【作者】 邹祖全；

【导师】 李铎； 张晓宏；

【作者基本信息】 浙江大学 ， 食品科学， 2009， 博士

【摘要】 恶性肿瘤严重威胁着人类的健康和生命,其中肺癌是死亡率和发病率都居于第一位的恶性肿瘤。结肠癌和原发性肝癌也是常见的恶性肿瘤,其发病率和死亡率也都居于恶性肿瘤的前列。目前对于恶性肿瘤的治疗方法还是传统的手术、化疗和放疗,但由于传统的肿瘤治疗方法无靶向特异性,在杀死肿瘤细胞的同时往往也会导致正常组织的损伤,从而有很强的副作用。随着肿瘤分子生物学的发展,针对恶性肿瘤的分子事件的靶向治疗越来越受到人们的重视。靶向治疗以及复合疗法将为恶性肿瘤的治疗开辟新的治疗途径。近年来的研究发现,PI3K/Akt/mTOR和Ras/Erk信号通路在肿瘤细胞生长、增殖、侵袭和转移中起关键作用,阻断细胞内相应的信号通路能抑制肿瘤细胞生长甚至促进肿瘤细胞凋亡。PI-103是由加州大学旧金山分校的研究人员合成的一种PI3Kα/mTOR抑制双靶点抑制剂,U0126是MEK激酶的抑制剂,两种抑制剂目前都处于细胞实验阶段,还没有用于临床肿瘤治疗。其中PI-103购自德国Merck公司,U0126购自Sigma公司。本文研究了PI-103单用以及和MEK抑制剂U0126、化疗药物紫杉醇和多不饱和脂肪酸DHA联用在非小细胞肺癌、肝癌和结肠癌中的抗癌效果。结果如下：1.研究发现在非小细胞肺癌A549细胞和H460细胞中,双抑制剂PI-103有显著的抗癌活性。PI-103在PIK3CA基因(该基因编码PI3K蛋白的催化单元p110α)突变的H460细胞有更明显的抗肿瘤作用。在A549和H460细胞中,PI-103和MEK抑制剂U0126联用其药物联用指数CI＜1,表明这两种药物有协同诱导细胞生长抑制的作用。PI-103和U0126协同诱导细胞生长抑制作用主要是通过阻断细胞内PI3K/Akt/mTOR和Ras/Erk通路,而且PI-103阻断了因雷帕霉素抑制nTOR而导致的Akt活化。研究发现PI3K/Akt/mTOR和Ras/Erk通路会共同作用于mTOR激酶。而且发现PI-103和U0126联用能同时抑制核糖体蛋白s6和真核起始因子4B(eIF4B)的磷酸化,从而抑制翻译复合物的组装和翻译起始,最终协同诱导细胞生长抑制,解释了为什么PI-103和U0126能协同诱导细胞的生长抑制。2.在A549细胞和H460细胞中,PI-103诱导了G0／G1期细胞周期停滞,和在A549细胞中相比,PI-103在H460细胞中诱导更明显的细胞周期停滞。PI-103和U0126联用协同诱导了G0／G1期细胞周期停滞。PI-103和U0126联用协同诱导的G0／G1期细胞周期停滞与细胞内G0／G1期相关周期调节蛋白改变有关,如细胞周期激酶抑制因子p21和p27上调,以及细胞周期蛋白cyclin D1和cyclin E1下调。3.PI-103不仅在A549和H460细胞中协同加强了紫杉醇的细胞生长抑制作用,而且也协同加强了紫杉醇诱导的细胞凋亡。PI-103和紫杉醇联用诱导的细胞凋亡与细胞内凋亡蛋白协同改变有关,紫杉醇和PI-103联用和单用相比,肿瘤抑制因子p53和促凋亡蛋白Bad显著上调,抗凋亡蛋白Bcl-2显著下调。这些凋亡相关蛋白的改变诱发了线粒体凋亡相关途径,从而导致细胞凋亡。PI-103作为第一种合成的PI3Kα/mTOR双抑制剂,其单用或者与U0126、紫杉醇联用都在非小细胞肺癌细胞A549和H460中显示了明显的抗癌活性。表明了复合治疗是一种有效的肿瘤治疗策略。我们的研究为吉非替尼抗性的非小细胞肺癌提供了一种崭新的治疗方案。4.同时我们也研究了PI-103在结肠癌细胞系CaCO2和肝癌细胞系HepG2中的抗癌效果,发现PI-103单用也能明显地抑制CaCO2和HepG2细胞的生长。而且PI-103和U0126联用也能协同诱导细胞生长抑制。在HepG2细胞中,PI-103和U0126也协同诱导了G0／G1期细胞周期停滞。PI-103同样能协同加强化疗药物紫杉醇诱导的细胞凋亡作用。也说明PI-103在结肠癌和肝癌中,是一种潜在的靶向治疗药物。我们的研究也同样为结肠癌和肝癌的临床治疗提供了潜在的治疗方案。5.DHA的抗癌活性与脂质过氧化有关,脂质过氧化产物对细胞有毒性从而抑制肿瘤细胞生长。另一方面DHA发生脂质过氧化因而减少了膜上DHA的水平,导致膜上磷脂酰丝氨酸减少,抑制了细胞内PI3K/Akt信号通路转导,从而抑制肿瘤细胞生长。血清能明显拮抗DHA的抗癌作用,10%的血清下,DHA反而促进肿瘤细胞生长,可能是血清中的生长因子或者牛血清白蛋白阻碍了膜上富集的DHA发生脂质过氧化反应,从而促进了细胞存活通路PI3K/Akt的信号转导。PI-103通过阻断磷脂酰丝氨酸介导的PI3K/Akt信号传导,有效地拮抗了DHA在正常培养下对A549细胞的生长促进作用。我们的研究进一步阐明了DHA的抗癌机制,而且为DHA作为一种辅助抗癌药物的研发具有一定的指导意义。更多还原

【Abstract】 Lung cancer is the leading cause of morbility and mortality of malignant tumors which seriously threat our health and lives. Colon cancer and primary hepatic carcinoma are also two common malignant tumors, which are characterized with high morbility and mortality. The major therapeutics of malignant tumors is surgery, traditional chemotherapy and radiotherapy. Traditional tumor therapies show high side-effects due to theirs non-specificity, which not only kill tumor cells but do harm to normal tissue. With the development of tumor molecular biology, tumor targeted therapy become increasingly important for cancer therapy, targeted therapy and compound therapy will provide several promising strategies for cancer therapy. In the recent years, a series of studies have shown that PI3K/Akt/mTOR and Ras/Erk signaling pathways play a key role in growth, proliferation, invasion and metastasis of cancer cells. Blockade of the two pathways will induce growth inhibition and apoptosis. The novel PI3Ka/mTOR dual inhibitor was synthesized by scientists in University of California in San Francisco, U0126 is the MEK kinase inhibitor. These two inhibitors are being used in preclinical experiments. PI-103 was purchased from Merck company, and U0126 from Sigma company. In the present study, the antitumor activity of PI-103 alone or in combination with U0126, paclitaxel and DHA was observed in non-small lung cancer cell line A549 and H460, colon caner cell line CaCO2 and hepatic carcinoma cell line HepG2. The results were presented as follows:1. PI-103, the novel PI3Kα/mTOR dual inhibitor showed high antitumor effects in A549 and H460 cells. H460 cells with activating mutations of PIK3CA, the gene encoding the p110αcatalytic subunit of PI3K, were more sensitive to low-dose PI-103. The CI value of PI-103 in combination with U0126 was less than one, suggesting that the combination of PI-103 and U0126 acted synergistically to induce growth inhibition. The synergistic antitumor effects were derived from concomitant inhibition of the PI3K/Akt/mTOR and Ras/Erk signaling pathways. Additionally, Akt phosphorylation induced by mTOR inhibition with rapamycin was inhibited by PI-103. We also found that mTOR functions downstream of Akt and Erk. The phosphorylation of ribosomal protein s6 and the eukaryotic translation initiation factor 4B (eIF4B) was concomitantly inhibited by PI-103 and U0126, which may cooperatively inhibited translation initiation complex assembly and translation initiation. This data explained why blockade of the PI3K/Akt/mTOR and Ras/Erk shows synergistic antitumor effects.2. G0/G1 arrest was induced by PI-103 in A549 and H460 cells, and H460 cells were more sensitive to PI-103. PI-103 synergized with U0126 to induce G0/G1 arrest in A549 and H460 cells. Our results showed that PI-103 or U0126 exerts its effects on cell cycle progression via the upregulation of p21 and p27, and the simultaneous downregulation of cyclin D1 and cyclin E1. The synergistic antiproliferative effects of PI-103 in combination with U0126 were derived from markedly up-regulating p21 and p27 protein levels, and significantly inhibiting the expression of cyclin D1 and cyclin E1.3. PI-103 not only potentiated paclitaxel-induced growth inhibition but also enhanced paclitaxel-induced apoptosis in A549 and H460 cells. Apoptosis induced by paclitaxel and PI-103 was related to alterations of apoptosis proteins. Compared with treatment of paclitaxel alone, the combination of PI-103 and paclitaxel significantly up-regulated tumor suppressor P53 and pro-apoptosis protein Bad, and simultaneously down-regulated anti-apoptosis protein Bcl-2. Alterations of these apoptosis-related proteins finally triggered mitochondrial-related apoptosis pathway and led to cell apoptosis. Our study showed that compound therapy is the most effective tumor therapeutics, and provide a potential therapy strategy for gefitinib-resistant non-small cell lung cancer.4. High antitumor activity of PI-103 was also showed in CaCO2 and HepG2 cells. PI-103 in combination with U0126 also showed synergistic antiproliferative effects with induction of G0/G1 arrest in CaCO2 and HepG2 cells. We also observed that Paclitaxel-induced apoptosis was enhanced by PI-103 in HepG2 cells. Our study suggested that PI-103 alone or in combination with other kinase inhibitors, or chemotherapy agents is a promising therapeutics for treating colon cancer and hepatic carcinoma.5. The antitumor effects of Docosahexaenic Acid (DHA) were associated with lipid peroxidation. Lipid peroxidation may decrease DHA levels in cell membrane and lead to reduced phosphatidylserine in intracellular membrane, which blocks the PI3K/Akt signaling pathway and leads to cell growth inhibition. Antitumor effects of DHA were significantly antagonisted by serum. High-concentration DHA promoted cell growth in A549 cells in culture with 10% FBS. It is possible that growth factors or BSA impedes the lipid peroxidation of DHA enriched in plasma membrane and promotes the signaling of the PI3K/Akt pathway, which is attenuated by PI-103. Our study further elucidated the antitumor mechanism of DHA and provided meaningful directions for development of DHA as an adjuvant antitumor agent.更多还原