【作者】 李丹；

【导师】 刘云鹏；

【作者基本信息】 中国医科大学 ， 内科学， 2008， 博士

【摘要】 目的胃癌是当今世界范围内第二位常见的恶性肿瘤。在我国,胃癌是肿瘤死亡的主要原因之一。虽然根治性手术后早期胃癌的5年生存率超过90%,但是晚期患者的预后较差,5年生存率不足10%。在我国,大约2/3的患者确诊时已属晚期,不能手术切除,放化疗是治疗晚期胃癌的主要手段。虽然阿霉素、氟尿嘧啶、多西紫杉醇及草酸铂等化疗药物的应用使晚期病人的生存期有所提高,但总体疗效仍不理想。蟾蜍灵（Bufalin）是传统中药蟾酥的有效成分之一,国外及我们的研究显示它可以诱导多种白血病细胞和一些实体瘤细胞凋亡,并下调Bcl-2、c-myc、WT-1等肿瘤相关基因的表达,活化丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号转导通路。但其诱导凋亡的确切机制仍不清楚。磷脂酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）/Akt信号转导通路在维持细胞生存和抑制细胞凋亡中起关键作用。活化的Akt可通过磷酸化Bad、Caspase-9和活化转录因子NF-κB等发挥抗凋亡作用。许多细胞毒药物都可通过抑制PI3K/Akt通路诱导细胞凋亡。但PI3K/Akt通路在蟾蜍灵诱导肿瘤细胞（特别是胃癌细胞）凋亡中的作用目前尚未见报道。为深入研究蟾蜍灵诱导肿瘤细胞凋亡的作用机制,本实验以人低分化胃粘液腺癌细胞株MGC803为模型,对Bcl-2家族、Survivin和Caspase-3等凋亡相关蛋白以及PI3K/Akt信号转导通路在蟾蜍灵诱导细胞凋亡过程中的作用进行了研究,同时对PI3K/Akt通路上游分子泛素连接酶Cbl-b在此过程中的调控机制进行了初步探讨。材料与方法1、采用MTT法测定细胞活力,绘制增殖曲线。2、采用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。3、采用瑞氏-姬姆萨染色,光学显微镜下观察细胞形态学变化并测定分裂指数。4、采用Rhodamine 123单染法检测线粒体跨膜电位。5、采用Western blot检测Bcl-2、Bax、Survivin、Caspase-3,、Akt、p-Akt和Cbl-b蛋白的表达。6、统计学处理:每次实验重复3次,数据以（?）±s表示,采用SPSS13.0软件进行t检验和单因素方差分析。实验结果1、蟾蜍灵以时间、剂量依赖性方式抑制MGC803细胞增殖,24h、48h和72h抑制细胞增殖50%的药物浓度(IC₅₀)分别为96.03nmol/L、29.72nmol/L及12.31nmol/L。20nmol/L蟾蜍灵作用MGC803细胞24h后,流式细胞仪结果显示出现明显的G₂/M期阻滞,G₂/M期细胞百分数为41.43±4.85%,对凋亡无明显影响。形态学表现为M期阻滞,细胞出现双核及停滞于分裂中期。此时,分裂指数由对照组的3.57±0.32%增加到15.97±1.96%（P＜0.01）。80nmol/L蟾蜍灵作用MGC803细胞24小时后,出现明显的亚二倍体凋亡峰（sub-G₁峰）,细胞凋亡率为18.76±2.42%,形态学可见典型的凋亡改变,表现为细胞膜完整,细胞质固缩,胞核固缩或碎裂,可见凋亡小体。2、80nmol/L蟾蜍灵作用MGC803细胞12h和24h后,线粒体跨膜电位显著降低,Rhodamine 123的平均荧光强度由对照组的2124.32±86.03分别下降至1502.44±84.09和1129.43±113.68（P＜0.01）。20nmol/L和80nmol/L蟾蜍灵作用MGC803细胞24h,Bax蛋白表达分别上调至对照组的1.27和1.80倍,Bcl-2蛋白表达分别下调至对照组的75%和82%,Bax/Bcl-2比值分别增加至对照组的1.69和2.20倍;Survivin蛋白表达分别上调至对照组的2.21和2.27倍。80nmol/L蟾蜍灵作用MGC803细胞24h,Caspase-3出现裂解活化。3、80nmol/L蟾蜍灵分别作用MGC803细胞1～16h,p-Akt蛋白在1h和8h表达轻微上调,至16h完全消失,而总的Akt蛋白水平没有变化,提示蟾蜍灵可抑制PI3K/Akt信号通路。预先用25μmol/L LY294002（PI3K特异性抑制剂）处理MGC803细胞1小时,然后加入80nmol/L的蟾蜍灵继续作用24 h,MTT结果显示细胞增殖率由单药蟾蜍灵组的51.26±4.10%下降至39.89±3.10%（P=0.01）,流式细胞仪分析显示细胞凋亡率由18.46±2.76%增加至31.40±4.77%（P=0.015）,而LY294002单独作用24h仅轻度抑制MGC803细胞增殖,诱导少量细胞凋亡,说明LY294002增强了蟾蜍灵抑制细胞增殖和诱导凋亡的作用。Western blot解析结果显示,蟾蜍灵单药组Survivin蛋白表达为对照组的1.87倍;LY294002预处理后,Survivin蛋白表达降至对照组的1.33倍,说明LY294002部分逆转蟾蜍灵对Survivin表达的上调。而应用20μmol/LPD98059（ERK特异性抑制剂）预处理MGC803细胞,未增强蟾蜍灵抑制细胞增殖和诱导凋亡的作用,间接提示PI3K/Akt通路在蟾蜍灵诱导MGC803细胞凋亡过程中的作用更为重要。4、80nmol/L蟾蜍灵分别作用MGC803细胞1～16h,Western blot解析结果显示,Cbl-b蛋白在1h和8h无明显变化,16h时增加至对照组的2.83倍,Cbl-b上调的时间与Akt活性被抑制的时间一致。25μmol/LLY294002预先处理MGC803细胞1h后,加入80nmol/L蟾蜍灵继续作用1h和8h,Cbl-b蛋白表达分别下降至对照组的51%和44%,同时Akt活性被抑制。5、2μmol/L钙动员阻断剂CsA预先处理MGC803细胞30min,然后加入80nmol/L蟾蜍灵继续作用16h,Western blot解析结果显示,CsA单药组对Cbl-b无明显影响,蟾蜍灵单药组Cbl-b蛋白表达上调至对照组的2.23倍,应用CsA预处理后,Cbl-b蛋白表达下调至对照组的1.20倍,提示蟾蜍灵引起的钙内流可能是Cbl-b上调的原因之一。为进一步证实这一假设,本实验应用钙离子载体A23187和钙动员阻断剂CsA分别或联合处理MGC803细胞,检测Cbl-b蛋白的表达。Western blot解析结果显示,1μmol/L A23187和2μmol/LCsA单独或联合处理MGC803细胞16h,A23187单药组Cbl-b蛋白上调至对照组的2.40倍;A23187与CsA联合作用组,Cbl-b蛋白表达为对照组的1.83倍,说明CsA部分逆转A23187上调Cbl-b的作用;而CsA单药组对Cbl-b表达无明显影响,提示在MGC803细胞内Cbl-b受钙的正向调节。结论1、蟾蜍灵抑制胃癌MGC803细胞增殖,在低浓度时诱导细胞M期阻滞,较高浓度时诱导凋亡。蟾蜍灵在诱导MGC803细胞凋亡过程中,降低线粒体跨膜电位,上调Bax蛋白表达,下调Bcl-2蛋白表达,活化Caspase-3,同时上调Survivin蛋白表达。2、蟾蜍灵在诱导胃癌MGC803细胞凋亡过程中抑制PI3K/Akt通路。预先抑制PI3K/Akt通路可增强蟾蜍灵抑制MGC803细胞增殖、诱导其凋亡的作用,并可部分逆转蟾蜍灵对Survivin的上调作用。3、蟾蜍灵在诱导胃癌MGC803细胞凋亡过程中上调Cbl-b蛋白表达,钙动员阻断剂CsA部分逆转蟾蜍灵对Cbl-b的上调作用。4、在胃癌MGC803细胞内,Cbl-b受钙的正向调节。更多还原

【Abstract】 ObjectiveGastric cancer is the second most common cancer in the world,and still remains one of the major causes of cancer death in China.Although 5-year survival rate of early gastric cancer is over 90%after surgery,but the prognosis for advanced gastric cancer that can’t undergo surgery is poor,with a 5-year survival rate of less than 10%.About 2/3 patients with gastric cancer is often diagnosed at an advanced stage in China,and they can only accept chemoradiotherapy.Although various chemotherapeutic agents, such as epirubicin,fluorouracil,docetaxel and oxaliplatin,have been used for advanced gastric cancer and the survival has improved,but the efficacy of chemotherapy is limited.Bufalin is one of the major active components of Chan Su,a traditional Chinese medicine.Other and our studies showed that bufalin induced apoptosis in some human leukemia and solid cancer cell lines.It has been reported that bufalin downregulated Bcl-2,c-myc,WT-1 and activated mitogen-activated protein kinase（MAPK） singling pathway during cell apoptotic progression.However,the exact mechanism of bufalin-induced apoptosis is still less clear.Many studies showed that phosphatidylinositol 3-kinase（PI3K）/Akt signaling pathway played a critical role in controlling the balance between cell survival and apoptosis.Activated Akt（p-Akt） phosphorylated Bad,Caspase-9 and I-kappaB kinase （IKK）,thus promoting survival and blocking apoptosis in some cancer cells.Some cytotoxic drugs induced cancer cell apoptosis by inhibiting the PI3K/Akt pathway.But the effect of bufalin on PI3K/Akt pathway during apoptosis of tumor cells,especially in gastric cancer cells,remains unknown. In this study,we investigated the mechanism of proteins related to apoptosis,such as Bcl-2 family,Survivin and Caspase-3,and PI3K/Akt singling pathway in bufalin-induced gastric cancer MGC803 cell apoptosis,we also investigated the regulatory mechanism of ubiquitin ligase Cbl-b,an upstream regulator of PI3K,in bufalin-induced apoptosis.Materials and Methods1.The effect of bufalin on MGC803 cell proliferation was measured using MTT assay.2.Cell cycle phase distribution and hypodiploid DNA was determined by flow cytometry with PI.3.Morphological analysis was performed by cytospin preparation with Wright-Giemsa stain.The slides were observed under a light microscope and the mitotic index was determined.4.Mitochondrial transmembrane potential was determined by flow cytometry with Rhodamine 123.5.Expression of Bcl-2,Bax,Survivin,Caspase-3,Akt,p-Akt and Cbl-b was analyzed by Western blot.6.Statistical analysis.All values are expressed as means±SEM.The differences of the results between two groups were evaluated by Student’s t-test.The data from three or more groups were evaluated by one-way analysis of variance（ANOVA） followed by SNK test.P＜0.05 was considered to be statistically significant.Results1.MTT assay showed that bufalin inhibited MGC803 cell proliferation in timeand dose-dependent manner.The inhibiting concentration of 50%cell growth(IC₅₀) at 24,48 and 72 h was 96.03nmol/L,29.72nmol/L and 12.31nmol/L,respectively.An increase in G₂/M phase was observed after the cells exposed to 20nmol/L bufalin for 24h and the percentage of cells in G₂/M phase was 41.13±4.85%.The morphological analysis demonstrated that most of the cells were in a binucleate state without cell division and some were in metaphase after exposure to 20nmol/L Bufalin for 24 h, while the mitotic index increased from 3.57±0.32%to 15.97±1.96%（P＜0.01） compare with the untreated control group.When the cells were treated with 80nmol/L bufalin for 24h,sub-G1 peak（representing apoptosis） emerged and the percentage of sub-G1 phase was 18.76±2.42%.The morphological analysis demonstrated that the apoptotic cells became rounded in shape and their nuclei exhibited a fragmented morphology, forming apoptotic bodies.2.After exposure to 80nmol/L bufalin for 12 and 24 h,the mitochondrial transmembrane potential was much lower than that of the untreated control cells and the fluorescence intensity of Rhodamine 123 decreased from 2124.32±86.03 to 1502.44±84.09 and 1129.43±113.68（P＜0.01）,respectively.After exposure to 20nmol/L and 80nmol/L bufalin for 24 h,the expression of Bax was increased to 1.27 and 1.80 times of the untreated control cells and Bcl-2 was decreased to 75%and 82%of the control cells.The radio of Bax/Bcl-2 was increased to 1.69 and 2.20 times of the untreated control cells,respectively.Meanwhile,the expression of Survivin was increased to 2.21 and 2.27 times of the untreated control cells.The cleavage of Caspase-3 was observed when the cells were treated with 80nmol/L bufalin for 24 h.3.After 80nmol/L bufalin treatment for 1-16 h,Akt phosphorylation increased slightly at 1 h and 8 h and then was inhibited completely at 16 h.The total Akt level was unchanged during treatment.MGC803 cells were pretreated with PI3K specific inhibitor LY294002（25μmol/L） and then exposed to 80nmol/L bufalin for 24 h,MTT assay demonstrated that the cell proliferation reduced from 51.26±4.10%to 39.89±3.10%（P=0.01） and the percentage of apoptofic cells increased from 18.46±2.76%to 31.40±4.77%（P=0.015） compare with treatment with bufalin alone. LY294002 treatment alone reduced cell proliferation and induced apoptosis very slightly.Western blot analysis showed that the upregulation of Survivin by bufalin was suppressed partially when the cells were pretreated with LY294002.In contrast,the ERK inhibitor PD98059 had no significant effect both on cell proliferation inhibition and apoptosis induced by bufalin in MGCS03 cells.Taken together,these results indicate that the PI3K/Akt pathway,but not ERK pathway,plays a crucial role in bufalin-induced apoptosis in MGC803 cells.4.Treatment with 80nmol/L bufalin for 1～16 h,the expression of Cbl-b was unchanged at 1h and 8h,and at 16h the expression of Cbl-b was 2.83 times over the untreated control cells.The time of upregulation of Cbl-b was identical with that of downregulation of p-Akt.Pretreatment with 25μmol/L LY294002 for 1h and then exposure to 80nmol/L bufalin for 1h and 8h,Akt phosphorylation was inhibited and the expression of Cbl-b reduced to 51%and 44%of the untreated control cells.5.Pretreatrnent with 2μmol/CsA for 30min,an inhibitor of calcium mobilization, and then exposure to 80nmol/L bufalin for 16h,CsA reversed bufalin-induced upregulation of Cbl-b and CsA alone did not affect the expression of Cbl-b.These results imply calcium mobilization by bufalin maybe is one of the reasons of increase of Cbl-b.To further certify the hypothesis,the expression of Cbl-b was determined after the cells were exposed to calcium ionophore A23187 and/or CsA.The expression of Cbl-b was 2.40 times over the untreated control cells after the cells were exposed to 1μmol/L A23187 for 16h.After the cells were exposed to 1μmol/L A23187 and 2μmol/L CsA for 16h,the expression of Cbl-b was 1.83 times over the untreated control cells.These results indicated CsA partially reversed A23187-induced upregulation of Cbl-b.Conclusion1.Bufalin inhibited MGC803 cell proliferation.It induced M phase arrest of cell cycle at low concentration,but induced apoptosis at high concentration in MGC803 cells.Bufalin-induced apoptosis accompanied with the collapse of mitochondrial transmembrane potential,the upregulation of Bax and Survivin,the downregulation of Bcl-2 and the activation of Caspase-3 in MGC803 cells.2.Bufalin inhibited activation of Akt during MGC803 cell apoptotic progression and LY294002 significantly enhanced bufalin-induced proliferation inhibition and apoptosis in MGC803 cells.LY294002 partially reversed bufalin-induced upregulation of Survivin.3.Bufalin upregulated Cbl-b during MGC803 cell apoptotic progression and CsA partially reversed bufalin-induced upregulation of Cbl-b.4.The expression of Cbl-b was upregulated by calcium mobilization in gastric cancer MGC803 cells.更多还原