptide synthesis method of fluorenylmethoxycarbonyl-amino acid and TBTU/HOBt coupling, and the solid phase carrier is Rink resin, which is synthesized on a PS3 automatic synthesizer (PTI), and the methoxycarbonyl group is synthesized during the synthesis.
  • the amino acid was used in a 5-fold excess (0.5 mm), and the coupling time of each synthetic residue was 30 minutes.
  • the terminal fluorenylmethoxycarbonyl group was removed with 20% piperidine/dimethylformamide (volume ratio). group.
  • the peptide resin was sufficiently washed with methanol.
  • the polypeptide was precipitated with cold diethyl ether and washed several times, and then vacuum-dried to obtain a crude synthetic peptide.
  • the cytotoxicity is detected by MTT assay, and the solid tumor is detected by a tumor-bearing experiment in nude mice. Health Long inhibition.
  • the burrowing tarantula was collected in the Hami area of Xinjiang and was artificially raised in the animal room of the Protein Chemistry Laboratory of the College of Life Sciences, Hunan Normal University.
  • the crude toxic was collected by electric stimulation, and the crude lyophilized was stored at 20 ° C after lyophilization.
  • the nude mouse was purchased in Shanghai.
  • the chromatographic peaks of the markers showed a single molecular weight peak with a single isotope molecular weight (M+H + ) of 2886. 754 Da.
  • the primary structure of the polypeptide of the present invention was determined by Edman degradation to be 24 amino acid residues, without cysteine residues, and the molecular weight of the experimental assay and the theoretically calculated molecular weight determined the C-terminal amidation modification of the polypeptide.
  • the polypeptide of the present invention contains only 24 amino acid residues and is relatively easy to synthesize.
  • polypeptide of the invention selectively kills cancer cells
  • the toxicity of the polypeptide of the present invention to Hela, HT1080, HNE1, CNE1 and JB6 cell lines was determined by MTT assay. 40 micromolar of the polypeptide of the present invention has obvious killing effect on five cell lines (see Fig. 3), wherein the toxicity to HT1080 and Hela cells is the strongest, and the toxicity to JB6 cells is relatively weak, respectively killing 98%. And 36% of the cells, indicating no The same cells are not sensitive to the polypeptide of the present invention, HT1080 and Hela are fibrosarcoma and cervical cancer cell lines, respectively, and JB6 is an immortalized mouse epithelial cell line, which can be subcultured, but no malignant proliferation of cancer cells. Features. The above results indicate that the polypeptide of the present invention has a certain selectivity for killing cancer cells.
  • the toxicity of the polypeptide of the present invention to normal cells was examined by a hemolysis test, and it was revealed that 200 ⁇ mol of the polypeptide of the present invention can only lyse about 20% of red blood cells, indicating that the polypeptide of the present invention is weak to normal cells.
  • the toxicity of the polypeptide of the present invention was tested. Subcutaneous injection of the mouse at 200 mg/kg body weight did not show significant toxicity (within 48 hours), indicating that the polypeptide of the present invention is less toxic and more resistant to cancer cells. Strong selectivity.
  • the mechanism of killing cancer cells by the polypeptide of the present invention was detected by Annexin V-fluorescein isothiocyanate/propidium iodide double staining, and it was found that the polypeptide of the present invention can induce apoptosis of Hela cells, as shown in FIG.
  • the drug-treated HeLa cells were less apoptotic, and 20 ⁇ mol of the polypeptide of the present invention resulted in apoptosis of about 25.6% of HeLa cells, indicating that the polypeptide of the present invention kills cancer cells by inducing apoptosis.
  • polypeptide of the present invention inhibits tumor growth in nude mice
  • the above experiments show that the polypeptide of the present invention has a strong inhibitory effect on cancer cells, but it is still necessary to determine whether the polypeptide of the present invention has an effect against solid tumors in vivo, and a nude mouse tumor-bearing model is used for research.
  • the experimental results show that the polypeptide of the present invention can significantly inhibit the growth of solid tumors.
  • the solid tumor volume of the control group and the administration group was similar, and during the time of the drug intervention, the tumor volume of the saline control group rapidly increased, indicating that it was The tumor continued to grow, but the tumor volume of the drug-administered group did not change significantly.
  • the mechanism by which the polypeptide of the present invention inhibits tumor growth is examined using tumor sections hematoxylin and eosin staining tissue staining methods.
  • the cells of the tumor tissue were closely arranged, and the nucleus staining was light blue.
  • the tumor tissue cells were arranged to be evacuated, the nucleus was concentrated, and the staining was dark blue, indicating that the polypeptide of the present invention induced tumor cell apoptosis and inhibited its growth.
  • the polypeptides of the present invention have strong inhibitory effects on cancer cells and anti-solid tumors, but it is still necessary to confirm the degree of inhibition of tumor cells by different sequences of polypeptides, and the Hela cell activity assay is used for comparative study.
  • the inhibition rate of the sequence SEQ NO. 1-SEQ SEQ NO. 4 to Hela cells at a concentration of 40 ⁇ 1/ ⁇ is about 90%, at a concentration of 2 ( ⁇ mol/L).
  • the inhibition rate of Hela cells was about 50%, and the inhibition rate of Hela cells was only about 2% at a concentration of 5 mol/L.
  • the sequence SEQ NO. 1-SEQ SEQ NO. 4 inhibited Hela cells.
  • the activity is equivalent, almost no difference; and the sequence SEQ NO. 5 can inhibit more than 80% of Hela cells at 4 ( ⁇ mol/L, 20 ⁇ 1/ ⁇ , 5 mol/L, the activity is more
  • the sequence SEQ NO. 1 - sequence SEQ NO. 4 is increased by about 10-fold.
  • the sequence SEQ NO. 1 and the sequence SEQ N0. 5 dissolve 19% and 37% of red blood cells at 200 ⁇ 1, respectively (see Figure 8), indicating the sequence SEQ The hemolytic activity of ⁇ 0.5 is stronger than the sequence SEQ N0. 1.
  • the anti-cancer active peptide of the present invention can be obtained in a large amount by high-efficiency and low toxicity by separation and purification and artificial chemical synthesis in natural venom, and the inventors have demonstrated through extensive pharmacological studies that the polypeptide of the present invention is developed for the treatment of diseases such as lung cancer, liver cancer, cervical cancer, and the like. Solid tumor drugs have a good application prospect.

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Description

一种抗癌活性肽
技术领域
本发明涉及一种抗癌活性肽， 具体涉及一种从新疆穴居狼蛛粗毒中分离的具有抗癌活性 的多肽。
背景技术
多肽抗癌药物是近年来抗癌药物研发的热点， 但由于活性低和毒性较大等因素的影响， 至今应用于临床的多肽抗癌药物甚少， 远不能满足抗癌药物市场日益增长的需求， 因此如何 获得一种活性高毒性小的抗癌多肽物质， 以满足大量开发抗癌药物的需求， 是目前亟待解决 的问题。
发明内容
本发明旨在于提供一种高效低毒的抗癌活性肽。
本发明提供的这种抗癌活性肽， 其氨基酸序列为：
Arg Lys Gly Trp Phe Lys Ala Met Lys Ser lie Ala Lys Phe lie Ala Lys Glu Lys Leu Lys 1 5 10 15 20
Glu His Leu-NH2 (多肽 C一末端酰胺化修饰）
本发明抗癌活性肽为 24肽， 从新疆穴居狼蛛粗毒中分离纯化获得， 也可以由人工化学合 成。 这种多肽能诱导细胞凋亡杀灭多种癌细胞， 并能抑制癌细胞增殖， 但对正常细胞和动物 毒性较弱； 同时还可抑制低氧诱导因子 HIF a转录活性， 从而抑制肿瘤组织血管再生， 有效 的抑制实体瘤生长； 其抗癌活性具有高效低毒的特点， 对于开发治疗肺癌、 肝癌、 子宫颈癌 等实体瘤的药物有着较好的应用前景。 由于能通过人工化学合成技术进行大规模制备， 为将 来药物开发中低成本和高产率的制备奠定了基础。
本发明涉及活性肽， 除了编码涉及的 SEQ NO. 1序列外， 还应包括编码对本发明活性肽分 子的氨基酸进行一个或多个的氨基酸替换、 插入或者缺失所得到的功能类似物也能达到本发 明活性肽的效果。 因此， 本发明也包括编码与 SEQ NO. 1所示的氨基酸序列至少 90%的同源性。 特别是下述的氨基酸序列： SEQ NO. 2序列
Lys G ly Trp Phe Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys G lu Lys Met Lys G lu H i s Leu - NH2
SEQ NO. 3序列
Lys G ly Trp Phe Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys G lu Lys Leu Lys G lu H i s Leu - NH2
SEQ NO. 4序列
Trp Phe Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys G lu Lys Leu Lys SEQ NO. 5序列
Lys Ala Met Lys Ser He Ala Lys Phe He Ala Lys - NH2
SEQ NO. 2为从新疆穴居狼蛛粗毒分离的一个类似物， 与序列只有两个氨基酸残基不同， 即序列 1N -末端残基 Arg缺失， 以及 Leul 9突变为 Met l 9。 但两者活性相同。
序列 SEQ NO. 3 - 5来自于序列 SEQ NO. 1 , 通过对于序列 SEQ NO. 1开展分子设计和抗癌 活性研究， 获得的变构体， 其中序列 SEQ NO. 3和 SEQ NO. 4活性与序列 SEQ NO. 1相同， 序列 SEQ NO. 5活性提高了约 10倍， 但同时具有较序列 SEQ NO. 1强的溶血活性。
附图说明
图 1.穴居狼蛛粗毒反相一高效液相色谱分离纯化图谱；
图 2.化学合成本发明抗癌活性肽反相高效液相色谱分离纯化图谱。
图中箭头标记的为目的峰。
图 3.采用 MTT法检测本发明多肽的细胞毒性， 40 微摩尔本发明多肽对五种细胞株都有 明显的杀灭作用。
图 4. 异硫氰酸荧光素一 Annexin V/碘化丙啶双染法检测显示本发明多肽诱导 Hela细胞 凋亡。 为双变量流式细胞仪散点图； 其中右下象限为凋亡早期细胞。
图 5-图 8. 本发明多肽有效抑制肿瘤生长，其中：
图 5.肿瘤生长体积变化监测， ·为给药组， 0为对照组， 结果显示对照组持续生长， 而给 药组肿瘤生长抑制；
图 6. 左图显示给药前以及最后一次给药后体积变化， 图中显示给药组和对照组所有裸 鼠的体积平均值， 黑色为给药前， 灰色为给药后； 右图为对照组和给药组肿瘤重量对比， 对 照组肿瘤的平均重量约为给药组的 5倍；
图 7. 五种多肽抑制 Hela细胞活性比较。
图 8.序列 SEQ NO. 1和序列 SEQ NO. 5溶血活性比较。
具体实施方式
本发明抗癌活性肽首先通过高灵敏度的的分析型反相高效液相色谱技术从新疆穴居狼蛛 毒液中分离纯化得到， 通过 Edman降解技术测定了本发明多肽的氨基酸序列， 表明它与现有 的抗癌活性肽的氨基酸序列相似性较低， 是一个全新的多肽分子。 具体操作如下：
将新疆穴居狼蛛粗毒干粉用双蒸水溶解配成 5毫克 /毫升的毒素溶液， 8000转 /分钟离心 5 分钟， 上清用密理博公司一次性过滤器 (0. 22 微摩尔）过滤后， 置于 4 ° C保存。 粗毒直接 用分析型反相高效液相色谱 （Vydac, C18, 218TP54, 4. 6 x 250毫米）分离， 上样量为 0. 5毫 克， 洗脱液分别为 0. 1%三氟乙酸 /水 和 0. 1%三氟乙酸 /乙氰 （B液）， 洗脱梯度： 0— 60 分钟， 0— 60 % B 液， 1 毫升 /分钟， 40 ° C。 在波长为 215 纳米下收集峰成分， 基质辅助解 离飞行时间质谱测定分子量后冷冻干燥， 得到本发明抗癌活性肽干粉。
本发明抗癌活性肽可以通过人工化学合成， 具体操作如下：
采用芴甲氧羰基-氨基酸和 TBTU/HOBt偶联的固相多肽合成方法合成本发明多肽，固相载 体为 Rink树脂， 在 PS3自动合成仪（PTI )上合成， 合成过程中芴甲氧羰基-氨基酸的用量为 5倍过量（0. 5毫米）， 每个合成残基的偶联时间为 30分钟， 用 20%的哌啶 /二甲基甲酰胺（体 积比） 去除末端芴甲氧羰基基团。 全部氨基酸偶联完成并除去最后一个氨基酸残基的芴甲氧 羰基-基团后， 用甲醇充分洗涤肽树脂。然后将冻干后的肽树脂放入带筛板的 BI0-RAD反应柱 中， 使用裂解试剂 K (配方为三氟乙酸： 水： 苯酚： 苯甲硫醚： 二巯基乙烷 = 82. 5 ： 5 ： 5： 5 ： 2. 5 (体积比） ）， 反应 2小时完成侧链保护基团的去保护并从树脂上裂解下来。 用冷乙 醚沉淀多肽并洗涤多次后， 冷冻真空干燥得到合成的多肽粗品。 通过半制备型反相高效液相 色谱 （反相柱： 10 X 250毫米依利特 C18柱； 线性梯度 B液 15 % _45 % 30分钟； 流速 3毫 升 /分钟） 分离收集洗脱峰冻干后备用。
为了验证本发明多肽 （包括从新疆穴居狼蛛粗毒获得的天然多肽或者人工合成多肽） 是 否具有抗癌活性， 采用 MTT法检测其细胞毒性， 以及采用裸鼠荷瘤实验检测其对实体瘤的生 长抑制作用。
1、 主要仪器和材料
穴居狼蛛采集于新疆哈密地区， 并在湖南师范大学生命科学学院蛋白质化学研究室动物 房人工饲养。 通过电剌激方法采集粗毒， 粗毒冻干后保存于一 20° C。 裸鼠购买于上海。 所有 测序试剂均来自美国应用生物系统公司； 固相化学合成试剂购买于美国 Chemassist公司； MTT [3- (4，5-二甲基一 2—噻唑）一 2， 5—二苯基溴化四唑]、 异硫氰酸荧光素购自于美国西格 马公司； Annexin V—异硫氰酸荧光素 /碘化丙啶双染试剂盒来购买于南京凯基公司； 小牛血 清购买于四季青公司； DMEM和 RPMI 1640培养基购自于 Gibico公司； 其它试剂均为国产分析纯 试剂。 癌细胞株： Hela、 HT1080、 JB6和 HNE1。 仪器： 基质辅助激光解吸电离飞行时间质谱、 491A型气相测序仪， 荧光显微镜、 荧光分光光度计、 流式细胞仪。
2 、 实验结果分析
2. 1 分离纯化穴居狼蛛粗毒及其生物性质分析
利用分析型反相高效液相色谱分离纯化穴居狼蛛粗毒（见附图 1 )， 标记的色谱峰经质谱 鉴定显示单一分子量峰， 其单同位素分子量 (M+H+)为 2886. 754Da。 通过 Edman降解测得本发明 多肽的一级结构为 24个氨基酸残基， 没有半胱氨酸残基， 比较实验测定分子量和理论计算分 子量确定该多肽 C一末端酰胺化修饰。 序列分析结果显示该多肽的一个典型特征便是含有 7个 赖氨酸残基，其理论 pi约为 10. 78，而且 lys较规则的分布于整个氨基酸序列形成所谓的 ΚΧΠΚ (Χ 代表疏水性氨基酸残基)模体； 圆二色光谱显示本发明多肽能够形成 α—螺旋构象， 通过绘制 本发明多肽的螺旋轮可以清晰的显示在 α—螺旋一侧为极性氨基酸残基， 而其相反的一侧由 疏水性氨基酸组成， 因而是一个双亲性的 α—螺旋。
2. 2 本发明多肽的人工化学合成
由于穴居狼蛛粗毒量较少， 而且本发明多肽在穴居狼蛛粗毒中的含量也比较低， 因而需 要通过化学合成获得足够量的样品。本发明多肽只含有 24个氨基酸残基， 合成相对容易。 我 们以 0. 1 毫米规模合成了本发明多肽， 经质谱鉴定显示纯度较高， 其单同位素分子量 (Μ+Η+) 为 2886. 621Da (见附图 2)， 与天然多肽分子量相同。
2. 3 本发明多肽选择性杀灭癌细胞
通过 MTT分析法测定了本发明多肽 对 Hela、 HT1080、 HNE1、 CNE1和 JB6五种细胞株的 毒性。 40微摩尔 本发明多肽对五种细胞株都有明显的杀灭作用（见附图 3)，其中对 HT1080、 Hela细胞的毒性最强， 而对 JB6细胞毒性相对较弱， 分别杀灭 98 %和 36 %的细胞， 说明不 同的细胞对本发明多肽敏感性不相同， HT1080和 Hela分别为纤维肉瘤和宫颈癌细胞株， 而 JB6 为永生化的小鼠上皮细胞株， 该细胞株可以传代培养， 但没有癌细胞的恶性增殖特点。 上述结果表明本发明多肽对癌细胞的杀灭作用有一定的选择性。
为了进一步验证， 采用溶血实验检测本发明多肽对于正常细胞的毒性， 结果显示 200微 摩尔 本发明多肽只能裂解约 20 %的红细胞， 说明本发明多肽对于正常细胞毒性较弱。 在整 体动物水平， 我们也检测了本发明多肽的毒性， 以 200 毫克 /kg体重皮下注射小鼠没有出现 明显的毒性反应 （48小时内）， 表明本发明多肽毒性较低， 对于癌细胞具有较强的选择性。
采用 Annexin V—异硫氰酸荧光素 /碘化丙啶双染法检测了本发明多肽杀灭癌细胞的机 制， 发现本发明多肽可诱导 Hela细胞凋亡， 如附图 4所示， 未经药物处理的 Hela细胞凋亡 较少， 而 20 微摩尔本发明多肽处理后可导致约 25. 6%的 Hela细胞凋亡， 说明本发明多肽通 过诱导细胞凋亡杀灭癌细胞。
2. 4 本发明多肽抑制裸鼠荷瘤生长
上述实验表明本发明多肽具有较强的抑制癌细胞作用， 但仍需要确定本发明多肽是否具 有在体内抗实体瘤的作用， 采用裸鼠荷瘤模型开展研究。 实验结果显示本发明多肽可明显抑 制实体瘤生长。 如附图 5所示， 在给药前 （0天）， 对照组和给药组的实体瘤体积大小相似， 而在药物干预的时间内， 生理盐水对照组的肿瘤体积快速增大， 表明其肿瘤持续生长， 而给 药组肿瘤体积没有明显变化， 其中一只裸鼠肿瘤在给药 3次后完全消失， 说明肿瘤生长被抑 制。 在第 10天给药完成， 对照组的肿瘤体积是给药组的 5倍 （P〈0. 05) (见附图 6) ，处死裸 鼠后剥离实体瘤， 发现给药组的肿瘤明显小于对照组， 通过称重表明对照组的肿瘤重量是给 药组的 5倍 （P〈0. 03) (见附图 6)， 上述结果表明本发明多肽可有效的抑制肿瘤生长。
采用肿瘤切片苏木精和伊红染色组织染色方法检测本发明多肽抑制肿瘤生长的机制。 对 照组肿瘤组织细胞排列紧密规则， 细胞核染色为浅蓝色， 而给药组肿瘤组织细胞排列疏散， 细胞核浓缩， 染色为深蓝色， 说明本发明多肽诱导肿瘤细胞凋亡而抑制其生长。
2. 5 本发明多肽抑制 Hela细胞的活性比较
上述实验表明本发明多肽均具有较强的抑制癌细胞及抗实体瘤的作用，但仍需确认不同 序列的多肽对肿瘤细胞的抑制程度， 采用 Hela细胞活性测定进行比较研究。 （见附图 7) 由图可知， 序列 SEQ NO. 1-序列 SEQ NO. 4在 40μηιο1/ί的浓度下对 Hela细胞的抑制率 均为 90%左右， 在 2(^mol/L的浓度下对 Hela细胞的抑制率均为 50%左右， 在 5 mol/L的浓度 下对 Hela细胞的抑制率仅 2%左右， 由此可知序列 SEQ NO. 1-序列 SEQ NO. 4抑制 Hela细胞的 活性相当， 几乎差别不大； 而序列 SEQ NO. 5在 4(^mol/L、 20μηιο1/ί、 5 mol/L的浓度下， 均 能对 80%以上的 Hela细胞起到抑制作用， 活性较序列 SEQ NO. 1 -序列 SEQ NO. 4提高了约 10倍。 序列 SEQ NO. 1和序列 SEQ N0. 5在 200μιηο1时分别溶解 19%和 37%的红细胞 （见附 图 8)， 表明序列 SEQ Ν0. 5的溶血活性强于序列 SEQ N0. 1。 工业实用性
通过在天然毒液中分离纯化和人工化学合成可以大量的获得高效低毒的本发明抗癌活性 肽， 发明人通过大量的药理学研究表明本发明多肽对于开发治疗诸如肺癌、 肝癌、 子宫颈癌 等实体瘤的药物有着较好的应用前景。

Claims

权利要求
、 一种抗癌活性多肽其氨基酸序列为： SEQ NO. 1 编码的序列， 及包括编码对活性肽分 子的氨基酸进行一个或多个的氨基酸替换、 插入或者缺失所得到的功能类似物， 或是 包括编码与 SEQ NO. 1所示的氨基酸序列至少 90%的同源性。
、 根据权利要求 1所述的一种抗癌活性多肽，其特征在于所述多肽其氨基酸序列为： SEQ NO. 2编码的序列。
、 根据权利要求 1所述的一种抗癌活性多肽，其特征在于所述多肽其氨基酸序列为： SEQ NO. 3编码的序列。
、 根据权利要求 1所述的一种抗癌活性多肽，其特征在于所述多肽其氨基酸序列为： SEQ N0. 4编码的序列。
、 根据权利要求 1所述的一种抗癌活性多肽，其特征在于所述多肽其氨基酸序列为： SEQ 能 N0. 5编码的序列。
PCT/CN2008/071804 2007-07-31 2008-07-30 Polypeptides having anticancer activity WO2009015602A1 (en)

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