人参皂苷Rg3对小鼠B16黑素瘤细胞侵袭、转移及MMP-9表达的影响

目的:观察人参皂苷Rg3(简称Rg3)对小鼠黑素瘤细胞B16的肺转移、侵袭能力及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响,探讨其抗肿瘤转移的作用机制。方法:体内建立小鼠自发肺转移和实体瘤模型,观察腹腔注射不同剂量的Rg3(对照组给予0.9%氯化钠溶液)后,肺部肿瘤转移灶的数目,并检测实体瘤中MMP-9蛋白的表达情况。采用Boyden小室侵袭实验及免疫细胞化学染色法检测Rg3对肿瘤细胞体处侵袭能力及MMP-9表达的影响。结果:采用不同剂量的Rg3(0.3、1.0和3.0mg/kg)治疗后,小鼠肺部转移灶的数目均较少,肿瘤组织中MMP-9的表达水平降低,与对照组比较差异有统计学意义(P<0.05)。在体外,2.5和5.0μg/mLRg3治疗组中侵袭穿过人工基膜的B16细胞数目明显少于对照组(P<0.01),且5.0μg/mLRg3可抑制肿瘤细胞中MMP-9的表达。结论:Rg3能够抑制小鼠黑素瘤细胞的肺转移,其抗肿瘤转移作用可能是通过降低肿瘤细胞中MMP-9的表达水平及细胞侵袭能力来实现的。     

木鳖子醇提物对黑素瘤B16细胞增殖的抑制及其可能机制

目的：观察木鳖子醇提物(ethanol extract from cochinchina momordica seed, CMSEE)对黑素瘤B16细胞增殖的影响,初步探讨其作用机制。方法：MTT法和克隆集落形成实验检测CMSEE对小鼠黑素瘤B16细胞生长的影响,倒置相差显微镜和瑞氏吉姆萨染色观察细胞形态学变化,流式细胞技术（FCM）检测B16细胞周期分布和凋亡率的变化,比色法检测CMSEE对B16细胞黑素生成和酪氨酸酶活性的影响,RTPCR法检测CMSEE对B16细胞中Cmyc、〖STBX〗P38和Tyr〖STBZ〗基因表达的影响。结果：CMSEE（10～100 mg/L）明显抑制B16细胞的增殖(P<0.05, P<0.01),呈质量浓度和时间依赖性。10～40 mg/L CMSEE处理后,B16细胞呈现典型的细胞分化形态,细胞周期阻滞于G0/G1期,黑素产生和酪氨酸酶活性增加(P<0.01),Cmyc基因表达下调,〖STBX〗P38和Tyr〖STBZ〗基因表达上调;100 mg/L CMSEE处理后的B16细胞呈现凋亡形态变化\[凋亡率达(37.2±329)%\],黑素生成减少(P<0.05),酪氨酸酶活性降低(P<0.01)。结论：CMSEE体外对B16细胞增殖有明显的抑制作用,其机制与其低剂量诱导分化和高剂量诱导凋亡有关。     

木鳖子醇提物诱导小鼠黑素瘤B16细胞分化的作用机制

目的:探讨木鳖子醇提物(ethanol extract of cochinchina momordica seed,CMSEE)诱导黑素瘤B16细胞分化的作用机制。方法:CMSEE处理B16细胞后,瑞氏染色法观察B16细胞的形态变化;比色法检测B16细胞的黑素含量。Westernblotting检测经CMSEE和p38、ERK通路抑制剂SB203580、SD98059分别处理后B16细胞中p-p38、p-ERK表达的变化。结果:CMSEE可诱导B16细胞分化,使细胞的黑素量含量明显升高(P<0.01),10、20、40μg/ml CMSEE处理组B16细胞的黑素量D值分别为(0.057±0.007)、(0.173±0.005)和(0.249±0.002),而对照组为(0.037±0.002)。20μg/ml CMSEE处理B16细胞后,p-p38蛋白表达水平明显升高,处理60 min时的相对表达量最高,为对照组的5.6倍;而p-ERK表达水平明显降低(P<0.01),处理60 min时的相对表达量为对照组的25%倍。p38通路抑制剂SB203580可阻断CMSEE对B16细胞的分化诱导作用,而ERK通路抑制剂SD98059可增强CMSEE对B16细胞的分化诱导作用。结论:p38通路和ERK通路的活化参与了CMSEE对B16细胞的分化诱导作用。     

基质金属蛋白酶抑制剂与肿瘤侵袭转移

基质金属蛋白酶抑制剂(TIMP)是基质金属蛋白酶(MMP)的特异性抑制剂,对抑制肿瘤的侵袭转移起重要作用.TIMP的表达水平与肿瘤预后具相关性,在肿瘤治疗上有潜在的重要价值.     

MMP-9与结直肠癌侵袭转移的相关性及临床研究进展

基质金属蛋白酶(MMP)-9是MMP家族相对分子质量最大的MMP,主要来源于巨噬细胞和中性粒细胞,MMP-9与结直肠癌的发生及侵袭转移相关,也可以为结直肠癌的临床预防及治疗提供新的途径.     

木鳖子单体化合物对羟基桂皮醛诱导小鼠黑素瘤B16细胞的分化及其机制

探讨从中药木鳖子中提取的单体化合物对羟基桂皮醛（p-hydroxylcinnamaldehyde,PHD）对小鼠黑素瘤B16细胞分化的影响及其可能的作用机制。方法: 以10、20、40 μmol/L PHD作用B16细胞,设空白对照及福司克林（Forskelin）阳性对照组。磺酰罗丹明（suphrhodamineB,SRB）法检测PHD对B16细胞生长的抑制率,平板克隆集落形成实验检测细胞的克隆形成能力,Giemsa 染色观察细胞形态,比色法检测细胞中黑色素含量和酪氨酸酶（tyrosinase,Tyr）活性,划痕愈合实验检测细胞的迁移能力,Western blotting检测Tyr、酪氨酸酶相关蛋白1（tyrosinase protein,Trp1）及MAPK信号转导通路中p-P38、p-JNK和p-ERK1/2的表达。结果：PHD对黑素瘤B16细胞具有明显的增殖抑制作用,10、20、40 μmol/L PHD作用B16细胞48 h后,其增殖抑制率与对照组相比明显增加[（12.78±0.87）%、（37.70±2.28）%、（42.17±4.18）% vs 0,P<0.05],20、40 μmol/L PHD组增殖抑制率与Forskelin组相比明显增加[（37.70±2.28）%、（42.17±4.18）% vs（22.00±1.13）%,P<005]。40 μmol/L PHD处理B16细胞24、48、72 h后,B16细胞呈典型分化形态,黑色素含量及Tyr活性与对照组相比均明显增加[（0097±002）、（0.13±0.04）、（0.15±0.05）vs（0.085±0.003）;（1.11±0.31）、（1.43±0.05）、（1.67±0.49）vs (0.64±010）,P<005];细胞集落形成能力和迁移能力都明显降低（均P<0.05）。与空白对照组相比,PHD处理组细胞Tyr和Trp1的表达明显增加（P<0.05）,MAPK信号通路中p-P38和p-JNK的表达水平也明显增加（P<0.05）,而p-ERK活性差异则无统计学意义（P>0.05）。结论：PHD通过上调MAPK信号通路中p-P38和p-JNK的活性而对小鼠黑素瘤B16细胞产生明显的增殖抑制,并在形态和功能上诱导黑素瘤细胞的分化。     

化学诱导小鼠肝癌模型中CD133的动态变化

目的:检测肝癌干细胞标志CD133在化学诱导C57BL/6J小鼠肝癌过程中各时期的动态变化.方法:通过化学法[二乙基亚硝胺(diethylnirtosamine,DEN)/四氯化碳(carbon tetrachloride,CC14)/乙醇]诱发50只C57BL/6J雄性小鼠肝癌,对照组为45只正常C57BL/6J雄性小鼠.观察小鼠成瘤情况和生长状态,对每2周定期处死小鼠获得的组织标本分别进行病理学切片观察,并采用实时荧光定量PCR、免疫组织化学法、蛋白质印迹法和FCM法分别检测CD133 mRNA和蛋白的表达情况.结果:化学诱导20周后,成功诱发出小鼠肝癌.实时荧光定量PCR和FCM检测结果显示,第8周后诱癌组小鼠肝组织中CD133的表达高于正常对照组,差异有统计学意义(P＜ 0.05或P＜0.001);随着诱癌时间的增加,CD133的表达量呈上升趋势,第16周时诱癌组CD133的表达明显高于前期诱癌组(P＜0.001).蛋白质印迹法检测结果显示,诱癌组CD133蛋白从第4周起出现弱表达,随着诱癌时间的递增,CD133蛋白表达量逐渐增多.免疫组织化学检测结果显示,诱癌组第8周CD133出现弱阳性,第16周出现中等阳性,第20周出现强阳性;而正常对照组无表达.结论:肝癌干细胞标志CD133参与了肝癌的发生、发展全过程,随着诱癌进展其表达量逐渐增加.     

Marimastat对裸鼠原位种植人胃癌生长和转移抑制的实验研究

目的 探讨基质金属蛋白酶抑制剂Marimastat对胃癌生长和转移的抑制作用。方法 采用人胃腺癌细胞株SGC—7901完整组织块棵鼠原位种植，建立胃癌转移模型。移植7天后开始腹腔内注射Marimastat，隔天1次，剂量为0mg/kg(对照组)、10mg/kg、30mg/kg、60mg/kg(治疗组)，共用8周，第9周处死动物，测量原位肿瘤大小、抑瘤率、肿瘤微血管密度(MVD)、MMP—9的高表达率、观察肿瘤转移情况。结果 治疗组原位肿瘤瘤重明显较对照组轻，肿瘤肝脏转移、腹膜转移、肿瘤组织微血管密度和肿瘤组织MMP—9高表达率与对照组比较，均有显著性差异(P＜0．05)，且与Marimastat剂量呈正相关。结论 Marimastat对体内胃癌的生长及转移均有抑制作用。     

高温抑制舌癌细胞侵袭及转移机理的实验研究

目的探讨高温治癌与基质金属蛋白酶系统之间的关系,以期阐明高温抑制肿瘤细胞侵袭及转移的机理.方法对舌癌细胞Tca-8113进行体外43℃加温,通过免疫组化、流式细胞术及明胶酶谱活性分析等方法进行分析,以研究加温后该舌癌细胞MMP-9、MMP-2表达量及其活性的改变.结果加温与未加温细胞MMP-9、MMP-2均有表达,但加温后的细胞MMP-9和MMP-2蛋白表达量减少,并且加温后的细胞MMP-9和MMP-2的活性较未加温细胞明显下降.结论高温能通过调节基质金属蛋白酶(MMP-9、MMP-2)的表达量及其活性来抑制肿瘤细胞的侵袭、转移能力.     

基质金属蛋白酶-9对结直肠癌侵袭转移的影响

 基质金属蛋白酶-9（MMP-9）是结直肠癌发生发展的重要促进基因,能够诱导结直肠癌细胞增殖、血管新生和肿瘤浸润,导致结直肠癌的侵袭转移。MMP-9对结直肠癌侵袭转移的影响既可独自完成又可与其他基因联合完成,是结直肠癌晚期转移的重要蛋白酶。     

木鳖子提取物体外抗肿瘤活性的初步研究

目的：观察木鳖子（cochinchina momordica seed,CMS）水提物（water extract of CMS,CMSWE）和醇提物（ethanol extractof CMS,CMSEE）对肿瘤细胞生长的影响,探讨CMSEE的抗肿瘤机制。方法：制备CMSWE和CMSEE。MTT法检测CMSWE和CMSEE对人黑色素瘤B16、肺癌细胞A549、乳腺癌细胞MDA-MB-231、食管癌细胞TE-13,以及正常人外周血淋巴细胞（PBMC）生长的影响,计算细胞生长抑制率;光镜下观察细胞形态变化;瑞氏-吉姆萨染色观察B16细胞凋亡形态学变化。流式细胞技术（FCM）检测B16细胞凋亡率和细胞周期分布。结果：CMSEE（25—300mg／L）对B16、A549、MDA-MB231和TE-13细胞增殖均有明显的抑制作用（P〈0．01）,对PBMC的增殖没有明显影响。CMSWE（25～300mg／L）对A549、TE-13、MDA-MB231、PBMC的生长没有明显影响,仅对B16细胞的增殖有较弱的抑制作用,且各浓度组抑制率均小于相同浓度的CMSEE（P〈0．01）。经CMSEE处理后,B16细胞显示典型的凋亡形态变化,凋亡率明显增高（P〈0．01）,G0／G1期细胞明显增加,S期、G／M期细胞减少（P〈0．01）。结论：CMSEE体外对人肿瘤细胞增殖有明显的抑制作用,其抗肿瘤机制可能与阻滞肿瘤细胞周期和诱导凋亡有关。     

Bromelain proteases suppress growth, invasion and lung metastasis of B16F10 mouse melanoma cells

The thiolprotease bromelain, isolated from pine apple stem, was suggested for use in adjuvant tumor therapy. This study examined the in vitro effects of crude bromelain, bromelain F9 and papain on B16F10 mouse melanoma cell lung colonization, in vitro cell proliferation, invasion through matrigel and CD44 expression. In vitro treatment of the melanoma cells with bromelain F9 and papain before i.v. injection into mice prevented lung colonization. The lung weight at day 20 was significantly reduced from 5.1% (untreated cells) to 1.6% (bromelain F9 treated cells). Papain was as effective as bromelain F9. However, there was no difference in the lung weight between bromelain F9 treated and the untreated group at day 27. Protease removal and further incubation of the B16F10 cells retained their capacity to induce lung tumor metastases. The proteases inhibited growth of the melanoma cells in a dose dependent manner. Crude bromelain was most active with a half maximal value of 7.5 mu g/ml. However, the antiproliferative effects did not correlate with the proteolytic activity. In a matrigel invasion assay, the proteases reduced the invasive capacity of the melanoma cells maximally by about 30%. Using flow cytometry, the proteases were found to reduce the CD44 density, present on the melanoma cells, to a different degree: crude bromelain was more active than bromelain F9 and papain, which had higher proteolytic activity. Crude bromelain was most active in abolishing the CD44 re-expression after protease treatment.     

Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.     

Effects of DL-alpha-difluoromethylornithine on the growth and metastasis of B16 melanoma in vivo

The effects of DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth of experimental mouse B16-F10 melanoma cells were investigated. DFMO (3%) in drinking water was administered to B16-F10 melanoma-bearing mice. At 24 days, B16-F10 melanomas in DFMO-fed mice weighed 75% less than those in control mice (p less than 0.001). DFMO reduced putrescine and spermidine levels in B16-F10 melanoma by 98% and 84%, respectively, and prolonged the mean survival time from 25.9 +/- 1.2 to 35.7 +/- 2.2 days (p less than 0.001). The effects of DFMO on experimental metastasis were also investigated. DFMO treatment resulted in a significant decrease in pulmonary metastasis induced by i.v. injection of B16-F10 melanoma cells.     

Effects of theophylline treatment on mouse B-16 melanoma cells in vitro

The effects of theophylline treatment on mouse B-16 melanoma cell growth, metabolism, and membrane antigen expression in vitro were studied. Theophylline treatment inhibited DNA synthesis and the cell growth rate, and caused an elevation of intracellular cAMP levels. Cells treated with theophylline became elongated and assumed a normal fibroblast-like morphology. Theophylline treatment of B-16 cells also reduced the levels of tumor specific antigen and H-2 antigen detectable on the cell membrane.     

Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells.

PURPOSE: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. EXPERIMENTAL DESIGN AND RESULTS: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. CONCLUSIONS: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.     

Effects of a calcium-antagonist (flunarizine) on the in vitro growth of B16 mouse melanoma cells

Flunarizine, a drug which binds to calmodulin, was found to inhibit both the growth rate and the survival fraction of a B16 mouse melanoma cell line cultured in vitro. Both of these effects, that are dose and time-dependent, occurred at doses which in vitro cause a 50% inhibition of the calmodulin-activated cyclic nucleotide phosphodiesterase prepared from the tumor. Cyclic AMP was increased in treated B16 melanoma cells, while DNA synthesis was inhibited, though the DNA content/cell was increased. The possible mechanisms of the drug's action are discussed herein.