中国科学院昆明动物研究所机构知识库

胰腺癌是一种死亡率高、侵袭性强的恶性肿瘤。由于其异质性和缺少早期干预,当诊断出时已经发生转移。同时,临床上使用的主流化学治疗药物由于缺乏靶向性,在发挥药效的同时产生诸多不良反应。本研究以金环蛇 (Bungarus fasciatus)抗菌肽Cathelicidin-BF为模板,利用其特殊的构象,进行改造和修饰,保留其抗肿瘤活性同时降低其细胞毒作用,获得一个15个氨基酸的衍生多肽LZ1。 体外实验中，本研究发现LZ1可以显著抑制诸多胰腺癌细胞系的增殖，其中对PANC-1细胞24 h的IC50为17μM，对正常细胞系的毒性较弱，表明其对肿瘤细胞具有选择性。此外，LZ1还能显著抑制PANC-1细胞的侵袭。随后，本研究应用一系列机制实验研究LZ1抗肿瘤的作用机制。MDC染色表明LZ1可以提高自噬泡的数量，此外western blotting，电镜以及免疫荧光的结果也提示LZ1可以提高自噬水平，其对细胞周期没有影响。LZ1并不能抑制和干扰PANC-1的复制和分裂，而能诱导其死亡。同时这种死亡不是凋亡坏死，而是自噬性死亡。接着，本研究证明LZ1诱导的细胞自噬是完全自噬途径。 为了找到LZ1可能发挥选择性的作用靶点，本研究应用pull-down技术发现了LZ1可能和一个分子量55 kDa的剪切形式的C-terminal truncated NCL(TNCL)相互作用，又进行了蛋白互作实验和基因敲降技术来验证这种结合的可靠性，推测LZ1的选择性是由NCL介导的。这是个全新而且具有选择性的靶标。最后，本研究还考察了LZ1的体内药效，并且同一线药物吉西他滨进行了比较。在原位移植瘤胰腺癌裸鼠模型上，LZ1剂量依赖性的减少了肿瘤的体积和重量，并且延长了裸鼠的生存率。相近药效情况下，LZ1体内毒性远小于吉西他滨。同时，还对LZ1的血浆稳定性和体内药物代谢动力学特征进行了考察。本研究的结果表明LZ1是药效显著，机制明确，靶点新颖，安全可控的治疗胰腺癌的候选分子药物。

Pancreatic cancer is a highly aggressive and lethal malignancy. Due to heterogeneity and lack of early preventation, it is most likely to be at an advanced or metastatic stages at time of diagnosis. Meanwhile, the clinical chemotherapeutical agents exhibit treatment benefit accompanied by many adverse effects because of non-targeted cytotoxic effects. We adopted approaches of substitution of amino acids and C-terminal modification to reserve anti-tumor activity and reduce cytotoxic reaction, based on antimicrobial peptide Cathelicidin-BF from Bungarus fasciatus as atemplate, then obtained one derived peptide composed of 15 amino acids, named LZ1. In pharmacodynamics assays, LZ1 significantly inhibited different types of pancreatic cancer cell lines in vitro. The IC50 of LZ1 on PANC-1 is 17μM. Moreover,its toxicity on normal cell lines is relatively very low, indicating its selectivity on cancer cells. In addition, LZ1 significantly prevented the invasion of PANC-1.Subsequently, we applied a series of assays to explore the anti-tumor mechanism of LZ1. MDC staining showed LZ1 increased the number of autophagic vacuoles. In addition, the results of western blotting, electron microscopy and immunofluorescence indicated that LZ1 increased autophagy level. However, LZ1 had no effect on cell cycle. We conclude that LZ1 may not inhibit and interfere with the replication and division of PANC-1, but could induce cell death. Moreover, the cell death induced by LZ1 is autophagic-programmed and is not associated with apoptosis or necrosis.Next,we found that the autophagy induced by LZ1 was through a complete autophagic pathway. In order to investigate that possible target that results in LZ1 selectivity, we applied pull-down technique. The results showed that LZ1 bound to a 55-kDa cleaved form of Nucleolin (NCL), named C-terminal truncated NCL (TNCL). Several protein interaction and gene knockdown experiments carried out to verify the reliability of this interaction, suggesting the selectivity of LZ1 towards cancer cells is mediated by NCL.This is a new target provided with selectivity. Furthermore, we examined efficacy of LZ1 in vivo, compared to the first-line treatment agent gemcitabine. LZ1 reduced the tumor volume and weight in a dose-dependent manner in orthotopictransplantable nude mice model of pancreatic cancer. LZ1 also prolonged the survival rate of the treated nude mice. Above all, the toxicity of LZ1 in vivo is far less than that of gemcitabine in the term of similar efficacy. Evaluation of plasma stability and pharmacokinetics of LZ1 in BALB/c-nude mice with LC-MS/MS analysis was also completed. In summery, LZ1 is a potential therapeutic candidate agent for pancreatic cancer with potent effect, clear mechanism, novel target and safe outcome.