【作者】 许春森；

【导师】 黄鹤光； 谢捷明；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 博士

【摘要】 南瓜蛋白(Cucurmosin CUR)是本课题组从南瓜的瓜瓤中提取的一种新的Ⅰ型核糖体失活蛋白,前期的研究发现CUR能显著诱导黑色素瘤B16和白血病K562等多种肿瘤细胞凋亡和分化。胰腺癌是一种临床表现隐匿、发展迅速和预后极差的消化道常见恶性肿瘤。本课题从体外检测南瓜蛋白的抗肿瘤活性开始,然后观察其诱导PANC-1细胞凋亡和增加吉西他滨的化疗敏感性作用,并通过小鼠胰腺癌原位移植模型测定CUR的体内抗瘤活性,继而在基因水平初步探讨其诱导凋亡和逆转胰腺癌的多药耐药的分子机制,为CUR潜在的抗胰腺癌临床应用提供理论和实验依据。目的体内外观察CUR对胰腺癌PANC-1细胞的生长抑制作用,并初步探讨其诱导凋亡和逆转多药耐药的分子机制。方法1.体外MTT法观察CUR对PANC-1细胞生长抑制情况,制作NOD/SCID小鼠胰腺癌原位移植模型,体内观察CUR对胰腺原位移植瘤的抑瘤率。2.电子显微镜、流式细胞仪、ELISA等方法观察CUR作用后PANC-1细胞超微结构的改变以及凋亡发生率的影响。3.荧光显微镜观察CUR作用后线粒体膜电位(ΔΨm )的变化,western blot检测CUR作用后细胞caspas-3、caspase-9、bcl-2、bax、smac、survivin蛋白以及细胞浆细胞色素C的变化。4.体外MTT法观察CUR联合吉西他滨对PANC-1细胞生长抑制作用,western blot检测CUR作用后P-170、MDRP蛋白表达的变化。结果1.体外CUR对PANC-1细胞生长的影响,不同剂量CUR (0.3125、0.625、2.5、5.0、10、20、40、80ug/ml)作用于PANC-1细胞24h后,细胞增殖抑制率分别为9.2、14.4、20.2、32.0、36.4、44.1、50.3和58.6%,而作用48h的细胞增殖抑制率分别为25.1、35.6、39.7、46.5、52.9、57.0、64.5和72.6%,作用72h的抑制率则为31.4、51.5、60.1、67.2、75.2、85.7、91.0和93.4%。随着作用时间的延长,其抑制率明显增加, 24,48,72h的半数抑制浓度(IC50)分别为40.24μg/m L、8.24μg/m L和1.17μg/m L,呈现明显的时间依赖性。结果提示,南瓜蛋白在体外能以剂量和时间依赖方式明显地抑制PANC-1细胞生长。2.体内观察CUR对小鼠胰腺原位移植瘤的抑瘤率,0.125mg/kg、0.25mg/kg和0.5mg/kg CUR的抑瘤率分别为45.2%、50.0%和59.7%,表明不同浓度的CUR对胰腺原位移植瘤的生长均有抑制作用。3. CUR对胰腺癌PANC-1细胞周期和凋亡的影响:①CUR处理后,胰腺癌细胞核变小,皱缩,染色质凝集,出现核碎裂。②0、2.5μg/mL、10μg/mL、40μg/mLCUR作用PNAC-1细胞72h, G0/G1期的比例分别为46.56±5.08、53.33±5.05、67.50±6.50和77.00±6.73%,随着药物浓度增加,G0/G1期比例逐渐升高。40μg/mL南瓜蛋白及等量培养液作用PANC-1细胞24、48和72h,G0/G1期的比例分别为56.60±6.65、67.83±6.76和77.00±6.73%,随着作用时间的延长,G0/G1期比例逐渐升高。表明CUR作用PANC-1细胞后,细胞周期阻滞于GO/GI期。③0、2.5、10、40μg/mL的CUR作用胰腺癌PANC-1 72小时的凋亡率分别为2.50士0.13%、8.30士1.23%、23.40士2.45%和48.50士3.65%(P<0.05),随着药物浓度的升高,PANC-1细胞的凋亡率逐渐增加,呈浓度依赖性。40μg/mLCUR作用24、48和72h,PANC-1细胞凋亡率分别为16.51士2.97、38.51士2.38和48.50士3.65%(P<0.05),随着作用时间延长,PANC-1细胞的凋亡率逐渐增加,呈时间依赖性。结果提示,CUR在体外能以剂量和时间依赖方式诱导PANC-1细胞凋亡。④0、2.5、10、40μg/mL的南瓜蛋白作用胰腺癌PANC-1 72小时的caspase-3活性分别0.009、0.011、0.035和0.065酶活力单位。结果显示,随着CUR浓度提高,caspase-3活性显着增高。4.线粒体凋亡通道的分子变化:0、2.5,10和40ug/mLCUR处理PANC-172h后,ΔΨm逐渐下降,细胞色素C释放到细胞浆明显增加,caspase-3和caspase-9的活力逐渐增强,smac、bax蛋白表达逐渐增加,而survivin和bcl-2蛋白表达无明显变化。5. CUR的化疗增敏作用,GEM单用时(72h)的IC50=36.76nmol/L ,与CUS合用时的GEM的IC50=12.14nmol/L,CUR逆转多药耐药倍数3.02。western blot检测CUR作用后PANC-1细胞多药耐药P-170蛋白表达逐渐减弱,作用呈剂量依赖性,而MDRP蛋白表达无明显变化。结论: CUR具有较强的体内、外抗肿瘤活性;且以剂量和时间依赖方式诱导PANC-1细胞凋亡;其机制可能与CUR通过激活PANC-1细胞促凋亡蛋白bax和smac表达增加,致细胞色素C释放到细胞浆增多,活化caspase-9和caspase-3,通过线粒体内在途径诱导细胞凋亡;CUR增加吉西他滨的化疗敏感性,其可能的机制是CUR下调胰腺癌的多药耐药P-170蛋白的表达。更多还原

【Abstract】 Cucurmosin(CUR), a new ribosome-inactivating proteins(RIP), exhibites anti-tumor and apoptosis-inducing effects both in vitro and in vivo on the human chronic myeloid leukemia K562 cell line and the murine B16 melanoma cell line. Pancreatic cancer is a devastating disease characterized by a very poor prognosis due to the low rates of early diagnosis and rapid progression, with most patients dying within 12 months after diagnosis. The goal of this study was to examine its anti-pancreatic cancer effects both in vitro and in vivo and explore the underlying molecular mechanisms with the aim of providing theoretical rationale and experimental evidence for its potential clinical application.ObjectiveTo investigate the anti-tumor effect of CUR on human pancreatic cancer PANC-1 cells in vitro and in vivo, and to identify the molecular mechanism of CUR on inducing apoptosis and chemo-sensitization.Methods1. MTT assay was used to determine the effect of CUR on the growth of PANC-1 cells in vitro, orthotopic transplantation NOD/SCID mouse model of pancreatic cancer was established to observe the anti-tumor effect of CUR in vivo.2. Electron microscopy, flow cytometric analysis and enzyme linked immunospecific assay were used to examine the apoptosis-inducing effect of CUR.3. Fluorescent staining was used to examine the change of mitochondrial membrane potential, and western blot was used to determine the protein level of caspase-3 , caspase-9, bcl-2 , bax, survivin ,smac and cytoplasmic cytochrome-c in PANC-1 cells after CUR treatment.4. MTT assay was used to detecte the cytotoxic effect of the different concentrations of CUS with GEM , and western blot was used to determine the protein level of P-170 and MDRP after CUR and GEM treatment.Results1. In vitro anti-tumor activity of CUR, PANC-1 cells were treated with 0.3125, 0.625, 2.5, 5.0, 10.0, 20.0, 40.0 and 80μg/mL CUR and cell viability was measured at 24, 48 and 72 hours using MTT assay. The inhibitory rates of the cell growth for 24 hours were 9.2, 14.4, 20.2, 32.0, 36.4, 44.1, 50.3 and 58.6% respectively. When treated for 48 hours , the inhibitory rates were 25.1, 35.6, 39.7, 46.5, 52.9, 57.0, 64.5 and 72.6% respectively. When treated for 72 hours, the inbitory rate was 31.4, 51.5, 60.1, 67.2, 75.2, 85.7, 91.0 and 93.4%.The 50% inhibitory concentrations (IC50) of CUR for 24, 48, and 72h were 40.24μg/mL, 8.24μg/mL and 1.17μg/mL respectively. CUR significantly inhibite the proliferation of PANC-1 cells in dosage- and time-dependent manner in vitro.2. In vivo anti-tumor activity of CUR, Orthotopic transplantation NOD/SCID mouse model of pancreatic cancer was established .Treatment with CUR at the dose of 0.125, 0.25 and 0.5mg/mL inhibited the growth of pancreatic cancer PANC-1 xenografs by 45.2%, 50.0% and 59.7% respectively. The anti-tumor results in vivo demonstrate that CUR was remarkably active against PANC-1 xenografs in a dose-dependent manner.3. Effects of CUR on cell cycle and apoptosis of human pancreatic cancer PANC-1 cells.①After exposure to 10ug/mL CUR for 24h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken or fragmentation nucleus by transmission electron microscopy.②Flow cytometric analysis with PI staining was used to detecet cell cycle changing after CUR intervention. Being exposed to 0, 2.5, 10.0, and 40.0ug/mL of the CUS for 72h, the percentage of G0/G1 phase cells was 46.56±5.08, 53.33±5.05,67.50±6.50 and 77.00±6.73 respectively. Being exposed to 40.0μg/mL of the CUS for 24, 48 and 72 hours, the percentage of G0/G1 phase cells was 56.60±6.65, 67.83±6.76 and 77.00±1.73%respectively, These data show that CUR induces the accumulation of PANC-1 cells in the G0/G1 phase of the cell cycle in a dose-dependent and time-dependent maner.③Flow cytometry combined with annexinv-FITC/PI staining showed that CUR induced apoptosis. When PANC-1 cells were incubated with 0 , 2.5,10,40μg/mL CUR for 72h , the rate of apoptosis were 2.50士0.13, 8.30士1.23, 23.40士2.45 and 48.50士3.65%. Being exposed to 40.0μg/mL of the CUS for 24, 48 and 72h, the rate of apoptosis was 16.51士2.97,38.51士2.38 and 48.50士3.65%. Significiant difference was found among them. Thus these data indicate that CUR induces the apoptosis of PANC-1 cells in a dose-dependent and time-dependent maner.④Enzyme linked immunospecific assay was used to detecet caspase-3 activity after CUR treatment. Being exposed to 0, 2.5, 10.0, and 40.0ug/mL of the CUS for 72h, the caspase-3 activity was 0.009,0.011,0.035 and 0.065 units. So the caspase-3 activity was enhanced by CUR in a dose-dependent maner.4 molecular mechanism of CUR inducing apoptosis. Western blot analysis indicate that treatment of PANC-1 cells with CUR.(2.5, 10, 40ug/mL) for 72h results in release of mitochondrial cytochrome C and increase expression of bax, smac, caspase-3 and caspases-9 protin level in dose-dependent maner , but survivin、bcl-2 protein level was not significiant altered by CUR .5 The 50% inhibitory concentration (IC50) of GEM decrease from 36.76 nmol/L to 12.14 nmol/Lwhen together with CUS, reversal fold is 3.02. Western blot analysis indicate that treatment of PANC-1cells with CUR(2.5,10,40ug/mL) for 72h results in a decrease of P-170 protein level in dose-dependent maner but MDRP protein level was not significiant altered by CUR .Conclusion1. CUR has superior anti-tumor efficacy on human pancreatic cancer PANC-1 cells in vitro and in vivo via cell apoptosis and G0/G1 cell cycle arrest. CUR induced the apoptosis of PANC-1 cells via the up-regulation of smac and bax gene, leading to release of mitochondrial cytochrome C and then activation of caspase-9 and caspase-3. Thus CUR induces apoptosis via the intrinsic pathway. CUR has a chemo-sensitization effect with gemcitabine on pancreatic cancer cell line PANC-1, which maybe associated with down-regulation of p-170 protein.更多还原