【作者】 张慧平；

【导师】 李晓明；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 博士

【摘要】 喉癌(laryngeal carcinoma)是头颈部常见的恶性肿瘤,其发病率及死亡率均呈逐年上升的趋势,严重威胁着人类的健康与生命。虽然手术为主,放疗与化疗为辅的综合治疗方法,使患者的5年生存率有所提高。但对于那些晚期或复发、转移的患者预后仍旧很差,手术丧失时机,而传统放化疗又由于其严重的毒、副作用常使得患者不能耐受,从而使患者的治疗受到极大的限制。因此,积极寻求喉癌治疗的新策略成为广大耳鼻咽喉科研究者面临的新课题。泛素-蛋白酶体通路(Ubiquitin-Proteasome Pathway,UPP)是真核细胞中内源性蛋白质选择性降解的重要途径,是调节细胞多种生物学过程如细胞周期运转、增殖、凋亡、信号传递、基因转录等的关键机制。它与人类恶性肿瘤的发生、发展密切相关。阻断UPP可以促使与肿瘤细胞增殖、分化、凋亡密切相关的多种蛋白质代谢发生紊乱,从而诱导细胞凋亡。于是蛋白酶体抑制剂作为一种新型抗肿瘤靶向治疗药物受到人们的广泛关注。硼替佐米作为第一进入临床的蛋白酶体抑制剂已经被美国食品药品监督局(FDA)批准用于难治性及复发性多发性骨髓瘤及套细胞淋巴瘤。关于肺癌及淋巴瘤的研究也已经进入临床试验阶段,但蛋白酶体抑制剂在喉癌中的研究甚少,其作用机制尚不完全清楚。信号转导与转录激活因3(STAT3,signal transducer and activator of transcription-3)是信号转导通路中的重要因子,是EGFR、IL-6/JAK、Src等多个致癌性酪氨酸激酶信号通道的汇聚的焦点,与人类恶性肿瘤的发生、发展和演进关系密切。有报道指出蛋白酶体抑制剂可诱导结直肠癌细胞中STAT3的激活。在喉癌中,蛋白酶体抑制剂对STAT3的影响尚无相关报道。我们前期的研究表明STAT3的激活与喉癌的发生、发展密切相关,抑制STAT3的激活可抑制喉癌细胞的恶性增殖及诱导凋亡。于是本研究以体外培养的人喉鳞癌Hep-2细胞株为研究对象,应用四甲基偶氮唑蓝(MTT)法、流式细胞仪、Western Blot、基因转染等技术探讨了蛋白酶体抑制剂对喉癌细胞的作用、可能机制及对STAT3的影响,并进一步联合pshSTAT3及常规化疗药物顺铂诱导喉癌细胞凋亡作用的观察,为寻找更加有效的分子靶向、多靶点联合治疗途径以及喉癌的临床治疗提供理论基础。本实验分为以下三部分:第一部分蛋白酶体抑制剂MG-132诱导喉癌细胞凋亡及对STAT3的影响目的:通过检测不同浓度及不同作用时间的蛋白酶体抑制剂MG-132对体外培养的人喉鳞癌Hep-2细胞株的增殖、周期分布、凋亡的作用及相关蛋白表达的情况,探讨蛋白酶体抑制剂对喉鳞癌细胞的作用、相关机制及对STAT3的影响。方法:1.以Hep-2细胞为实验对象,四甲基偶氮唑蓝(MTT)法检测不同浓度(0、1、2.5、5、10、20μmol/L)MG-132作用24h、48h及2.5μmol/L MG-132作用0h、12h、24h、48h、72h后细胞的增殖情况。2.流式细胞技术(FCM)检测不同浓度(0、1、2.5μmol/L)MG-132作用48h的细胞凋亡及2.5μmol/L MG-132作用0h、12h、24h、48h、72h后细胞凋亡及周期分布情况。3. Western blot检测MG-132(2.5μmol/L)作用0h、12h、24h、48h后,Hep-2细胞的p21、cyclinD1、CDK4、Bcl-2、STAT3、p-STAT3蛋白表达水平。结果:1. MTT检测显示:MG-132可以有效抑制Hep-2细胞的增殖,在本实验范围内呈浓度(24h:r=0.925,P<0.01;48h:r=0.944,P<0.01)及时间依赖性(r=0.945,P<0.01);24h的半数抑制量(IC50)=20μmol/L;48h的半数抑制量(IC50)=2.5μmol/L。2. FCM结果显示:细胞凋亡率随MG-132作用时间的延长及浓度的递增而上升,同样具有量效(r=0.842,P<0.01)及时效性(r=0.888,P<0.01)。Hep-2细胞的周期分布发生变化,处于G0/G1和G2/M期的细胞比例上升,S期的细胞比例下降(P<0.01)。3. Western blot:p21蛋白水平随着MG-132作用时间的延长而表达显著增强,cyclinD1有中等程度的下调。MG-132作用12h时,可观察到p-STAT3蛋白表达明显增强且维持在一个稳定的水平,Bcl-2蛋白也随之逐渐增强,CDK4及STAT3蛋白水平在不同时间点无明显变化。结论:1.蛋白酶体抑制剂可以有效抑制人喉鳞癌Hep-2细胞在体外的增殖并且诱导其发生凋亡,随作用浓度及时间的不同而变化,呈浓度及时间依赖性。2.蛋白酶体抑制剂可以诱导Hep-2细胞发生G0/G1和G2/M期的双重阻滞。3.蛋白酶体抑制剂对喉癌细胞作用的机制之一与p21蛋白水平的上调及cyclinD1蛋白水平的下调相关。4.蛋白酶体抑制剂在发挥有效作用的同时导致了关键信号分子STAT3蛋白的激活及抗凋亡家族成员Bcl-2蛋白水平的上调,从而在一定的程度上降低了药物的效能。第二部分蛋白酶体抑制剂联合pshSTAT3对喉癌细胞增殖与凋亡的影响目的:研究短发夹RNA沉默STAT3能否增强蛋白酶体抑制剂MG-132对喉癌细胞的抗肿瘤作用。方法:1.构建及扩增pshSTAT3,并经酶切和测序鉴定。2.研究分五组:⑴细胞对照组:未经任何处理的Hep-2细胞;⑵阴性对照质粒组:转染阴性对照质粒的Hep-2细胞;⑶MG-132组:经2.5μmol/L MG-132处理的Hep-2细胞;⑷联合组:阳性重组质粒转染+MG-132(2.5μmol/L)处理的Hep-2细胞;⑸pshSTAT3组:转染阳性重组质粒的Hep-2细胞。3.利用MTT和FCM两种方法分别检测不同实验组Hep-2细胞的增殖活性及凋亡率的情况。4. Western blot检测不同实验组Hep-2细胞p-STAT3蛋白表达的情况。结果:1.经酶切及测序结果证实,成功构建阳性重组质粒pshSTAT3。2.重组质粒转染Hep-2细胞情况的观察:真核表达质粒和阴性对照质粒转染Hep-2细胞后6h即可在荧光显微镜下观察到绿色荧光,转染后24h,绿色荧光逐渐增多增强,荧光倒置显微镜下观察细胞转染效率在80%以上,FCM检测转染效率为85.76%。3. pshSTAT3增强了MG-132对Hep-2细胞的增殖抑制作用:将阳性重组质粒或阴性对照质粒转染Hep-2细胞,转染后24h,联合或不联合2.5μmol/L MG-132,再继续培养48h,MTT法检测各组的细胞增殖抑制率。结果发现阴性对照质粒组和细胞对照组细胞的体外增殖活性无明显差异(P>0.05);联合组能显著抑制Hep-2细胞的增殖活性(抑制率:62.23±1.25%),与MG-132组、pshSTAT3组及阴性对照质粒组相比,差异均有统计学意义(P<0.01)。4. pshSTAT3增强了MG-132对Hep-2细胞诱导凋亡的作用:细胞转染后24h加2.5μmol/L MG-132,再继续培养48h,FCM检测各组的细胞凋亡率,结果显示联合组细胞出现明显的凋亡峰,其凋亡率(55.80±3.12%)显著高于MG-132组(41.20±3.21%)和pshSTAT3组(22.13±1.84%),差异具有统计学意义(P<0.01);阴性对照质粒组和细胞对照组间无明显差异(P>0.05)。5. pshSTAT3与MG-132对p-STAT3表达的影响:Western blot检测显示细胞对照组与阴性对照质粒组两组之间的p-STAT3蛋白水平无明显差异;MG-132组p-STAT3蛋白表达显著增强;联合组及pshSTAT3组p-STAT3蛋白表达明显减弱。结论: pshSTAT3可以抑制MG-132诱导的STAT3蛋白的激活,从而提高了蛋白酶体抑制剂MG-132对喉鳞癌细胞的抗肿瘤作用。第三部分蛋白酶体抑制剂MG-132联合化疗诱导喉癌细胞凋亡的观察目的:探讨蛋白酶体抑制剂MG-132能否增强常规化疗药物顺铂(DDP)对喉鳞癌细胞的毒性作用。方法:取对数期生长的Hep-2细胞分为四组:对照组、MG-132组、DDP组、DDP+ MG-132组。利用MTT和FCM两种方法分别检测不同实验组细胞的增殖及凋亡情况。结果:1. MG-132联合DDP对Hep-2细胞增殖活性的影响:不同浓度DDP(10、20、40、80μmol/L)单用或联合1μmol/L MG-132作用于Hep-2细胞48h后结果显示:在每个浓度水平上DDP组与DDP+MG-132组比较均有统计学意义(P<0.01,P<0.05)。10μmol/L DDP+MG-132组与20μmol/L DDP组、20μmol/L DDP+MG-132组与40μmol/L DDP组比较均具有统计学差异(P<0.01,P<0.05)。DDP单独作用的IC50=62.22μmol/L;当与1umol/L MG-132联合作用时,其IC50下降至27.79μmol/L。2. MG-132联合DDP诱导Hep-2细胞凋亡作用的观察:10μmol/L DDP单用或联合1μmol/L MG-132作用于Hep-2细胞48h后,FCM检测结果显示DDP+MG-132组诱导细胞凋亡作用最强,与对照组、DDP组、MG-132组相比均具有显著性差异(P<0.01)。结论:蛋白酶体抑制剂能显著增强常规化疗药物顺铂对喉鳞癌Hep-2细胞株的毒性作用,两者联合可以在有效作用发挥的同时降低DDP的应用剂量,从而减轻临床上常见的毒副作用。更多还原

【Abstract】 Laryngeal carcinoma is a common malignant tumor of the head and neck. Recently, the incidence and mortality of laryngeal carcinoma are increasing gradually and threatens human being’s health and life seriously. Although the comprehensive treatment options including surgery, radiation therapy and chemotherapy have improved the 5-year survival rate in recent decades, the prognosis for patients with advanced, relapsed or metastatic disease is poor. Moreover, traditional radiation therapy and chemotherapy are associated with considerable adverse toxicities that can not be tolerated by patients. The effective treatment has been extremely limited. Therefore, to seek novel and valid therapeutic strategies for laryngeal cancer becomes a new challenge of the head and neck researchers.The ubiquitin-proteasome pathway (UPP) is the major system responsible for degradation of intracellular proteins in eukaryotes. By controlling the levels of key proteins, it plays an essential role in multiple cellular processes, including cell cycle progression, proliferation, apoptosis, signal transduction and transcription. It is closely related to human malignancies. Blockade of UPP creates an imbalance of various regulatory proteins that control the cell function of proliferation, differentiation and apoptosis, which induces the apoptotic pathways within the cell, indicating that the proteasome inhibitor could be used as an attractive novel anticancer drug. Bortezomib (Velcade, PS-341) was the first proteasome inhibitor to receive approval from the US Food and Drug Administration for the treatment of relapsed or refractory multiple myeloma and mantle cell lymphoma. In lung cancer and lymphoma, Clinical trials with bortezomib alone or in combination are ongoing. But little has been reported of the effect of the proteasome inhibitor on laryngeal cancer cells, and the molecular mechanism is unclear so far. Signal Transducer and Activator of Transcription-3 (STAT3) is an important factor of signal transduction pathway, and is the focus of EGFR, IL-6/JAK, Src and other tyrosine kinase signal carcinogenicity channel convergents. It is constantly activated in great quantities in human tumor cells and links closly with the occurrence, development and evolution of human malignant tumor. It has been reported that the proteasome inhibitor can activate STAT3 in colorectal cancer cells. There has been no report about the effects of the proteasome inhibitor on the STAT3 protein expression in laryngeal carcinoma by now. Our previous studies have shown that the activation of STAT3 is closely related to the occurrence and development of laryngeal cancer and the suppression factor can inhibit the malignant proliferation and induced the apoptosis of cancer cells. In this study, we used human laryngeal squamous carcinoma cell line Hep-2 cultured in vitro, and MTT, Flow cytometry (FCM), Western Blot, gene transfer techniques to explore the role and the molecular mechanisms of the proteasome inhibitor MG-132. The effect of MG-132 on STAT3 was also investigated. Further more, MG-132 was combined with pshSTAT3 or cisplatin, a conventional chemotherapy drug, to observe the changes of the proliferation and apoptosis in cells. This may be helpful to find more effective molecular targets, multi-target combination therapy strategies, as well as the clinical treatment options of laryngeal cancer. This experiment scheme was separated into three parts:Part one The proteasome inhibitor MG-132 induced apoptosis and its effect on STAT3 in laryngeal carcinoma cell line Hep-2Objective: By testing the effects of MG-132 on proliferation, cell cycle distribution, apoptosis and related protein expression at different times and concentrations, we explored the role, the molecular mechanism or the effect of the proteasome inhibitor on STAT3 in human laryngeal squamous cell carcinoma cell line Hep-2 cells.Methods:1. Hep-2 cells were treated with different doses (control, 1, 2.5, 5, 10, 20μmol/L) of MG-132 for 24h or 48h. In addition, Hep-2 cells were treated with 2.5μmol/L MG-132 for varying lengths of time (control, 12h, 24h, 48h, 72h). After treatment, the effect of growth suppression on cells was evaluated with MTT assay.2. Hep-2 cells were treated with varying doses (control, 1, 2.5, 5μmol/L) of MG-132 for 48h, FCM was used to detect cell apoptosis. In addition, the cells were treated with 2.5μmol/L MG-132 for varying lengths of time (control, 12h, 24h, 48h or 72h). After treatment, cell apoptosis and cell cycle were detected by FCM.3. Western blotting was used for detecting the protein expression level of STAT3, p-STAT3, Bcl-2, p21, cyclinD1, CDK4 in cells at control, 12h, 24h, 48h after treatment.Results:1. MTT assays showed that MG-132 can effectively inhibit the proliferation of Hep-2 cells in a dose- (24h:r=0.925,P<0.01;48h:r=0.944,P<0.01) and time-dependent manner(r=0.945,P<0.01). The 50% inhibition concentration (IC50) at 24h is 20μmol/L, and at 48h is 2.5μmol/L.2. FCM demonstrated that apoptosis rate increased with the increasing of concentration and the time, and in a concentration- (r=0.842,P<0.01) and time-dependent manner (r=0.888,P<0.01). Analysis of cell cycle showed that the percentage of cells in G0/G1 and G2/M phase were increased and decreased in S phase cells (P<0.01).3. Western blot showed that MG-132 caused marked up-regulation of p21 protein. Meanwhile, a moderate down-regulation of cyclinD1 was observed in Hep-2 cells. Cells were treated with MG-132 for 12h, resulting in significantly up-regulation of the p-STAT3 protein and maintained at a stable level. The Bcl-2 protein was also moderately up-regulated following MG-132 treatment. The expression levels of STAT3 and CDK4 were not appreciably changed.Conclusion:1. Proteasome inhibition by MG-132 decreases proliferation and increases apoptosis in human laryngeal squamous cell carcinoma cell line Hep-2. 2. Proteasome inhibition by MG-132 induces cells cycle arrest in G0/G1 and G2/M phase.3. The mechanism of MG-132-induced antitumor effects on Hep-2 cells involves in up-regulation of p21 and down-regulation of cyclinD1.4. Proteasome inhibition by MG-132 led to activate of the key signaling molecule STAT3 protein and up-regulation of the anti-apoptotic family member Bcl-2 protein, which might limit the efficacy of the anticancer agents. Part Two Antiproliferative and proapoptotic effects of combination of proteasome inhibitor with pshSTAT3 on laryngeal carcinoma cellsObjective: To explore if inhibition of activated STAT3 by transfecting plasmid expressing short hair pin RNA (shRNA) can enhance the antitumor effect of the protease inhibitor MG-132 on human laryngeal carcinoma cells.Methods:1. Recombinant plasmid expressing short hairpin RNA targeting STAT3 was constructed and identified by enzyme digestion and sequencing. 2. Experiments were divided into five groups: control group, pshNeg group, MG-132 group, combined group, pshSTAT3 group. The recombinant expression plasmid (pshSTAT3) and negative control plasmid (pshNeg) were transfected into Hep-2 cells by Lipofectamine 2000. After 6 hours, the medium was changed, and cells were observed under inverted fluorescence microscopy. Twenty-four hours after the transfection, cells were observed again. Subsequently, the cells were left untreated or treated with 2.5μmol/L MG-132 for 48h.3. After treatment, cell proliferative activity and cell apoptosis rate were detected in different groups by MTT assay and FCM.4. After treatment, the protein expression of p-STAT3 in all groups was detected by Western blot.results:1. The recombinant expression plasmid pshSTAT3 was constructed successfully and confirmed by restriction enzyme digestion and sequencing results.2. The observation of transfection efficiency after recombinant plasmid transfection: After transfection with pshSTAT3 and pshNeg for 6h, green fluorescence was observed in Hep-2 cells under fluorescence microscopy. Green fluorescence gradually increased and transfection efficiency was above 80% at 24 hour post-transfection, and the transfection efficiency was 85.76% by FCM.3. The pshSTAT3 enhanced the inhibition effect of MG-132 on proliferation in Hep-2 cells. After treatment, MTT assay showed that the proliferation ability of Hep-2 cells was not altered in control group and pshNeg group (P>0.05). The pshSTAT3 combined with 2.5μmol/L MG-132 significantly inhibited proliferation of Hep-2 cells compared with the other group (inhibition rate: 62.23±1.25%, P <0.01).4. The pshSTAT3 enhanced the proapoptotic effect in Hep-2 cells induced by MG-132. After treatment, FCM showed that apoptosis rate of the pshSTAT3 plus 2.5μmol/L MG-132 group (55.80±3.12%) was significantly higher than that of the MG-132 group (41.20±3.21%) or the pshSTAT3 group (22.13±1.84%) (P<0.01). There was no significant difference between the control group and the pshNeg group (P>0.05).5. Expression of p-STAT3 after treatment. Western blot showed that up-regulation of p-STAT3 protein expression was observed in the MG-132 group, with its down-regulation in the combined group and the pshSTAT3 group. The expression levels of p-STAT3 were not appreciably changed in the control group and the pshNeg group.Conclusion: The shRNA targeting STAT3 can inhibit MG-132-induced p-STAT3 protein expression up-regulation; thereby significantly enhance the antitumor effect of protease inhibitor MG-132 on human laryngeal carcinoma cells.Part Three Observation on the antiproliferative and proapoptotic effects of proteasome inhibitor MG-132 combined with chemotherapy in laryngeal carcinoma cells Objective: Cisplatin (DDP) is commonly used as a frontline chemotherapeutic agent. Our study is to determine if proteasome inhibitor MG-132 can enhance the cytotoxicity of DDP in human laryngeal squamous carcinoma cells.Methods: Hep-2 cells in logarithmic phase were divided into four groups: control group, MG-132 group, DDP group, MG-132+DDP group. After 48h, cell viability in each group was determined by MTT assay, and the apoptosis rate was detected by FCM.Results:1. Varying doses of DDP with or without MG-132 affected the proliferation of Hep-2 cells. The cells were treated with varying concentrations of DDP (10, 20, 40, 80μmol/L) with or without 1μmol/L MG-132 for 48h. MTT assay showed that the combination of MG-132 with DDP can significantly enhance the proliferation inhibition of the cells at the same dose level (P<0.01, P<0.05). The cellular proliferation of 10μmol/L DDP+MG-132 group was significantly lower than that of 20μmol/L DDP group (P<0.01). Similarly, there was a significant difference between 20μmol/L DDP+MG-132 group and 40μmol/L DDP (P<0.05). The IC50 value of DDP was 62.22μmol/L, and the value descended to 27.79μmol/L when combined with 1μmol/L MG-132.2. DDP plus MG-132 increased the apoptosis of Hep-2 cells. The cells were treated with 10μmol/L DDP alone and with 1μmol/L MG-132 for 48h. FCM results demonstrated that the combination of DDP plus MG-132 can markedly increased the apoptosis rate (P<0.01).Conclusion: Proteasome inhibitor can enhance the cytotoxicity of the chemotherapy drug cisplatin in Hep-2 cells. The combination of two drugs potentiated the anticancer effect on Hep-2 cells.更多还原