【作者】 董小锋；

【导师】 李杰；

【作者基本信息】 山东大学 ， 外科学， 2014， 博士

【摘要】 目的：肝细胞肝癌(Hepatocellular carcinoma, HCC)在男性癌症死亡率中居于第二位,肝癌患者的5年的生存率只有11%,HCC的致病因素主要包括慢性病毒性肝炎、长期过量饮酒、肝硬化、食用含有黄曲霉素B1或者亚硝胺的食物等,其中慢性病毒性肝炎及长期过量饮酒导致肝硬化并进一步发展成为肝癌被认为是肝细胞肝癌发生的主要因素。早期的肝癌患者并无明显的临床症状加之诊疗条件及医疗技术的限制,肝癌患者在发现时多数已处于病程的中晚期,仅约1/3患者有手术切除的机会,并且多数肝癌患者在局部切除术后往往伴有无法避免的肝内转移,预后效果差；HCC的局部治疗(肝癌射频消融治疗、肝动脉介入化疗栓塞及局部酒精治疗等)只能暂时缓解肝癌患者的临床症状,对延长患者的生存期及改善预后并无明显作用；肝移植因其高昂的费用及供体来源的限制,无法在中晚期肝癌患者中得到广泛应用；肝癌易产生化疗耐受,对放疗治疗亦缺乏敏感性；索拉菲尼(sorafenib)作为一种多蛋白激酶抑制剂,是目前公认的对肝癌患者治疗有效的分子靶向药物,其具有抗血管生成和促进肝癌细胞凋亡的双重作用,在Ⅲ期大型临床试验中发现其延长肝癌患者的中位生存期可达2-3个月,但因其高昂的价格及药物耐药性的产生,在临床上尚未无法得到广泛应用。因此,寻找肝癌治疗的新方法及新的靶点蛋白是目前肝癌治疗研究中的热点。环氧合酶-2(cyclooxygenase-2, COX-2)过表达于多种肿瘤组织内,是前列腺素合成中的限速酶,可调控花生四烯酸到前列腺素的转化,进而发挥其生物学活性。COX-2可影响到肿瘤生长的各个方面,包括细胞增殖、迁移、侵袭、凋亡及耐药基因的表达等,COX-2高表达的肿瘤患者对药物治疗的反应更低,患者预后更差。研究发现,与癌旁组织、正常肝组织比较,COX-2在肝癌组织中的表达明显增高,COX-2高表达的肝癌患者其预后更差,提示COX-2蛋白可能为肝癌治疗中潜在的靶点蛋白。美洛昔康(meloxicam)是COX-2的特异性抑制剂,其可有效抑制COX-2的蛋白活性,我们前期的研究结果发现,meloxicam可抑制肝癌细胞的增殖、诱导细胞周期捕获及促进细胞凋亡的发生,但其分子调控机制尚缺乏相应的研究。前列腺素E2(prostaglandin E2, PGE2)是在COX-2过表达的肿瘤中受COX-2调控合成的含量最高的前列腺素,PGE2可通过与细胞膜表面的受体结合(如PGE2受体EP2等),发挥其生物活性。细胞自噬是细胞的一种自我消化过程,由于其可诱导细胞的死亡,亦称为Ⅱ型程序化细胞死亡；因细胞类型及所受外界刺激的差异,细胞自噬亦可拮抗药物对肿瘤细胞的促凋亡作用,维持肿瘤细胞的生存,细胞自噬的激活被认为是细胞生存过程中起调控作用的双刃剑。肿瘤细胞的自噬与凋亡之间存在多节点,多层面的交叉调控,探讨自噬与凋亡间的共调控蛋白,凋亡与自噬之间的相互调控机制是目前研究自噬与凋亡关系的热点。然而,细胞自噬的激活在meloxicam调控的肝癌细胞生长中的关系及作用尚需进一步探讨。本研究中,我们设计实验探讨meloxicam抗肝癌的分子机制,研究自噬与凋亡在1neloxicam介导的肝癌的治疗中的相互调控作用,并对相关的分子机制研究,从而为meloxicam在临床的应用提供实验依据和联合用药的理论基础。方法：选取5种最常见的肝癌细胞系—SMMC-7402、Bel-7402、HepG2、 SMMC-7721及Huh7,应用Western blot筛选出COX-2高表达的肝癌细胞株,并检测PGE2受体EP2在肝癌细胞株中的表达。为进一步筛选出对1neloxicam治疗敏感的肝癌细胞株,应用CCK-8细胞活力实验检测COX-2高表达肝癌细胞株对meloxicam治疗的敏感性。选择对meloxicam治疗敏感的肝癌细胞株,应用细胞迁移、侵袭、粘附及克隆形成实验,检测meloxicam对所选肝癌细胞株迁移、侵袭、粘附及克隆形成能力的影响。为进一步探讨meloxicam引起肝癌细胞迁移、侵袭及克隆形成能力的调控机制,应用Western blot检测meloxicam、PGE2及rh-MMP-2对与肝癌细胞侵袭、迁移、粘附及克隆形成密切相关蛋白E-钙粘素(E-cadherin)的影响,检测meloxicam对与E-钙粘素(E-cadherin)降解密切相关的基质金属蛋白酶1/2(matrix metalloproteinase1/2, MMP-1/2)表达的调控作用;通过酶联免疫吸附实验ELIS A (Enzyme Linked Immunosorbent Assay)检测meloxicam或(和)PGE2对E-cadherin的降解产物可溶性钙粘素(soluble E-cadherin, sE-cadherin)的影响,检测PGE2及可溶性基质金属蛋白酶-2(recombinant human MMP-2protein, rh-MMP-2)对E-cadherin的降解产物sE-cadherin的影响；通过real-time RT-PCR, RT-PCR检测meloxicam、PGE2及rh-MMP-2对E-cadherin mRNA表达的影响。肿瘤细胞的迁移、侵袭与凋亡能力的改变是细胞生存过程中的共同事件,共同调控着肿瘤细胞的活性,为进一步探讨迁移、侵袭及凋亡蛋白之间的相互关系,应用Western blot检测meloxicam、PGE2及rh-MMP-2对凋亡信号通路PI3K/AKT中AKT蛋白磷酸化激活的影响。流式细胞仪Annexin V/PI双染法检测肝癌细胞株HepG2, SMMC-7721细胞经meloxicam, meloxicam+PGE2、 meloxicam+rh-mmp-2处理后,与对照组比较细胞凋亡率的变化。为进一步检测内源性及外源性凋亡信号蛋白在meloxicam介导的肝癌细胞凋亡中的作用机制,应用Western blot检测meloxicam对内源性凋亡相关蛋白Bcl-2, Bax, Bcl-xl, mcl-1及survivin蛋白表达的影响；Western blot检测meloxicam, PGE2及AKT激活的特异性抑制剂MK-2206对凋亡蛋白survivin、mcl-1、Bax的调控作用；Western blot检测meloxicam和(及)PGE2对外源性凋亡通路中关键蛋白Fas-L、Fas蛋白表达的调控。细胞自噬的激活是(肿瘤)细胞拮抗药物治疗及外界不良刺激的的一种自我保护机制。为进一步探讨自噬在meloxicam治疗肝细胞肝癌中的作用,分别通过Western blot蛋白分析、吖啶橙(acridine orange, AO)及单丹磺酰尸胺(monodansylcadaverine, MDC)荧光染色检测细胞自噬激活的标志性蛋白LC3-Ⅱ、自噬小体、自噬溶酶体的形成,探讨meloxicam对肝癌细胞自噬激活的影响。分别应用细胞自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)、氯喹(chloroquine, CQ)抑制meloxicam诱导的肝癌细胞自噬激活后,流式细胞仪Annexin V/PI双染法检测细胞白噬在meloxicam介导的肝癌细胞凋亡中的作用。应用Western blot进一步检测细胞自噬对受meloxicam调控的凋亡相关蛋白Bax, mcl-1, survivin及Fas-L表达的影响。结果：Western blot蛋白检测结果示：COX-2蛋白在肝癌细胞系Bel-7402, SMMC-7721及HepG2细胞中呈高表达状态,而在肝癌细胞系SMMC-7402、Huh7中呈低表达状态。选取COX-2高表达的肝癌细胞株Bel-7402, SMMC-7721及HepG2,经CCK-8细胞活力检测发现在COX-2高表达的肝癌细胞株HepG2, Bel-7402及SMMC-7721中,meloxicam在浓度为80μM时对HepG2,SMMC-7721及Bel-7402细胞活力具有最强的抑制作用,且敏感性依次为HepG2>SMMC-7721>Bel-7402。PGE2的受体EP2蛋白在SMMC-7402、 Bel-7402、HepG2、SMMC-7721、Huh7细胞系中均有表达,且在肝癌细胞株HepG2、 SMMC-7721中表达量最高。肝癌细胞系HepG2、SMMC-7721细胞及meloxicam在浓度为80μM,被选择用于后续实验研究。细胞侵袭、迁移、粘附及克隆形成实验发现,与对照组比较,经meloxicam处理的肝癌细胞株(HepG2、SMMC-7721),细胞的迁移、侵袭、粘附能力明显降低,克隆实验中集落形成数显著减少。Western blot蛋白检测发现肝癌细胞经meloxicam (80μM)处理后,细胞间粘附蛋白E-cadherin表达增多,细胞上层培养液中sE-cadherin浓度降低,而sE-cadherin是E-cadherin胞外域降解后的可溶性的蛋白片段(80KD),表明meloxicam可能是通过调控E-cadherin降解相关的蛋白酶的表达／活性来实现对E-cadherin的调控,继而对细胞的迁移、侵袭、粘附及克隆形成能力进行调控。我们进一步研究发现meloxicam可抑制MMP-2在肝癌细胞中的表达,而加入PGE2后,meloxicam对MMP-2的抑制作用失,表明meloxicam主要是通过COX-2依赖的途径实现对MMP-2蛋白表达的调控。Real time RT-PCR、标准RT-PCR检测meloxicam对MMP-2mRNA调控,其检测结果与meloxicam对MMP-2蛋白的调控一致,表明meloxicam主要在转录水平,通过COX-2依赖的途径对MMP-2蛋白表达进行负调控；melxociam亦可以通过COX-2依赖的途径,在转录水平对E-cadherin的表达进行调控。在含有meloxicam的肝癌细胞培养液中加入rh-mmp-2后发现,meloxicam对E-cadherin表达的正调控作用消失,表明meloxicam在蛋白水平对E-cadherin表达的调控,亦是通过COX-2依赖的途径来实现的。AKT蛋白激酶(Protein Kinase B, PKB)的磷酸化激活与否是细胞凋亡过程中的重要事件,细胞的迁移、侵袭等行为与细胞凋亡的发生为非独立的、交叉重叠的过程,AKT蛋白可能为此过程中的关键调控蛋白。Western blot蛋白检测发现经meloxicam处理72小时的肝癌细胞,AKT蛋白的磷酸化激活受到明显抑制；而在meloxicam处理的肝癌细胞中,分别加入PGE2及rh-MMP-2后,发现PGE2及rh-MMP-2均可拮抗meloxicam对AKT激活的抑制作用,而rh-MMP-2可增强对AKT的激活具有正调控作用的sE-cadherin在细胞上清液中的浓度,表明meloxicam对AKT蛋白激活的调控是通过COX-2/PGE2/E-cadherin的途径来实现的。流式细胞仪Annexin V/PI双染法凋亡检测发现PGE2, rh-MMP-2均可拮抗meloxicam对肝癌细胞的促凋亡作用。Western blot蛋白检测示,meloxicam可增强促凋亡蛋白Bax, Fas-L表达,降低抗凋亡蛋白mcl-1, survivin的表达,而对抗凋亡蛋白Bcl-2, Bcl-xL的表达无明显调控作用。通过加入AKT蛋白激酶激活的特异性抑制剂MK-2206、COX-2蛋白激酶活性产物PGE2发现,meloxicam对抗凋亡蛋白mcl-1, survivin的调控主要是通过对AKT激活的抑制来实现的；meloxicam对促凋亡蛋白Bax, Fas-L的表达调控是通过COX-2/AKT非依赖的途径来实现的。吖啶橙(AO),单丹磺酰尸胺(MDC)荧光染色及Western blot蛋白分析发现,经meloxicam处理72小时后,肝癌细胞中自噬小体及自噬溶酶体明显增多,细胞自噬激活的标志性蛋白LC3-Ⅱ表达增强,表明meloxicam诱导了肝癌细胞自噬的激活；应用细胞自噬的特异性抑制剂3-MA后,肝癌细胞中自噬溶酶体、自噬小体显著减少,细胞自噬激活标志蛋白LC3-Ⅱ表达降低,meloxicam诱导的细胞自噬受到有效抑制。为进一步检测细胞自噬在meloxicam诱导的肝癌细胞凋亡中的作用,Annexin V/PI双染法流式细胞技术测凋亡发现,细胞自噬特异性抑制剂3-MA/CQ单独应用时对肝癌细胞凋亡并无明显作用,meloxicam与细胞自噬激活抑制剂3-MA/CQ联合应用组中,细胞凋亡率显著增高,表明细胞自噬激活在meloxicam诱导的肝癌细胞凋亡中为拮抗因素。Western blot蛋白检测结果发现,细胞自噬激活后可通过将meloxicam诱导生成的Bax蛋白吞噬,拮抗meloxicam对肝癌细胞凋亡的诱导作用,此过程是通过COX-2非依赖的机制来实现的。结论：1. Meloxicam通过COX-2/PGE2依赖的机制抑制了肝癌细胞中MMP-2表达,抑制了其介导的E-cadherin降解,降低了sE-cadherin的生成；2. sE-cadherin可通过HER/IGF-1途径,促进AKT蛋白的磷酸化激活,继而调控肝癌细胞中抗凋亡蛋白mc1-1和survivin表达；3. Meloxicam通过COX-2非依赖的机制引起了肝癌细胞中促凋亡蛋白Bax, Fas-L表达的上调,促进了肝癌细胞的凋亡；4. Meloxicam诱导的细胞自噬可通过吞噬、降解促凋亡蛋白Bax来发挥其拮抗肝癌细胞凋亡的作用。意义：1.在肝细胞肝癌中,研究了meloxicam通过COX-2依赖及非依赖的机制,发挥其促进肝癌细胞凋亡的作用；2.在肝细胞肝癌中,研究了meloxicam诱导的细胞自噬对细胞凋亡的拮抗作用；3.在肝细胞肝癌治疗中,为meloxicam与其他抗肿瘤药物的联合应用,提供了实验依据及理论基础。更多还原

【Abstract】 Background:Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death in men worldwide, and the five-year survival rate is only11%. The main pathogenic factors include chronic viral hepatitis, long time excessive drinking, cirrhosis, long-term consumption of food contaminated by nitrosamines or aflatoxin B1et al. Among all of the above factors, the change from chronic viral hepatitis/long time excessive drinking, cirrhosis to hepatocellular carcinoma is considered as the major process for the hepatocarcinogenesis. Because no obvious clinical symptoms were showed in early-phase of HCC, most of HCC patients were found at an advanced stage, only1/3patients had a chance for surgery resection and most patients were always accompanied with unavoidable intrahepatic or distant metastasis after surgery resection. The local treatment of HCC (including radiofrequency ablation, transcatheter arterial chemoembolization and percutaneous ethanol injection therapy et al) could only ameliorate the clinical symptoms and shows no significant improvement on the prognosis and median survival of HCC patients. Owing to the high fees and restrictions of donors, the application of liver transplantation was significant limited in the therapy of advanced HCC patients. HCC is extremely resistant to the chemotherapeutic drugs and is non-sensitive to radical therapy. Sorafenib is the unique first-line drug recommended for advanced HCC, but it has not been widely accepted as it only prolongs2-3month of survival of advanced HCC patients compared to placebo and and it is cost-prohibitive. Therefore, it is urgently needed to seek new therapy strategy and new targeted protein for the treatment of HCC. Cyclooxygenase (COX)-2, a rate-limiting enzyme in the synthesis of prostaglandin (PG), has emerged as an anti-cancer target protein, and it is overexpressed in many types of cancers including HCC. COX-2is closely related to the cell proliferation, migration, invasion, apoptosis and drug resistance and the COX-2overexpressed tumor patients show a worse prognosis. Compared with pericarcinomatous tissue and normal liver tissue, COX-2is highly expressed in hepatocellular carcinoma and HCC patients with highly expressed COX-2always showed a worse prognosis; the results indicats that COX-2is a potential target in the therapy of HCC. Meloxicam is a COX-2specific inhibitor and could effectively suppress the activity of COX-2and our previous studies indicated that meloxicam could effectively supress cell proliferation, induce cell apoptosis and cell cycle arrest, but the mechanisms particularly the molecular signaling pathways in its anti-cancer effects remain obscured. PGE2is the predominant product of COX-2, and is the most abundant among the PGs produced by COX-2-overexpressing tumors; PGE2activates diverse biological effects primarily through its binding to a family of receptors, such as prostaglandin E2receptor (EP2). The biological relevance of EP2-dependent signaling to the oncogenic effects of PGE2is corroborated by the impaired of tumor cell growth, invasion and metastatic dissemination in EP2-knock animals. Autophagy was initially referred as a self-digestion process, later was found to induce cell death and considered to be Type Ⅱ programmed cell death (PCD). Now it is accepted that autophagy acts as a double-edged sword for cancer cells depending on the cellular context and the stimuli. The autophagic pathway crosstalks with apoptosis, and the mutiple molecular nodes of crosstalks present many opportunities for therapeutic intervention making it a hot spot for cancer research. However, the role Autophagy in meloxicam-mediated apoptosis of cancer remains unknown.In this research, we will detect the molecular mechanism of meloxicam in the therapy of HCC and further detect the possible protein molecular nodes of crosstalks between autophagy and apoptosis and will provide some theories for the combination of meloxicam and other antitumor drugs. Methods:Five common hepatocellular carcinoma cells SMMC-7402, Bel-7402, HepG2, SMMC-7721and Huh7were selected and the COX-2, EP2protein expression was detected in by Western blot. CCK-8cell proliferation assay was used to detect the drug sensitivity of meloxicam in COX-2highly expressed hepatocellular carcinoma cells. Cell migration assay, cell invasion assay, cell adhesion assay and colony formation assay were used to detect the biological effects of meloxicam on COX-2highly expressed HCC cell line. The effects of meloxicam and (or) PGE2on the expression of cell adhesion protein E-cadherin and MMP-1/2were detected by Western blot. The effects of meloxicam and (or) rh-MMP-2on the expression E-cadherin were also detected by Western blot. ELISA assay was used to detect the concentration of sE-cadherin in the cell culture media directed by meloxicam, PGE2or rh-mmp-2. The effects of meloxicam, PGE2or rh-mmp-2on E-cadherin mRNA and MMP-2mRNA were also detected by both real-time RT-PCR and standard RT-PCR.Western blot was used to detect the effects of meloxicam, PGE2or rh-MMP-2on the phosphorylation activation of AKT. HCC cells were divided into control, meloxicam, meloxicam+PGE2, meloxicam+rh-mmp-2four groups and Annexin V-FITC/PI double staining was employed to quantify the apoptosis of HCC cells. The expression of meloxicam on the apoptosis associated protein Bcl-2, Bax, Bcl-xL, mcl-land survivin was detected by Western blot at the indicated time points. In order to further detect the role of extrinsic apoptosis pathway in meloxicam mediated HCC cell apoptosis, the expression of Fas and Fas-L was also detected by Western blot at the indicated time points, then the effects of meloxicam and (or) PGE2on Fas-L and Fas-L were also detected by Western blot.In order to detect the role of cell autophagy in meloxicam mediated HCC cell apoptosis, we detect the activation of cell autophagy by Western blot and fluorescence staining (AO/MDC staining) in HCC cells after treated by meloxicam for72hours. In the presense or absense of autopgagy specific inhibitors3-MA/CQ, annexin V-FITC/PI double staining was employed to quantify the apoptosis change of hepatocellular mediated by autophagy via flow cytometry. To further detect the functional relationship between autophagy and apoptosis, we next examined the expression of apoptosis-associated proteins including survivin, Mcl-1, Bax and Fas-L, which had been shown to be regulated by meloxicam in the presence or absense autophagy inhibitor3-MA.Results:Western blot analysis showed that the available HCC cell lines expressed different levels of COX-2protein, thus Bel-7402, SMMC-7721and HepG2cells intensely expressed COX-2, whereas SMMC-7721and Huh7cells showed weak expression of COX-2. CCK-8cell proliferation assay showed that meloxicam exhibited the strongest inhibitory activity at the concentration of80μM in COX-2highly expressed HCC cell lines. EP2protein was expressed in all of the five HCC cell line at defferent levels.Meloxicam could effectively suppress the migration, invasion, adhesion and colony formation ability of HCC cells. Meloxicam could effectively increase the full length E-cadherin expression both on mRNA and protein level and decrease the concentration of sE-cadherin in cell culture media. Our further research indicated that meloxicam supressed MMP-2expression both on mRNA and protein levels and the addition of PGE2could effectively reverse this effect. For a further study, we also found that meloxicam regulated the expression of E-cadherin both on mRNA and protein level and the addition of PGE2could almost completely reverse the effect of meloxicam on the regulation of E-cadherin. The effect of meloxicam on E-cadherin expression was disappeared when rh-mmp-2was added into the cell culture media indicating that meloxicam ragulated E-cadhein directly or indirectly in a COX-2dependent pathway.The AKT pathway plays a critical role in regulating cell growth, proliferation, survival and motility, which drive tumor progression. Meloxicam significantly inhibited the activation of AKT and both the addition of PGE2and rh-mmp-2could partly reverse the inhibitory effects indicating meloxicam suppressed AKT activation in a COX-2dependent pathway. Annexin V-FITC/PI double staining showed that both the addition of PGE2and rh-mmp-2attenuated the promoting apoptosis effects of meloxicam on HCC cells. Meloxicam increased the expression of proapoptotic protein Bax, Fas-L and decreased the expression of antiapoptotic protein mcl-1, survivin in a time dependent manner. Meloxicam regulated mcl-1and survivin majorly through the inhibition of AKT activation, in a COX-2dependent pathway, whereas meloxicam regulated the expression of Bax and Fas-L majorly through a COX-2/AKT independent pathway.Meloxicam induced autophagy activation was verified by AO/MDC staining for the detection of autophagosomes and Western blot for the detection of LC3-II and Beclin-1, the marker protein of autophagy. In a further study, we showed that inhibition of autophagy by3-MA/CQ had little effect on apoptosis, but synergized with meloxicam in inducing apoptosis of HepG2cells which was verified by Annexin V-FITC/PI double staining. The activation of autophagy could engulf the pro-apoptotic protein Bax and showed little effect the apoptitic related protein Fas-L, survivin and mcl-1.Conclusions:1. Meloxicam regulated MMP-2in COX-2/PGE2dependent pathway followed by the decreased degration of E-cadherin and decreased sE-cadherin expression.2. sE-cadherin could regulate the activation by binding with HER/IGF-1receptor and then regulate the expression of anti-apoptotic protein mcl-1and survivin.3. Meloxicam could significantly increase proapoptotic protein Bax and Fas-L expression in a COX-2independent pathway.4. Meloxicam induced the activation of cell autophagy and the activated autophagy attenuated the proapoptotic effects of meloxicam on HCC cells via engulfing the proapoptotic protein Bax.Significance:1. We revealed the mechanism of meloxicam in the therapy of HCC through both COX-2dependent and independent pathways.2. We verified that meloxicam induced cell autophagy could significantly attenuate its proapoptotic effects on HCC cells. 3. We provided a novel molecular theory for the possible combination of meloxicam and other anti-tumor drugs in the therapy of HCC.更多还原