【作者】 林婷婷；

【导师】 沈家骢； 刘俊秋；

【作者基本信息】 吉林大学 ， 高分子化学与物理， 2010， 博士

【摘要】 肿瘤坏死因子相关的凋亡诱导配体(TRAIL , tumor necrosis factor-related apoptosis-inducing ligand)是肿瘤坏死因子(TNF)超家族成员之一。研究表明,TRAIL与靶细胞表面的死亡受体结合能够特异性地诱导多种肿瘤,如白血病、乳腺癌、直肠癌、膀胱癌、黑色素瘤、恶性神经胶质瘤、肺癌、前列腺癌等细胞凋亡,而对正常细胞没有影响。其独特的生物学特点给肿瘤治疗带来了新的希望,被公认为最有前途的治疗肿瘤的蛋白。近来,TRAIL的临床应用出现瓶颈,因为多数肿瘤细胞产生了对TRAIL不同程度的耐受机制。研究者们提出设想,能否通过某种药物消除肿瘤细胞对TRAIL的耐受性,而对其介导的凋亡敏感。已经有大量实验表明,通过与其他药物的联合作用,可以显著的增强TRAIL杀伤肿瘤细胞的能力。谷胱甘肽过氧化物酶(GPx)模拟物对肿瘤细胞的增殖具有抑制作用。在本论文中,我们以乳腺癌细胞为研究对象,详细探讨了两种GPx模拟物:双硒桥联环糊精(2-SeCD)及双碲桥联环糊精(2-TeCD)作为TRAIL的敏化试剂联合诱导细胞凋亡的效应和相关机制。1.在细胞水平验证了2-SeCD可以作为TRAIL的敏化试剂首先以体外培养的乳腺癌细胞株MDA-MB-468与T47D细胞为例进行研究。结果表明, TRAIL单独作用的时候不能够杀伤乳腺癌细胞,而2-SeCD与其联合作用时,可以显著改变细胞的耐受性,恢复凋亡信号传导蛋白Caspase级联信号的传导,增加TRAIL诱导细胞凋亡的能力。通过对凋亡通路的研究,我们发现,2-SeCD的敏化机制在于剂量依赖性的上调死亡受体death receptor 5 (DR5)的表达含量,并抑制转录因子NF-κB亚基p65的核转移。2.建立裸鼠乳腺癌皮下移植瘤模型,论证2-SeCD做为TRAIL敏化药物的可行性在体外实验的基础上,我们通过建立MDA-MB-468乳腺癌荷瘤裸鼠模型,验证了2-SeCD与TRAIL联合应用治疗乳腺癌的药学效应。结果表明,药物联用可以显著抑制裸鼠体内肿瘤生长,并提高荷瘤鼠的生存质量。免疫组织化学染色结果显示,药物联合治疗组中细胞增殖抗原标记物Proliferating Cell Nuclear Antigen (PCNA)及Ki-67的表达含量减少,同时细胞凋亡标记物Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)的表达含量明显增多。最后,2-SeCD上调DR5蛋白表达和下调p65-NF-κB的表达同样在裸鼠荷瘤组织中得到证实。3.在细胞水平验证低剂量的2-TeCD可以作为TRAIL的敏化试剂低剂量的2-TeCD可以与TRAIL协同杀伤乳腺癌细胞,并且通过顺序添加的方式可以将2-TeCD敏化TRAIL的浓度降低到亚微摩尔的水平。通过Annexin V/PI双染实验,以及Caspase活性检测实验,我们进一步证明了TRAIL和2-TeCD联合抑瘤的效应是通过激活Caspase途径,诱导细胞凋亡而实现的。最后2-TeCD敏化TRAIL通路的机制在于剂量依赖性的上调死亡受体DR5蛋白表达,同时阻断NF-κB的活化通路。本研究结果证明GPx模拟物可以作为TRAIL的敏化试剂而应用于临床治疗中。更多还原

【Abstract】 Tumor necrosis factor(TNF)α–related apoptosis-inducing ligand(TRAIL)is a member of the TNF family of cytokines. It exerts cytotoxic effects on malignant cells without any harm to normal cells. To date,five members of the human TNF receptor(TNFR)superfamily have been identified that can bind TRAIL,including death receptors DR4(TRAIL-R1)and DR5(TRAIL-R2),decoy receptors DcR1(TRAIL-R3)and DcR2(TRAIL-R4),as well as soluble receptor osteoprotegerin(OPG). However,only death receptors contain cytoplasmic death domain that are required for transmitting a cytotoxic signal. Following TRAIL engagement with either DR4 or DR5,the ligated death receptors cluster and initiat the formation of the death-inducing signaling complex(DISC). The functional death-inducing signaling complex is minimally composed of death receptors,FADD and Caspase 8 or 10. Active Caspases 8 and 10 cleave and directly activate downstream effector Caspases(3,6,7),which could ultimately result in apoptosis. Meanwhile,TRAIL receptors signaling pathway also leads to the activation of the nuclear factor-κB(NF-κB),which operates as a negative regulator for DISC formation. The subunit of NF-κB could up-regulate anti-apoptotic genes,such as cellular c-FLIP and c-IAPs,which eventually thwart activation of Caspases.As a promising therapy agent for treatment of malignancies,TRAIL has been shown to induce apoptosis against breast carcinoma. Unfortunately,the majority of cell lines are insensitive to TRAIL-induced apoptosis,and the mechanism of the resistance has been attributed to dysfunction of different steps in the apoptosis pathways,as well as elevation of survival signals. The combination therapy was therefore envisaged,basing on the recent evidences that pretreatment of tumor cells with a chemotherapeutic agent could sensitize them to TRAIL. However,the combinations should be taken very carefully due to the potential toxicity to normal tissues. In this respect,it will be important to find a safe and effective agent to overcome TRAIL resistance in breast cancer therapy.Seleno-cyclodextrin(2-selenium-bridgedβ-cyclodextrin,2-SeCD),a synthetic seleno-organic compound with unique glutathione peroxidase(GPx)-like activity,has exhibited anti-oxidant,anti-inflammatory and anti-tumor properties with very low toxicity. It has been extensivelydemonstrated as a promising substitute for ebselen,a well known GPx mimic,with enhanced enzymatic activity and water solubility. 2-tellurium-bridgedβ-cyclodextrin(2-TeCD),another synthetic organotellurium compound,has exhibited anti-oxidant and anti-inflammatory activities in vitro. Furthermore,2-TeCD has been shown to exhibit anti-tumor effects on breast carcinoma through selective inhibiting of thioredoxin reductase(TrxR)which is overexpressed in tumor tissues. Considering the anti-tumor property of the two agents,we hypothesized that their combination with TRAIL could be a promising strategy in the treatment of refractory breast tumors.1. 2-SeCD sensitizes resistant human breast cancer cells to TRAIL-induced cytotoxicity in vitro.We examined MDA-MB-468 and T47D cells for TRAIL sensitivity using different concentrations of recombinant human soluble TRAIL and/or 2-SeCD. Cells did not exhibit growth arrest by TRAIL alone treatment,while significant decrease of cell viability was observed with a synergistic treatment of 2-SeCD and TRAIL. Using the accepted criterion that apoptotic cells are Annexin V–positive/Propidium iodide–negative,we found that more than 50 percents of cells were undergo apoptosis in both of the cancer cell lines,upon exposure to the combination of the two agents. This experiment suggests that the sensitization of TRAIL-induced cytotoxicity by 2-SeCD is associated with apoptosis.We next analyzed the Caspase-8 cleavage,a proximal event in the TRAIL-induced Caspase cascade. TRAIL alone failed to induce detectable Caspase-8 and Caspase-3 processing when compared to control. In contrast,cleaved-Caspases was clearly detected in both of the cancer cells when treated with the combination treatment of 2-SeCD and TRAIL. The immunoblotting results were further substantiated by Caspase activity assay. Compared to agent-alone group,the combination group significantly increased Caspase-3 and Caspase-8 activity in both of the MDA-MB-468 and T47D cells to about 4~6 times.The activation of the Caspase-8 in combined treatment suggests that 2-SeCD targets an early step in the TRAIL-induced apoptosis pathway; we therefore evaluated the effects of 2-SeCD on expression of TRAIL receptor DR4 and DR5. Using Western bolting,flow cytometric analysis,RT-PCR reactions , as well as iRNA techniques we extensively showed that 2-SeCD dose-dependently induced the expression of TRAIL receptors DR5,but not DR4 on both mRNA and protein levels. These data suggest that up-regulation of DR5 by 2-SeCD is essential for the ability of TRAIL to induce apoptosis in resistant breast cancer cells.2-SeCD treatment also suppressed TRAIL-induced nuclear factor-κB(NF-κB)prosurvival pathways by preventing cytosolic IκBαdegradation,as well as p65 nuclear translocation. Since traditional selenium compounds were not reported to target NF-κB activity in sensitizing TRAIL-resistant cancers,we hypothesize their unique GPx -like activity would be an attractive explanation. Therefore,inhibition of NF-κB nuclear translocation by 2-SeCD also contributes to its sensitization of TRAIL-induced apoptosis in breast cancer cells.2. 2-SeCD and TRAIL results in tumor growth inhibition and apoptosis in vivo.Based on the above findings,we next sought to evaluate whether the effect of 2-SeCD alone and in combination with TRAIL could inhibit tumor growth in vivo. MDA-MB-468 cells were subcutaneous injected into the right axilla of the female Balb/c nu/nu mice. TRAIL was ineffective in inhibiting the tumor growth; however,the combination of 2-SeCD and TRAIL exhibited significant inhibitory effects compared with other treatment groups. Immunohistochemical examination of tumor tissues revealed that 2-SeCD alone inhibited cell proliferation [proliferating cell nuclear antigen(PCNA)and Ki-67 staining] and induced apoptosis(TUNEL staining)in xenograft tumors. On the contrary,TRAIL had no significant effects compared with control group. Importantly,the combination of 2-SeCD and TRAIL were more effective in inhibiting tumor cell proliferation and inducing apoptosis than single agent alone. Finally,xenograft sections from each of the treatment groups were immunostained with anti-p65 and anti-DR5 antibodies. Treatment of mice with a combination of 2-SeCD and TRAIL significantly showed more expression of DR5 and less expression of p65-NF-κB proteins than that of mice treated with vehicles or TRAIL.For the first time,we present clear evidences that 2-SeCD,a GPx mimic,can overcome TRAIL resistance in vitro and in vivo; therefore this combination provides a powerful therapeutic option for human breast cancer treatment.3. Low doses of 2-TeCD could sensitizes resistant human breast cancer cells to TRAIL induced cytotoxicity in vitro.2-tellurium-bridgedβ-cyclodextrin(2-TeCD)shares the unique chemical characteristics with its selenium counterparts(2-SeCD),but with a much better GPx mimic activity. Despite the antioxidants properties,2-TeCD has been recently shown to exhibit anti-tumor effects on breast carcinoma with a very low concentration. However,the application of 2-TeCD should be taken very carefully due to potential toxic effects of tellurium on kidney,hepatic and nervous system. In this study,we reported that 2-TeCD,in a low dose,could convert the TRAIL resistant breast cancer cells to TRAIL sensitive. Importantly,by using a sequential treatment protocol,where cells were first treated with 2-TeCD for 24 hours followed by TRAIL treatment,we could reduce the doses of 2-TeCD for TRAIL-sensitization in the submicromolar range. Using the Annexin V/ propidium iodide staining and Caspase activity assay , we then reavealed that the sensitization of TRAIL-Induced cytotoxicity by 2-TeCD is associated with apoptosis. Finally,the mechanism of sensitization could also attribute to the fact that 2-TeCD dose-dependently induced the expression of TRAIL receptors DR5,as well as down-regulated the activity of NF-κB.更多还原