【作者】 宗爱珍；

【导师】 王凤山；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2013， 博士

【摘要】 恶性肿瘤已成为全球的主要致死疾病之一,而90%因肿瘤致死的患者机体内发生了肿瘤的转移。因此,研发抗肿瘤转移药物对提高癌症患者的存活率至关重要。由于于具有免疫增强和抗肿瘤活性且毒副作用小,天然多糖抗肿瘤生长和转移活性的研究已成为肿瘤药物研究热点之一。乌贼墨多糖SIP为本课组从曼氏无针乌贼(Sepiella maindronni)墨中，首次分离到的一种结构新颖的多糖。研究发现SIP硫酸化衍生物SIP-S能够抑制肿瘤细胞基质金属蛋白酶-2(MMP-2)的表达和活性，并能抑制肿瘤细胞侵袭和内皮细胞迁移,据此我们推测SIP-S具有抗肿瘤转移的活性。本课题不但深入研究了SIP-S在小鼠体内的抗肿瘤生长和转移的活性,而且于体外研究了其抗肿瘤生长和转移的活性作用机制1SIP-S体内抑瘤和免疫增强活性的研究本论文建立S180肉瘤细胞小鼠皮下移植瘤模型,并考察了SIP-S体内抗肿瘤和免疫增强的活性。单独用药考察结果表明,SIP-S具有显著的抗肿瘤活性和免疫增强活性。SIP-S单独应用能够显著抑制S180荷瘤小鼠的肿瘤生长,给药剂量为30、20和10mg/kg·d的模型动物体内的抑瘤率分别达到43.5、39.7和32·4%；与环磷酰胺(CTX)30mg/kg·d给药组相比,SIP-S各剂量组瘤重均无显著性差异(P>0.05),说明SIP-S具有显著的抗肿瘤活性。此外,CTX给药组的小鼠脾脏指数显著降低(P<0.01),而SIP-S给药组小鼠的脾脏指数和胸腺指数有轻微或显著升高,说明SIP-S具有免疫增强活性。联合用药考察结果表明,SIP-S同样具有良好的抗肿瘤活性。SIP-S(15mg/kg-d)与CTX(12.5mg/kg·d)联合应用时的抗肿瘤活性高于SIP-S15(mg/kg·d)及CTX (25mg/kg·d)单独用药的活性,并且对小鼠的免疫功能没有显著影响。从而证明SIP-S与CTX联用具有协同增效作用,其免疫增强活性是其抗肿瘤活性的作用机制之一。2SIP-S抗肿瘤活性机制的体外研究SIP-S体外对肿瘤细胞的生长具有良好的抑制活性,且成时间和剂量依赖性。采用四甲基偶氮唑蓝(MTT)比色法和细胞克隆形成实验检测了SIP-S的体外抗肿瘤活性。检测结果表明,500、100和20μg/mL的SIP-S对人卵巢癌SKOV3细胞有明显的增殖抑制作用,作用2天时抑制率分别为41.06%、32.48%和25.10%,而第7天时抑制率则分别提高到约为86.96%、67.39%和50.00%。结果说明SIP-S能够直接影响肿瘤细胞的生长,随着时间和剂量的增大,其对肿瘤细胞的抑制作用增强。Annexin V-FITC/PI结合流式细胞仪检测表明,500、100、20μg/mL和0μg/mLSIP-S作用SKOV3细胞,36h后细胞的早期凋亡比率分别为8.54%、3.66%、1.79%和3.18%,坏死细胞和凋亡晚期细胞比率分别为17.41%、6.44%、5.76%和3.90%,证明SIP-S能够诱导细胞凋亡。另外,Western blot结果表明SIP-S能明显抑制SKOV3细胞内聚腺苷二磷酸核糖聚合酶(PARP-1)的表达和上调促凋亡蛋白caspase-3、-8、-9和bax的表达水平,但是对bcl-2和caspase-10的表达无显著影响。该结果同时证明这种胞内调节活性呈一定剂量依赖性。以上结果说明SIP-S能够通过调节凋亡相关蛋白的表达诱导肿瘤细胞的凋亡,抑制肿瘤细胞的生长,发挥其抗肿瘤作用。3SIP-S抗肿瘤转移活性的研究SIP-S体内不但有抗肿瘤转移活性,还能抑制转移瘤的生长。通过建立小鼠黑色素瘤B16F10人工肿转移模型,本论文考察SIP-S的体内抗转移活性。结果表明,与生理盐水组相比,SIP-S30和15mg/kg-d剂量组均能显著抑制小鼠肺部转移结节数(P<0.01),抑制率分别为87.95和85.94%。此外,阴性对照组部分动物出现肺部转移结节生长过大致肺部完全受到侵袭以及肝转移现象,但药物组均未出现此种情况,表明SIP-S体内不但有抗肿瘤转移活性,还能抑制转移瘤的生长。HE病理切片可见药物组肿瘤转移结节细胞核异型较少,免疫组化检测发现药物组转移结节上碱性成纤维细胞生长因子(bFGF)及细胞间黏附分子-1(ICAM-1)的表达量较生理盐水组降低。这可能是SIP-S抗转移活性的作用机制之一。4SIP-S抗肿瘤转移机制的研究本论文推测SIP-S通过调节细胞黏附分子的表达和抑制乙酰肝素酶的活性来实现抗肿瘤转移的活性。分别采用MTT法、流式细胞术、酶联免疫法和Western blot检测,本论文分析了SIP-S对肿瘤细胞黏附能力、P-选择素/配体结合、乙酰肝素酶活性以及细胞黏附分子表达的影响。MTT检测结果表明,SIP-S能够较好的抑制SKOV3细胞与Matrigel胶的黏附,SIP-S500、100和20μg/mL浓度下作用40min后的黏附抑制率分别为65.10%、54.40%和38.41%,作用80min后的黏附抑制率分别为68.53%、58.27%和42.76%。流式细胞术检测显示SIP-S能够抑制重组P-选择素/Fc嵌合蛋白(P-Fc)与表达其配体P-选择素糖蛋白配体1(PSGL-1)的HL-60细胞的结合,SIP-S5和1mg/mL的抑制率分别为23.81%和20.19%。此外,SIP-S能够显著抑制乙酰肝素酶对底物硫酸乙酰肝素和细胞外基质的降解,SIP-S10和5μg/mL浓度下的抑制率大于60%,与阳性对照药肝素的抑制活性相当,说明SIP-S能显著抑制乙酰肝素酶的活性。Western blot检测发现SIP-S能明显抑制SKOV3细胞上黏附分子ICAM-1、转化生长因子β1(TGF-β1)、整合素β1(integrin β1)和人脐静脉内皮细胞融合细胞EA.hy926上E-选择素的表达,但是对SKOV3细胞上E-钙黏素、CD44V6和EA.hy926细胞上VCAM-1和的表达没有显著影响。以上结果皆证明SIP-S具有调节细胞黏附分子的表达抑制肿瘤细胞的黏附、抑制乙酰肝素酶的生物活性,我们推测这是SIP-S抗肿瘤转移的作用机制。5SIP-S抗血管生成活性的研究体外管腔形成实验表明SIP-S能够抑制内皮细胞管腔形成和血管内皮生长因子VEGF和bFGF的表达,从而发挥抗肿瘤生长和转移的活性。SIP-S500、100和20μg/mL作用一定时间,可见内皮细胞小管形成数目减少,且管腔不完整,与阴性对照组比较差异显著。建立鸡胚尿囊膜模型检测SIP-S体内对血管生成的影响,与生理盐水组相比,SIP-S2和0.4mg/mL能够显著抑制鸡胚尿囊膜上的新生血管生成(P<0.01)。以上结果证明SIP-S具有抗血管生成的活性。Western blot检测发现SIP-S能明显抑制SKOV3细胞上和内皮细胞上血管内皮生长因子VEGF和bFGF的表达,这可能是其抗血管生成作用的机制之一。血管生成在肿瘤生长和转移的过程中均发挥重要作用。因此,推测SIP-S的抗血管生成活性是其抗肿瘤生长和转移活性的作用机制之一。6结论和意义本课题采用一系列体内、体外的方法确定了SIP-S抗肿瘤生长和转移的活性并初步揭示了SIP-S该类活性的作用机制。研究表明SIP-S体内具有显著的抗肿瘤生长和转移活性。进一步研究发现SIP-S能够抑制肿瘤细胞生长、诱导肿瘤细胞凋亡、增强荷瘤小鼠免疫功能、缓解化疗药物CTX所致的免疫损伤、降低肿瘤细胞的黏附能力、抑制乙酰肝素酶的活性并抑制新生血管生成,这些活性是SIP-S抗肿瘤生长和转移的作用机制。本课题研究证明了SIP-S是一种具有良好的抗肿瘤生长和转移活性的多糖且确定了其部分作用机制。本课题的完成,不但在硫酸化多糖抗肿瘤发生及转移研究方面具有重要理论意义,而且相关结论为SIP-S在肿瘤治疗中的应用开发奠定了坚实基础,应用价值同样明显。更多还原

【Abstract】 Cancer is a leading cause of death worldwide, and metastasis is responsible for about90%of the deaths from cancer. Therefore, the development of antimestatic drugs is urgently needed to control human malignancies. Natural polysaccharides have become the focus of research due to the advantages such as immunoenhancing activity, antitumor activity, as well as few toxic and adverse effects. Previously, we isolated a new polysaccharide SIP from the ink of cuttlefish Sepiella maindroni. The previous studies demonstrated that sulfated SIP (SIP-S) significantly inhibited the expression as well as the activity of matrix metalloproteinase-2(MMP-2), and decreased the invasion of SKOV3cells as wll as the migration of ECV3O4cells. These results suggested that SIP-S may be a candidate compound for preventing tumor metastasis. In this study, we determined the anti-cancer and anti-metastatic activities of SIP-S in mice models, and proved the possible mechanisms of these activities.1SIP-S has anticancer and immunostimulatory activitiesThe subcutaneous transplanted model of S180sarcoma in mice was made to investigate the activities of anticancer and immunostimulatory of SIP-S.SIP-S has immunoenhancing as well as anticancer activities, and it is demonstrated that the immunoenhancing activity is one mechanism for its anticancer activity. SIP-S could significantly inhibit tumor growth in S180-bearing mice. The inhibition rates of30,20and10mg/kg·d of SIP-S were43.5%,39.7%and32.4%, respectively. There were no obvious difference between the mean tumor weight of SIP-S-treated groups and cyclophosphamide (CTX)-treated group. Besides, SIP-S could improve mice thymus and spleen index in S180-bearing mice and mice treated with CTX. The combination treatment of SIP-S with CTX showed higher anticancer potency than the monotherapy. These results showed that SIP-S has immunoenhancing and anticancer activities, and demonstrated that the immunoenhancing activity is one mechanism for SIP-S’s anticancer activity.2SIP-S could inhibit the growth and induce the apoptosis of tumor cellsSIP-S could induce apoptosis of cancer cells by regulating the expression of relevant protein, so as to inhibit the prolifieration of cancer cells.The anticancer activity of SIP-S in vitro was investigated with MTT assay and cells colony-forming experiment. The inhibition rates to SKOV3cell proliferation by incubating with500,100and20μg/mL of SIP-S for2days were41.06%,32.48%and35.10%, respectively. And, the inhibition rates to SKOV3cell colony formation with the same concenntrations of SIP-S for7days were86.96%,67.39%and50.00%, respectively. These results showed that SIP-S could inhibit tumor cells growth in vitro and the effects are associated with time and given doses.Flow cytometry combined with AnnexinV-FITC/PI staining showed that SIP-S could induce tomor cells apoptosis. The early apoptosis rates of SKOV3cells under the concentrations of500,100,20and0μg/mL for36h were8.54%,3.66%,1.79%and3.18%, respectively, and the necrosis and late apoptosis rates were17.41%,6.44%,5.76%and3.90%. Western blot analysis showed that SIP-S could suppress the expression of PARP-1and increase the expression of caspase-3,-8,-9and bax in tumor cells and the effect was associated with concentration, while it had no significant influence on the expression of bcl-2and caspase-10.3SIP-S shows anti-metastatic activity in vivo The antimetastatic activity of SIP-S in vivo was examined by using the model of artificial lung metastasis with mouse melanoma B16F10. Compared with the saline group, SIP-S (15and30mg/kg-d) administrated for12d markedly decreased B16F10pulmonary metastasis in mice models by85.94%and87.95%, respectively. Besides, SIP-S could significantly inhibit liver metastasis of B16F10and the growth of metastatic tumor. Immunohistochemistry showed that SIP-S decrease the expression levels of ICAM-1and bFGF in lung metastasis nodules.The metastatic tumor sections staining with HE showed that nuclear atypia rates in SIP-S-treated groups were decreased compared with the saline group. The expression of basic fibroblast growth factor (bFGF) and intercellular adhesion molecule-1(ICAM-1) in metastatic nodules of SIP-S-treated group was less than in that of saline group from immunohistochemical examination, which may be one of mechanisms of anti-metastatic activity of SIP-S in vivo.4SIP-S inhibits tumor cell adhesion and heparanase(HPA) activityIt is demonstrated that regulating the expression of cell adhesion molecules and inhibiting heparanase activity might be the anti-tumor metastasis mechanisms of the SIP-S.In order to explore the mechanism of anticancer activity of SIP-S, we detected the inhibitory effects of SIP-S on adhesive ability of SKOV3cells and the activity of HPA by MTT assay and enzyme-linked immunosorbent assay(ELISA assay), respectively. MTT assay showed that SIP-S could inhibit the adhesive ability of SKOV3cells to matrigel. The inhibition rate by treating with500,100and20μg/mL of SIP-S for40min were65.10%,54.40%and38.41%, while68.53%,58.27%and42.76%by treating with SIP for80min respectively. ELISA assay showed that SIP-S could significantly inhibit the degradation of HPA to its substrate heparan sulfate and the extracellular matrix. Compared with the positive control drug, the inhibition rates by treating with10and5μg/mL of SIP-S were over60%, which demonstrate SIP-S can significantly inhibit the activity of HPA. Flow cytometry assay showed that SIP-S could inhibit the P-selectin-mediated adhesion to HL-60cells. The inhibition rates with5and1mg/mL of SIP-S treatment were23.81%and20.19%, respectively. Western blot analysis showed that the expression of ICAM-1, integrin β1, TGF-β1and E-selectin in tumor cells and endothelial cells was significantly inhibited by SIP-S. But, there were no obvious change in the expression of E-cadherin and CD44V6in SKOV3cells and VCAM-1in EA.hy926cells.5SIP-S shows anti-angiogenic activityThe anti-angiogenic activity of SIP-S was detected both in vitro and in vivo. SIP-S could inhibit endothelial cell lumen formation, proven by tube formation experiments in vitro. The number of endothelial cell tube formation was significantly reduced after treatment of SIP-S500,100and20μg/mL for a few hours compared the control group. SIP-S inhibited neovascularization in chick chorioallantoic membrane (CAM) assay at the dose of2and0.4mg/mL. These results showed that SIP-S possess anti-angiogenic activity. Western blot analysis showed that SIP-S significantly decreased the expression of VEGF and bFGF in SKOV3and EA.hy926cells, which might be one of the mechanisms of anti-angiogenic effect of SIP-S.6ConclusionsIn this study, the anti-cancer and anti-metastatic effects and mechanisms of SIP-S were investigated. SIP-S has significant anti-cancer and antimetastic activities, which might be mediated by inducing tumor cell apoptosis, enhancing the immune function of tumor-bearing mice, suppressing the adhesive ability of tumor cells and inhibiting neovascularization in CAM.The success of this project will not only determine that SIP-S was a polysaccharide with good anti-cancer activity and anti-matastasis activity, but also uncover fundamental anti-metastasis mechanisms of SIP-S in vitro and in vivo.. The information we got will also improve and enhance the development of the sulfated polysaccharide based anti-cancer drug in the future.更多还原