【作者】 燕虹；

【导师】 毕勇毅； 唐定国；

【作者基本信息】 武汉大学 ， 病原生物学， 2009， 博士

【摘要】 背景：已有研究报道肿瘤干细胞(Cancer stem cells, CSCs)或肿瘤起始细胞对化疗天然耐药,或对化疗具有耐药性的肿瘤细胞表现出CSCs的表型特征。也有研究表明CD44+(α2β1hi)前列腺癌细胞含有高致瘤性和转移性的肿瘤干细胞,但是,是否这些肿瘤干细胞对化疗药物具有更强的抵抗力,以及是否在功能上参与前列腺癌的发展目前并不清楚。为了阐明这些重要的问题,我们开展了此项研究。目的：1.了解前列腺癌CSCs是否对化疗药物具有更强的抵抗力或长期化疗药物处理是否富集CSCs。2.从一个新的角度探讨CD44和／或CD44+的前列腺癌细胞在肿瘤发展中的作用。3.为临床有效治疗前列腺癌提供新思路。方法：1.选择含有一部分CD44+细胞的前列腺癌细胞株Du145,用两种临床药物etoposide和paclitaxel,以及三种实验药物stauro sporine、WP1102和WP1103(两种新的paclitaxel类似药)亚致死剂量长期处理该肿瘤细胞株,IC50实验用于检验这些药物选择的细胞是否真正具有耐药性和交叉耐药性。2.将建立的耐药细胞株移植到NOD/SCID小鼠皮下和前列腺,观察肿瘤发展,以评价耐药Du145细胞致瘤和转移能力。3.细胞增殖实验、BrdU掺入实验和克隆形成实验用于评价细胞的增殖速度及克隆效率；Western blotting和免疫荧光染色用于分析各种蛋白的表达水平。4.采用shRNA干扰技术使Du145细胞中CD44表达下降,再通过肿瘤发展实验评价CD44在前列腺癌发展中的功能性作用。结果：通过亚致死剂量的不同药物长期处理肿瘤细胞株,成功建立了各种类型的耐药Du145肿瘤细胞模型。耐药的Du145细胞移植到NOD/SCID小鼠皮下和前列腺,Du145-Paclitaxel(即对paclitaxel耐药的Du145细胞)和Du145-WP1103(即对WP1103耐药的Du145细胞)细胞致瘤能力完全丧失,Du145-VP16(即对VP16耐药的Du145细胞)、Du145-STS(即对STS耐药的Du145细胞)和Du145-WP1102(即对WP1102耐药的Du145细胞)三种耐药细胞致瘤和转移能力表现为不同程度的下降。通过该方法选择的耐药DLD1结肠癌细胞和一部分耐药UC14细胞也表现为致瘤能力下降。体外实验发现耐药Du145细胞增殖减慢,克隆效率下降。进一步分子机制研究发现Du145-Paclitaxel和Du145-WP1103细胞P27和P21表达明显增加,抗凋亡蛋白BCL-2表达下降,而其它三种类型耐药细胞P27和BCL-2表达均有所增加；Western blotting和免疫荧光染色结果显示Du145-Paclitaxel和Du145-WP1103细胞CD44无表达和/或CD44+细胞缺乏,Du145-VP16和Du145-STS细胞CD44表达减少和/或CD44+细胞减少,Du145-WP1102细胞CD44表达和CD44+细胞比例与对照Du145细胞无明显差别,但更多细胞CD44为弱阳性表达。有趣的是,CD44+细胞减少的程度与细胞再生肿瘤的能力密切相关,肿瘤细胞中CD44+细胞越少,其致瘤和转移能力越低。CD44 Knockdown试验进一步证实了Du145细胞中CD44表达下降可抑制肿瘤发生和发展。结论：1.一些耐药肿瘤细胞可能不一定富集CSCs或甚至缺乏CSCs。2.CD44和/或CD44+细胞与前列腺癌发展密切相关,耐药Du145细胞中CD44表达越低和/或CD44+细胞越少,致瘤和转移能力越低。3.CD44可作为临床开发治疗前列腺癌药物时的靶标参考,并应注意提高药物在体内到达CD44+细胞的效率。更多还原

【Abstract】 Background:Cancer stem cells (CSCs) or tumor-initiating cells have been reported to be resistant to therapeutics and therapy-resistant cancer cells have been shown to manifest phenotypic properties of CSCs. We have shown that CD44+(α2β1hi) prostate cancer cell population harbors highly tumorigenic and metastatic stem/progenitor cells, but whether these stem-like cancer cells are more resistant to chemotherapy drugs and are functionally involved in prostate cancer development remains unanswered. To address these important questions, we conducted the study.Objectives:1. To understand whether stem-like prostate cancer cells are more resistant to chemotherapy drugs or whether chronic drugs treatment enrich stem-like prostate cancer cells.2. To determine the functional roles of CD44 and/or CD44+cells in prostate cancer development.3. To provide new sight on efficiently treating prostate cancer in clinical.Methods:1. We chronically treated Du145 prostate cancer cells, which have a subset of CD44+ cells, with two clinically relevant drugs, etoposide and paclitaxel, and three experimental drugs, i.e., staurosporine and two novel paclitaxel analogs WP1102 and WP1103. IC50 assay was used to determine whether the drug-tolerant Du145 cells were actually drug-resistant to selecting drugs and also cross-resistant to non-selecting drugs.2. The limiting-dilution tumor experiments in NOD/SCID mice by injecting subcutaneously (s.c) or orthotopically in the dorsal prostate (DP) were carried out to evaluate the tumorigenicity and metastaticity of drug-tolerant cells.3. Proliferation assay and BrdU incorporation assay were used to measure cell proliferation. The clonal assay was carried out to measure the ability of cells, at the single-cell level, to founder a self-renewing clone. Western blotting and immunofluorescence staining experiments were carried out to measure the protein expressions including P27, P21, BCL-2, CD44, etc.4. To prospectively determine whether CD44 is causally involved in prostate cancer cell tumorigenicity, we infected parental Du145 cells with a lentiviral vector encoding CD44 shRNA (CD44-shRNA) or a non-silencing shRNA control vector (NS-shRNA). Then these cells were implanted either subcutaneously or orthotopically in the DP.Results:Chronic sublethal drug treatment led to generation of drug-tolerant and cross-resistant cancer cells. Surprisingly, when the drug-tolerant cells were implanted, either subcutaneously or orthotopically, into NOD/SCID mice, they showed much reduced tumorigenicity or were even non-tumorigenic. Drug-tolerant DLD1 colon cancer cells and some UC14 bladder cancer cells selected by this protocol also displayed reduced tumorigenicity.Drug-tolerant Du145 cells demonstrated low proliferative and clonogenic potential. Consistent with reduced cell proliferation, drug-tolerant Du145 cells showed increased levels of two important cyclin-dependent kinase inhibitors, p21 and p27, especially in Dul45-Paclitaxel and Du145-WP1103 cells, the two cell lines that completely lacked tumorigenicity. The p27 levels were also elevated in all three other drug-tolerant Du145 cell lines. In contrast to p21 and p27, Bcl-2, an anti-apoptotic protein, showed less again in the two non-tumorigenic Du145 lines, i.e., Du145-Paclitaxel and Du145-WP1103.Western blotting and immunofluorescence staining experiments showed that both Du145-Paclitaxel and Du145-WP1103 cultures completely lacked CD44 and/or CD44+ cells. Du145-VP16 and Du145-STS cultures also showed reduced CD44 protein levels and/or numbers of CD44+ cells. By contrast, Du145-WP1102 cells, which retained some tumor-initiating capacity, showed similar levels of CD44 protein expression to and slightly more CD44+ (many faintly positive) cells than parental Du145 cultures. The extent to which CD44 protein and/or CD44+ cells were ablated in drug-tolerant Du145 cultures appeared to correlate well with the level of reduction in their tumorigenic potential. Prospective knockdown of CD44 in Du145 cells inhibited tumor regeneration. Conclusions:Altogether, our studies reveal that:1. Some drug-resistant cancer cells are not necessarily, and may actually be depleted of, CSCs;2. CD44 and/or CD44+ cancer cells play causal roles in tumor regeneration;3. In exploring the new drugs to treat prostate cancer, it is possible to choose CD44+ prostate cancer cells as therapeutic target, and especially, efficient delivery of drugs in vivo should be considered to improve anti-tumor effects. V更多还原