新人工阳离子多肽AIK抗肿瘤活性的初步研究

Antitumor activity of a novel synthetic cationic peptide AIK: A preliminary observation

目的:研究新发现的人工阳离子多肽AIK在体内外对肿瘤细胞的抑制作用和杀伤机制。方法:MTS方法检测AIK对急性早幼粒白血病细胞HL-60的抑制作用,确定AIK最佳作用浓度及作用时间,并以此条件测定其对10株人肿瘤细胞(95C、95D、HL-60、He La、B95-8、HO-8910PM、HO-8910、SMMC-7721、U2OS和A549细胞)及人正常肝细胞HL-7702的抑制效应。流式细胞术检测AIK对宫颈癌He La细胞凋亡的影响,并经DAPI染色后荧光显微镜下观察细胞的形态学变化。制备小鼠荷肝癌H22细胞皮下移植瘤模型,将36只模型小鼠随机分为PBS阴性对照组、AIK低剂量组[8 mg/(kg·d)]、高剂量组[15 mg/(kg·d)]以及MTX阳性对照组[1 mg/(kg·d)],给予连续10 d瘤旁注射治疗,记录小鼠体质量并观察其活动状态;用药结束后,脊椎脱臼法处死小鼠,比较肿瘤质量及体积。结果:AIK对10种肿瘤细胞均有不同程度的杀伤活性,600μg/ml AIK作用24 h后对肺巨细胞癌95C、白血病HL-60和宫颈癌Hela细胞生长的抑制分别达(90.33±0.75)%、(89.06±1.28)%和(76.09±3.68)%。AIK处理组He La凋亡细胞[(4.88±0.57)%vs(0.51±0.19)%,P<0.05]及坏死细胞比例[(2.96±0.50)%vs(1.87±0.27)%,P<0.05]均显著高于对照组,400μg/ml AIK处理组50%以上的H22细胞出现胞膜破碎、细胞裂解等坏死表型。AIK高剂量组、低剂量组和MTX阳性对照组的移植瘤体积和质量均显著低于PBS阴性对照组(P<0.01)。4组小鼠体质量在治疗期间没有显著差异,但MTX组小鼠体质量在治疗第5天开始出现下降,其余3组在药物处理期间呈上升趋势。结论:AIK能够抑制多种肿瘤细胞的生长,诱导细胞凋亡和坏死;显著抑制肝癌H22细胞小鼠皮下移植瘤的生长,无明显不良反应。

Objective： To evaluate the antitumor activity of a novel synthetic cationic peptide designated as AIK, and to elucidate the underlying mechanism （s） in vitro and in vivo. Methods：In experiments in vitro, the optimal dosage and treatment duration for AIK to exert its maximal cell proliferation inhibitory activity were determined by MTS cell prolifera- tion assays in human leukemia HL-60 cells. And then, the antitumor activity of AIK at the optimal dosage and for the opti- mal duration of treatment was assessed in ten tumor cell lines （i. e. , 95C, 95D, HL-60, HeLa, B95-8, HO-8910PM, HO-8910, SMMC-7721, U2OS, A549） and one human normal liver cell line HL-7702. The effects of AIK on HeLa cell apoptosis and morphology were examined by flow cytometry and microscopy respectively. In experiments in vivo, mouse liver cancer H22 cells were transplanted into the armpits of male Kunming mice. Thirty-six mice that developed tumors of a similar size as confirmed 5 days after H22 cell transplantation were randomized to receive subcutaneously PBS （vehicle control）, low dose AIK （8 mg/[ kg· d ] ）, high dose AIK （ 15 mg/[ kg · d] ） and methotrexate （ 1 mg/[ kg- d] ） of as a positive control daily for 10 days, during which body weight was measured every day. Animals were sacrificed 24 h after the last treatment. Subcutaneous tumors were counted in each animal. Data on the weight and volume of the individual tumors were obtained and analyzed. Results： In vitro, AIK exhibited a potent eytotoxic activity against all types of cancer cells tested, in particular, the pulmonary giant cell carcinoma 95C cells, the leukemia HL-60 cells, and the cervical cancer HeLa cells. AIK treatment, as compared with vehicle control, resulted in significant increases in apoptosis （[4.88±0.57]% vs [0.51±0.191%, P〈0.05） and necrosis （[2.96 ±0.50]% vs [1.87±0.27%, P〈0.05） in HeLa cells. Microscopic assessment showed necrotic phenomena including dilated, fragmented cell membrane and cell lysis in more than 50% of AIK treated H22 cells. In vivo, tumor volume and weight were significantly smaller in both AIK-and methotrexate-treated animals than in PBS-treated animals （P 〈 0.01 ）. Body weight declined in methotrexate- treated animals but tended to increase in other 3 groups of animals 5 days after treatment. Nevertheless, the overall differ- ences in body weight changes between the 4 groups of animals failed to reach a statistical significance （P 〉 0.05 ）. Con- clusion ： The novel cationic peptide AIK possesses a potent antitumor activity both in vitro and in vivo, thus having a great therapeutic potential for various types of cancer.