【作者】 孙建国；

【导师】 吴平； 刘非燕；

【作者基本信息】 浙江大学 ， 生化与分子生物学， 2012， 博士

【摘要】 化疗是癌症临床治疗的重要手段,化疗药物通过直接杀死肿瘤细胞或者抑制肿瘤细胞的分裂增殖、促进细胞分化来达到治疗效果。化疗的缺陷是无差别地对正常细胞的杀伤作用。此外,肿瘤细胞对化疗药物的耐药性也是目前癌症治疗的一个主要障碍,近90%的转移性和复发癌症病人治疗失败都是由于耐药性引起的。因此寻找高效低毒的抗肿瘤药物以及逆转肿瘤细胞的耐药性(Drug Resistance)是治疗癌症的两个重要课题。本研究从蘑菇中提取到一种天然药物成分Clitocine(克力托辛),并发现该化合物能有效抑制多种肿瘤细胞的增殖。更为重要的是我们首次报道了Clitocine能有效诱导耐药性肿瘤细胞的凋亡以及逆转肿瘤细胞的多药耐药性。本文从分子生物学水平揭示了Clitocine的抗肿瘤生物活性的分子机理,并首次确定了该化合物是NF-κB的抑制剂,这一发现暗示了Clitocine可能具备更加广泛的生物活性。本文的主要研究结果如下：1、Clitocine能够诱导肿瘤细胞凋亡采用MTT法检测Clitocine对肿瘤细胞的毒性,结果显示该化合物对多种肿瘤细胞包括肝癌细胞HepG2、R-HepG2(耐药性HepG2)和SMMC-7721、宫颈癌细胞HeLa、胃癌细胞SGC-7901、子宫癌细胞MES-SA和MES-SA/Dx5(耐药性MES-SA)以及乳腺癌细胞MCF-7和Bcap37等都表现出较高的活性抑制作用,其中对肝癌的毒性最大。而且Clitocine对敏感性HepG2细胞和耐药性R-HepG2细胞的毒性很相近。进一步实验结果显示Clitocine能够诱导耐药性肝癌细胞凋亡,包括出现DNA片段化(DNA ladder)、磷脂酰丝氨酸(PS)外翻、Caspase激活、线粒体膜崩溃以及PARP的剪切等一系列凋亡的典型特征。此外,Clitocine诱导的肝癌细胞凋亡涉及细胞外与细胞内两条信号通路。2、Clitocine诱导的细胞凋亡通过Bcl-2家族成员受到调控蛋白免疫印迹(Western Blot)结果显示,Clitocine能够使Bcl-2家族成员抑凋亡蛋白Bcl-2下调而促凋亡蛋白Bad上调。利用RNA干扰技术(RNAi)表明Bad的上调是Clitocine诱导耐药性肝癌细胞凋亡的重要原因。3、Clitocine能够下调P-糖蛋白的表达以逆转耐药性肿瘤细胞的耐药性本文选用的两株耐药性细胞株R-HepG2和MES-SA/Dx5都是细胞膜泵P-糖蛋白(P-glycoprotein)过表达的,该蛋白的过表达是很多肿瘤细胞产生耐药性的重要原因；我们的数据显示Clitocine能够显著降低P-糖蛋白的表达水平。将Clitocine与阿霉素(Doxorubicin)配伍使用可有效增强耐药性细胞对阿霉素的敏感性,大幅提高低浓度下阿霉素对耐药性肿瘤细胞的杀伤作用。流式细胞术的数据显示,Clitocine能够增加阿霉素在细胞内的积累。因此,我们推测Clitocine能够逆转肿瘤细胞的耐药性与P-糖蛋白的表达被抑制密切相关。4、Clitocine通过下调NF-KB抑制P-糖蛋白的表达荧光定量PCR的数据显示,Clitocine下调了P-糖蛋白编码基因MDR1的mRNA水平。我们推测Clitocine是在转录水平调控MDR1基因的表达。因此,我们克隆了P-糖蛋白编码基因MDR1的启动子序列,并构建萤光素酶基因报告系统。利用将启动子分段分析的方法,确认Clitocine调控MDR1基因表达的特定启动子区域。结合转录因子预测软件分析,得到NF-κB可能是Clitocine的作用靶点。染色质免疫共沉淀(CHIP)技术确认了NF-κB能够直接结合到我们确认的启动子调控区域,结合位点的点突变以及NF-κB的过表达能够逆转Clitocine的调控作用。5、Clitocine能够抑制NF-κB的激活Clitocine能够抑制阿霉素诱导的NF-κB表达的增加,并降低NF-κB重要下游基因凋亡抑制蛋白家族成员XIAP的表达。利用电泳迁移率实验(EMSA),我们发现Clitocine能够有效抑制NF-κB的激活以及活化NF-κB向细胞核内的转运。6、Clitocine能够在动物模型中抑制耐药性肿瘤细胞的生长将耐药性肿瘤细胞R-HepG2接种到裸鼠皮下形成肿瘤,Clitocine能够显著抑制肿瘤细胞的生长。此外利用剥离的肿瘤组织进行免疫组织化学实验,结果表明Clitocine能够在体内抑制P-糖蛋白的以及NF-κB的表达。概括起来,Clitocine能够有效诱导耐药性肿瘤细胞的凋亡,并通过抑制NF-κB的来下调P-糖蛋白的表达,进而逆转肿瘤细胞的耐药性。我们首次证明了Clitocine是一种NF-κB的新型抑制剂,这说明该化合物可能具有较为广泛的生物活性,具备了重要了应用前景。更多还原

【Abstract】 Chemotherapy is a very important means during cancer clinical treatment. The chemotherapeutic agents can be used to treat tumor by killing cancer cells directly, inhibiting cancer cells proliferation and growth or activating cancer cells to differentiation. However, these toxic agents are unable to distinguish the cancer cells and normal cells. So, chemotherapy always brings in some side effect during clinical application such as too toxic to patients. Further more cancer cells can also develop drug resistance to drugs during chemotherapy. Resistance to chemotherapeutic drugs is currently a major problem in cancer therapy, accounting for treatment failure in over 90% of human patients with metastatic or recurrent cancer. Therefore, it is urgent to find novel anticancer agents which can kill cancer cells with higher efficiency and significantly circumvent their drug resistance. Here, we extracted a natural compound clitocine from mushroom Leucopaxillus giganteus and further study initially indicated that the compound can effectively inhibit the proliferation of many human cancer cell lines and overcome drug resistance in drug resistant cancer cells. At the present, we revealed the biological activities of clitocine and the underlying molecular mechanism as follows:1. Clitocine can induce apoptosis in human cancer cells. The anti-proliferation effect of clitocine on human cancer cell lines was assessed by the MTT assay, including hepatoma HepG2, R-HepG2 (drug resistant HepG2) and SMMC-7721 cells, human cervical cancer HeLa cells, human gastric cancer SGC-7901 cells, human uterine cancer MES-SA and MES-SA/Dx5 cells, human breast carcinoma MCF-7 and Bcap37 cells. The compound exerted highest cytotoxicity in hepatoma cells and the toxicity is similar in both sensitive and drug resistant cells. Further study showed that citcocine can induce apoptosis in hepatoma R-HepG2 cells which was confirmed by DNA ladder, PS externalization, caspase activation, mitochondria disruption and PARP cleavage. Additionally, both intrinsic and extrinsic signaling pathways were involved in the apoptosis induced by clitocine in R-HepG2 cells.2. Bcl-2 family members regulated the apoptosis induced by clitocine. The data of western blot assay indicated that clitocine can downregulate the Bcl-2 level and upregulate the Bad level. Furthermore, the Bad played an indispensable role in apoptosis induced by clitocine confirmed by using siRNA to silence the Bad mRNA.3. Clitocine can circumvent drug resistance of R-HepG2 cells by downregulating P-glycoprotein expression. Drug resistant R-HepG2 cells presented very low sensitivity to doxorubicin. However, combine using of clitocine and low dose doxorubicin can effectively kill cancer cells. The data from flow cytometry indicated that clitocine significantly increased the doxorubicin accumulation in the cancer cells. More interestingly, we found that clitocine can inhibit the P-glycoprotein expression in both two drug resistant cancer cells R-HepG2 and MES-S/Dx5 which were P-glycoprotein overexpressed.4. Clitocine inhibited P-glycopritein expression via downregulation NFκB. Cloned the P-glycoprotein coding gene MDR1 promoter to construct the dual reporter luciferase system. Truncation analysis of MDR1 promoter sequence was performed to determine the regulatory region of clitocine. Combining computational analysis for the putative transcription factor binding sites, it was confirmed that NFκB may be the target through which clitocine regulated the MDR1 expression. Additionally, the binding of NF-κB to the putative binding site within the MDR1 promoter was confirmed by chromatin immunoprecipitaition (CHIP) assay. Point mutation in the putative binding site of NF-κB and overexpression of this transcriptional factor can interestingly counteracted the clitocine effect on P-gp in R-HepG2 cells.5. Clitocine could inhibit the activation of NF-κB by doxorubicin. Clitocine could also suppress the expression of NF-κB and its’downstream genes such as XIAP even in presence of doxorubicin in RD cells. More importantly, clitocine significantly overcome the activation of NF-κB by doxorubicin which was characterized as nuclear translocation of NF-κB.6. Clitocine can inhibit the tumor growth in animal model. In order to find out if clitocine was also able to suppress the tumor growth in vivo, a nude mouse animal model subcutaneously injected with R-HepG2 cells was employed. After solid tumor formation, the tumors were treated with clitocine. Our compound can effectively inhibit the tumor growth. On the other hand, immunohistochemistry assay showed that the expressions of NF-κB p65 and P-gp were both suppressed by clitocine in vivo.Taken together, Clitocine can induce apoptosis in multidrug resistant cancer cells effectively and suppress the P-glycoprotein expression via inhibiting NF-κB. All our data indicated that Clitocine may possess various biological activities as a novel NF-κB inhibitor and reveal a potential application in future.更多还原