中药夏枯草对结肠癌细胞FasL基因表达和侵袭能力的影响

目的:研究夏枯草对人结肠癌SW480细胞FasLmRNA表达及对其侵袭能力的影响,为中药抗肿瘤提供实验依据.方法: 根据MTT法得到夏枯草对SW480细胞的半数有效抑制浓度(IC50),确定药物作用浓度.SW480细胞分别经夏枯草不同浓度(0.5IC50、IC50)作用后,应用逆转录-聚合酶链反应(RT-PCR)法检测夏枯草作用前后人结肠癌SW480细胞FasL mRNA的变化;应用Transwell细胞侵袭试验检测夏枯草对SW480细胞侵袭能力的影响.结果: 夏枯草处理后较处理前SW480细胞FasL mRNA表达水平均明显高于对照组(P<0.01);而且FasL mRNA表达水平随夏枯草作用浓度增加显著上调,夏枯草不同浓度组比较均有显著性差异(P<0.01);随夏枯草作用浓度升高,SW480细胞侵袭能力明显增强,不同浓度组比较均有显著性差异(P<0.01).结论: 夏枯草在一定时间内均可上调人结肠癌SW480细胞FasL mRNA的表达,而且这种上调作用在一定范围内呈剂量依赖性,可使结肠癌细胞的侵袭能力增强.     

芬太尼对结肠癌细胞株SW480增殖及侵袭的影响

目的：探讨不同浓度的芬太尼对结肠癌细胞株 SW480增殖和侵袭的影响。方法：选用不同浓度的芬太尼（0．5、5、50ng／ml）干预结肠癌细胞株 SW480,采用四甲基偶氮唑蓝（MMT）法检测细胞增殖,Transwell法测定细胞侵袭。结果：各组芬太尼孵育 SW480细胞48h 及72h 后,均能显著抑制细胞的增殖（P ＜0．05）,且呈浓度和时间依赖性。芬太尼孵育 SW480细胞48h,与对照组相比,各浓度组均浓度依赖性抑制凋亡（P＜0．05）。结论：芬太尼可抑制结肠癌细胞株 SW480的增殖及侵袭。     

肝细胞生长因子对结肠癌细胞SW620增殖、侵袭的影响

摘要：目的探讨肝细胞生长因子（Hepatocyte growth factor,HGF）对人结肠癌细胞株SW620的增殖和侵袭能力的影响。方法应用MTT法分析HGF对SW620细胞增殖的影响,确定最佳促增殖浓度;应用基底膜重建实验和电镜观察HGF对细胞侵袭能力的影响;应用PCR技术检测HGF对MMP-2 mRNA表达的影响。结果当HGF浓度≥10ng/ml时促进SW620增殖且呈质量浓度依赖性; HGF明显促进SW620 移行且呈质量浓度依赖关系;加入 HGF 后,SW620细胞内的微丝明显增多,MMP-2表达水平升高。结论HGF可以在体外促进人结肠癌细胞SW620的增殖和移行。     

槲皮素对人结肠癌SW480细胞体外侵袭能力的影响

目的：研究槲皮素对结肠癌SW480细胞的体外侵袭能力的影响,并探讨其可能的作用机制。方法：结肠癌SW480细胞经槲皮素处理后用Transwell小室的方法检测其体外黏附、侵袭和运动能力。明胶酶谱分析法检测SW480细胞分泌的基质金属蛋白酶活性。结果：结肠癌SW480细胞经槲皮素处理后,体外侵袭、运动能力呈剂量依赖性下降,SW480细胞MMP-2及MMP-9的分泌下降（P〈0.05）。结论：槲皮素在体外剂量依赖性地抑制结肠癌SW480细胞的转移能力,这可能与槲皮素抑制MMP-2及MMP-9的分泌有关。     

芦荟大黄素诱导胃癌细胞株MGC-803细胞凋亡药效学作用研究

目的:观察芦荟大黄素诱导人胃癌细胞株MGC-803凋亡药效作用研究.方法:用50、100、500、1000μmol/L浓度芦荟大黄素作用胃癌细胞株48h,倒置显微镜下观察细胞增殖情况;MTT法检测芦荟大黄素对细胞增殖的抑制作用;电镜观察、判定凋亡细胞;凋亡检测试剂盒检测细胞凋亡.结果:倒置显微镜下,随着芦荟大黄素作用浓度升高,细胞增殖能力随之下降;MTT法亦检测到上述结果;当芦荟大黄素作用浓度是1000μmol/L时,可检测到明显的凋亡细胞.结论:高浓度的芦荟大黄素可诱导胃癌细胞株MGC-803细胞的凋亡.     

芹菜素对人结肠癌细胞株SW480体外侵袭能力的抑制作用

目的:观测芹菜素对大肠癌细胞株SW480体外侵袭、粘附和运动能力的影响。方法:人工重组基底膜技术观察药物对细胞侵袭、运动和粘附的影响。结果:芹菜素处理后SW480细胞的侵袭、运动及粘附能力下降,并呈时间和剂量依赖性。结论:芹菜素多个层面抑制SW480细胞的侵袭、运动和粘附。     

芹菜素对人结肠癌细胞株SW480体外侵袭能力的抑制作用

目的：观测芹菜素对大肠癌细胞株SW480体外侵袭、粘附和运动能力的影响。方法：人工重组基底膜技术观察药物对细胞侵袭、运动和粘附的影响。结果：芹菜素处理后SW480细胞的侵袭、运动及粘附能力下降,并呈时间和剂量依赖性。结论：芹菜素多个层面抑制SW480细胞的侵袭、运动和粘附。     

YAP蛋白在结肠癌中的表达及其对SW480细胞增殖和侵袭的影响

目的:研究YAP蛋白在结肠癌中的表达及临床意义, 并初步探讨其对结肠癌细胞增殖、侵袭的影响。方法:免疫组化检测YAP蛋白在25例结肠癌组织及配对正常组织中的表达,并分析其与结肠癌临床病理特征之间的关系;用Real-Time PCR和 Western blot验证YAP-shRNA慢病毒载体转染效率;生长曲线实验和MTT法检测下调YAP表达对结肠癌细胞SW480增殖的影响;Transwell实验检测下调YAP表达对结肠癌细胞SW480侵袭的影响;Western blot检测下调YAP表达对CyclinD1、E-cadherin及Vimentin蛋白的影响。结果:YAP在结肠癌组织中的表达显著高于正常结肠组织（P＜0.05）,并且与淋巴结转移密切相关(P＜0.05);转染YAP-shRNA慢病毒载体的SW480细胞YAP的表达在mRNA 和蛋白水平明显降低（均P＜0.05）;下调YAP表达后结肠癌细胞增殖及侵袭明显减弱（均P＜0.05）;下调YAP 表达可抑制结肠癌细胞CyclinD1、E-cadherin及Vimentin蛋白的表达（均P＜0.05）。结论:YAP在结肠癌组织中高表达,且与淋巴结转移相关,下调YAP表达可抑制结肠癌细胞增殖及侵袭能力,预示YAP在结肠癌恶性进展中具有重要的作用。     

膜联蛋白A2对人结肠癌细胞SW620侵袭迁移的影响

目的:研究膜联蛋白A2(ANXA2)对人结肠癌细胞SW620侵袭迁移的影响.方法:采用siRNA技术沉默ANXA2在SW620细胞中的表达,实验分为干扰组、对照组和野生组,RT-PCR法检测各组ANXA2mRNA水平,Westem blot法检测各组ANXA2、纤维蛋白溶酶(PL)、基质金属蛋白酶-1(MMP-1)、血管内皮生长因子(VEGF)蛋白的表达,Transwell侵袭实验检测各组SW620细胞穿膜能力.结果:干扰组与对照组、野生组相比,ANXA2 mR-NA水平、ANXA2、PL、MMP-1、VEGF蛋白表达及SW620穿膜细胞数差异均有统计学意义(P＜0.05),而对照组和野生组相比上述各指标均无统计学差异.ANXA2蛋白和PL、MMP-1、VEGF蛋白水平、穿膜细胞数呈正相关(P＜0.05).结论:沉默ANXA2基因后可能通过下调PL、MMP-1及VEGF蛋白的表达,抑制SW620细胞侵袭迁移能力起到抗肿瘤作用.     

细胞因子上调大肠癌细胞Fas配体表达

目的：研究白介素１８（ＩＬ－１８）等内源性细胞因子对大肠癌细胞ＦａｓＬ表达水平的调控作用。方法：采用Ｗｅｓｔｅｒｎ蛋白印迹法测定细胞因子处理后大肠癌细胞的ＦａｓＬ表达水平。结果：在检测的６株大肠癌细胞中,全部表达ＦａｓＬ蛋白;ＴＮＦ－α和ＩＦＮ－γ显著上调ＤＬＤ－１ ＦａｓＬ蛋白的表达水平;ＴＮＦ－α和ＩＦＮ－γ以及乙酸肉豆佛波醇（ｐｈｏｔｂｏｌ１２－ｍｙｒｉｓｔａｔｅ １３－ａｃｅｔａｔｅ,ＰＭ     

Clinicopathological significance of Fas and Fas ligand expressions in esophageal cancer

Esophageal carcinomas have recently been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. However, the prognostic importance of Fas/FasL and their correlation with clinicopathological characteristics are yet to be delineated in this highly malignant carcinoma. Specimens from 106 esophageal squamous cell carcinoma patients were used for immuno-histochemical evaluation of Fas, FasL, and CD8 expressions. Fifty-two (49%) and 34 (32%) patients were positive for FasL and Fas, respectively. There were no associations between FasL expression and clinicopathological characteristics except lymph vessel invasion. Strong FasL expression correlated with significant (P < 0.001) decrease in tumor nest CD81 cells. However, neither FasL nor CD81 had any impact on patient survival. Strong Fas expression was correlated with depth of invasion (40.3% in pT1, T2 versus 20.5% in pT3, T4; P5 0.0308), histological differentiation (45.7% in well versus 25.4% in nonwell; P < 0.05), and lymph node metastasis (22.6% in positive versus 45.5% in negative; P < 0.01). Fas expression was one of the independent favorable prognosticators for patients’ survival (risk ratio, 3.26; P < 0.01) in esophageal SCC. Fas expression was an independent prognosticator for recurrencefree survival, whereas FasL expression did not influence the survival in esophageal squamous cell carcinoma. Down-regulation of tumor Fas may be the hallmark of immune privilege for the tumor, thus causing the patients’ poorer outcome. Tumor FasL may counterattack the host immune cells to such an extent that the prognosis is not affected.     

Fas ligand expression in human pancreatic cancer

Tumor cells escape immunologic rejection through diverse mechanisms. Fas and its ligand represent one such mechanism. Recently we reported that pancreatic cancer cell lines express Fas-ligand and kill lymphoid cells by Fas-mediated apoptosis in vitro. This study was designed to determine whether human pancreatic tumors express Fas-ligand in vivo, as a potential mediator of counterattacking the host immune cells, and to determine if Fas-ligand expression correlates with clinicopathologic characteristics and patient survival. Fas-ligand expression in 45 primary pancreatic ductal cancers and two hepatic metastases was determined using immunohistochemistry. Apoptotic cells within the tumor were assessed by immunohistochemistry for the presence of single stranded DNA. Immunohistochemistry showed that 37 (82%) of the primary tumors and both of the hepatic metastases expressed Fas-ligand. Tumor infiltrating lymphoid cells were frequently apoptotic, but the cancer cells were rarely apoptotic. Patients with Fas-ligand-positive tumor had significantly shorter survival times than Fas-ligand-negative. A multivariate analysis indicated that Fas-ligand expression and the histologic margin status were significant prognostic factors. These results suggest that the expression of Fas-ligand in human pancreatic cancers and the induction of apoptosis in the infiltrating lymphoid cells may be required to counterattack the host cytotoxic T-lymphocytic and natural killer cellular responses.     

Up-regulation of Fas ligand and down-regulation of Fas expression in oral carcinogenesis

An important molecule involved in delivering the death signal that initiates apoptosis is called Fas, or Apo-1, which sits on the cell surface. When another molecule called the Fas ligand (FasL) binds to it, Fas triggers a series of events inside the cell that leads to apoptosis. In order to investigate the mechanism of immune escape and the expression of Fas and FasL in oral premalignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs), a total of 64 samples were evaluated by an immunohistochemical method using a labelled streptavidin–biotin assay. These samples comprised nine hyperkeratotic and 24 oral premalignant lesions (nine of mild, moderate, and six of severe dysplastic lesions), and 24 OSCCs, together with seven healthy controls. The results demonstrated that the majority of invasive OSCCs showed down-regulation of Fas expression but up-regulation of FasL expression. These phenomena were also detected in OPLs. The results indicate that the expression of Fas and FasL is involved in oral carcinogenesis and this may be a mechanism by which the cancer cells evade the host immune assault. Perhaps, in future, Fas/FasL system may be used as a prognostic biomarker in predicting the behavior of oral premalignant lesions.     

术前化疗后结直肠癌组织中Fas/FasL和survivin的表达

目的探讨化疗后结直肠癌组织Fas/FasL和survivin的表达情况及两者之间的相关性。方法采用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记技术(TUNEL法)和免疫组化方法,测定化疗后和未化疗两组各16例结直肠癌患者肿瘤的原位凋亡(AI)、原位增殖(KI)及survivin表达,运用流式细胞仪测定肿瘤细胞的细胞周期、Fas/FasL表达,用双抗体ELISA法检测患者血清可溶性Fas/FasL(sFas/sFasL)表达。结果化疗组结直肠癌患者肿瘤细胞的AI及Fas/FasL均较对照组高,KI、G2/M期细胞、sFas及survivin较对照组低,差别有统计学意义(P<0.05)。结论化疗通过诱导结直肠癌细胞凋亡及阻止细胞进入G2/M期从而抑制癌细胞的生长。Fas/FasL系统与结直肠癌细胞凋亡、增殖调控有关。survivin阳性表达下降程度可作为化疗有效的标志之一。     

表达FasL的肝癌细胞通过Fas/FasL诱导T淋巴细胞发生凋亡

目的研究发现肿瘤组织中FasL阳性表达区肿瘤浸润淋巴细胞的凋亡率比FasL阴性区高,推测肿瘤细胞可通过增加FasL表达来促使肿瘤浸润淋巴细胞凋亡.本实验在体外验证肝癌细胞是否可以诱导T淋巴细胞发生凋亡.方法选择FasL表达阳性的肝癌细胞(效应细胞)与对Fas介导凋亡敏感的活化T淋巴细胞共培养,取3种效靶比20:1,10:1,5:1.生长曲线法,流式细胞术,荧光显微镜观察分别检测T淋巴细胞的凋亡情况.结果流式细胞仪检测结果显示,肝癌细胞接种浓度为0(对照),0.5×105/ml,1×105/ml,2×105/ml时,T淋巴细胞凋亡率为1.80±0.21%,5.49±0.17%,11.18±0.14%,18.22±0.11%.荧光显微镜观察结果表明,肝癌细胞接种浓度为0(对照),0.5×105/m1,1×105/ml,2×105/ml时,T淋巴细胞凋亡率为1.58±0.12%,5.22±0.13%,9.74±0.21%,19.33±0.18%.3种效靶比条件下,肝癌均能诱导T淋巴细胞发生凋亡.随着肝癌接种浓度的升高及培养时间的延长,T淋巴细胞凋亡率逐渐增加,每个实验组与对照组比较P值均＜0.01.结论肝癌细胞可以通过Fas系统诱导淋巴细胞发生凋亡,这为肝癌的免疫逃逸和反攻击提供了证据.     

Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-beta expression

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-beta1 (TGF-beta1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-beta1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-beta1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8-33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6-2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-beta1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.     

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).