Synthetic triterpenoids inhibit growth and induce apoptosis in human glioblastoma and neuroblastoma cells through inhibition of prosurvival Akt,NF-<Emphasis Type="Italic">κ</Emphasis>B and Notch1 signaling

Glioblastomas are high-risk primary brain tumors that are generally unresponsive or only weakly responsive to the currently available antineoplastic agents. Thus novel therapeutic strategies and agents are urgently needed to treat these incurable cancers. Oleanolic acid and ursolic acid are naturally occurring triterpenoids that have been used in traditional Asian medicine as anti-inflammatory and anti-cancer agents. Recently, synthetic oleanolic acid triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives have been shown to exhibit potent antitumor activity against diverse types of tumor cell lines, including leukemia, multiple myeloma, osteosarcoma, breast, lung, and pancreatic cancer cell lines; however, the anticancer activity of these agents for brain tumors has not been reported. In the present study, we investigated the apoptosis-inducing activity of CDDOs in glioblastoma (U87MG, U251MG) and neuroblastoma (SK-N-MC) cell lines. Cell growth/viability (MTS) and cytotoxicity (LDH release) assays demonstrated that glioblastoma cell lines are least sensitive to CDDO, but are highly sensitive to CDDO-Me and CDDO-Im at concentrations of 2.5–10 μM. CDDO-Im and CDDO-Me were equipotenent in their growth inhibitory activity. The primary mode of tumor cell destruction was apoptosis as demonstrated by significant increase in the number of hypo-diploid (sub-G0) cells and annexin V-FITC binding. Induction of apoptosis was associated with the activation of procaspases-3, -8, and -9, mitochondrial depolarization and the release of cytochrome c from mitochondria. Furthermore, CDDO-Me inhibited the levels of anti-apoptotic and prosurvival p-Akt, NF-κB (p65) and Notch1 signaling molecules. These studies provide rationale for clinical evaluation of these novel agents for the management of lethal brain neoplasms.     

TLR4、p-Akt在哮喘大鼠气道平滑肌迁移的作用及小青龙汤对其影响

目的探讨Toll样受体4(Toll-like receptor 4,TLR4)及p-Akt在哮喘发病中的作用及小青龙汤对气道平滑肌迁移的影响。方法 SPF级♂大鼠40只随机分为:正常对照组(A)、哮喘4周组(B)、哮喘8周组(C)、小青龙汤4周组(D)和小青龙汤8周组(E),每组8只。以卵清白蛋白(OVA)致敏与激发建立哮喘大鼠气道重塑模型。测定气道阻力;观察气道壁嗜酸性粒细胞侵润情况;图像分析软件测定支气管管壁面积、平滑肌面积和平滑肌至上皮下基底膜距离;免疫组织化学方法检测TLR4、p-Akt蛋白表达。结果 B组、C组、D组和E组大鼠的WAt/Pi、WAm/Pi较正常对照组均增加(P<0.01),间距/Pi减小(P<0.01);B组和C组大鼠的WAt/Pi高于E组,间距/Pi则较两组明显缩短(P<0.05);TLR4在B组和C组气道平滑肌上呈阳性或强阳性表达,表达量高于A组(P<0.01),C组亦高于B组(P<0.05),E组的表达量亦低于C组和D组(P<0.01)。p-Akt在B组、C组及D组的表达均高于A组(P<0.01),D组亦高于B组(P<0.05),E组低于B组和D组,与D组相比亦降低(P<0.05)。结论 TLR4和p-Akt参与哮喘气道平滑肌的迁移,小青龙汤可能通过抑制TLR4和p-Akt的表达在哮喘气道重塑方面发挥一定的作用。     

姜黄素抑制结肠癌SW480细胞的增殖及其机制

目的:研究姜黄素对人结肠癌SW480细胞增殖以及survivin、caspase-3和p-Akt表达的影响。方法:用不同浓度姜黄素(5～40μmol/L)作用SW480细胞24、48和72 h,MTT法检测姜黄素对SW480细胞生长的抑制作用,Western blotting法检测SW480细胞中survivin、caspase-3和p-Akt蛋白的表达。结果:姜黄素以剂量(5～40μmol/L)和时间(24～72 h)依赖的方式抑制SW480细胞的增殖(P<0.01),40μmol/L姜黄素作用72 h对SW480细胞生长的抑制率可达(75.86±3.93)%。姜黄素抑制SW480细胞中p-Akt、survivin蛋白的表达、促进caspase-3蛋白的表达,均呈时间和剂量依赖性;40μmol/L姜黄素作用SW480细胞48 h后p-Akt和survivin蛋白表达显著减少[(0.204±0.025)vs(0.367±0.035),P<0.01;(0.208±0.014)vs(0.385±0.034),P<0.01],caspase-3蛋白表达显著增加[(0.371±0.028)vs(0.127±0.023),P<0.01]。结论:姜黄素抑制SW480细胞增殖,其机制可能与抑制Akt磷酸化、下调survivin和上调caspase-3蛋白的表达有关。     

CD160在慢性淋巴细胞白血病中的生物学特征研究

目的 探讨CD160在慢性淋巴细胞白血病中的表达及临床意义.方法 应用流式细胞仪检测22名健康对照者、91例CLL患者、76例B细胞非霍奇金淋巴瘤CD160的表达.同时检测91例CLL患者和22名对照者PTEN、p-Akt的表达.结果 22名健康对照1例（4.5％）CD160呈弱阳性.91例CLL患者89例（97.80％） CD160阳性,远高于对照组,差异有统计学意义（x2=89.38,P＜0.01）.CLL患者PTEN蛋白较对照组表达明显减低,p-Akt表达较对照组显著增高,且CLL患者CD160表达水平与p-Akt表达水平呈正相关,而与PTEN表达水平呈负相关.76例淋巴瘤患者5例（6.58％） CD160阳性,分别为4例HCL,1例SMZL.48例MCL患者CD160均阴性.CD160在CLL中的阳性率远高于淋巴瘤,差异有统计学意义（x2=140.06,P＜0.01）.CLL与MCL免疫表型差异主要是CD23与FMC7的阳性率不同,CD160在CLL中的阳性率远高于MCL,差异有统计学意义（x2=130.51,P＜0.01）.四格表诊断性试验分析显示,CD160在CLL中的敏感度为97.80％,特异性为90.79％,准确度为94.61％.CD160在CLL患者高表达与临床预后无关.结论 CD160在CLL细胞中普遍高表达,有助于CLL的诊断和鉴别诊断.     

组织芯片结合免疫组化检测人结肠癌组织中PTEN/p-Akt1的表达及其临床意义

目的 研究结肠癌、腺瘤和相应癌旁正常结肠黏膜组织中PTEN、p-Akt1的表达及其临床意义.方法 取85例结肠癌、18例结肠腺瘤和9例癌旁结肠黏膜组织分别制成72点和104点两块组织芯片,采用免疫组化法检测结肠组织芯片中PTEN、p-Akt1的表达.结果 PTEN、p-Akt1在结肠癌、结肠腺瘤和正常结肠黏膜组织中的表达差异无统计学意义(P>0.05),在不同分化程度的大肠癌组织中PTEN的表达差异有统计学意义(P<0.05).与其他临床病理资料无关;p-Akt1的表达与大肠癌组织学分化程度及淋巴结转移有关(分别P<0.001和<0.05),与其他临床病理资料无关.结论 PTEN、p-Akt1畸变可能未参与正常大肠细胞早期恶性转化的过程,但在结肠癌发生、发展中可能起一定作用.     

c-Met和 p-Akt与胃癌血管生成拟态的关系

目的：探讨p-Akt蛋白与HGF／c-Met通路是否参与胃癌VM的形成及其可能的分子机制。方法按照入组标准与排除标准,收集胃癌患者共74例,正常对照患者16例。应用免疫组织化学法,检测肿瘤组织及正常对照组中c-Met受体、p-Akt蛋白的表达情况,应用CD-34和PAS双染法测定VM。结果 c-Met受体与VM表达呈显著正相关（γ＝0．277,P＜0．05）,p-Akt蛋白和VM的表达呈显著正相关（γ＝0．409,P＜0．05）,表明c-Met受体、p-Akt蛋白与胃癌VM的生成存在显著相关性。结论胃癌组织中存在血管生成拟态。 c-Met受体、p-Akt蛋白在胃癌VM发生中可能起重要作用。     

乳腺癌组织中p-Tyr、p-Akt和p-p70S6K的表达及其临床意义

目的 探讨p-Tyr、P-Akt和p-p70S6K在乳腺癌中的表达以及与临床病理参数的关系.方法 用免疫组化SP法检测120例乳腺浸润性导管癌,20例癌旁正常组织,20例导管内癌组织中p-Tyr、P-Akt和p-p70S6K的表达.结果 p-Akt表达与p-Tyr和p-p70S6表达呈正相关(r=0.211,P=0.021;r=0.199,P=0.030),在癌旁正常组织中无表达,在癌组织中呈高表达.P-Tyr、P-Akt和p-p70S6K表达与术后无瘤生存有关,差异具有统计学意义(分别P=0.019,P=0.002和P=0.044),与绝经状态、年龄、肿瘤大小和TNM分期无关(P>0.05).p-Tyr、P-Akt表达与ER表达呈正相关(r=0.252,P=0.006;r=0.229,P=0.012),p-Akt和p-p70S6表达与淋巴结转移有关,差异有统计学意义(分别P=0.004,P=0.022),p-Akt表达还与组织学分级有关,差异有统计学意义(P=0.040),与PR表达呈负相关(r=-0.201,P=0.028).结论 p-Tyr、P-Akt和p-p70S6K在乳腺癌的发生发展中起重要作用.p-Akt可作为判断乳腺癌预后的参考指标.     

Regulation of cell death and survival pathways in experimental glaucoma

This study investigates cell death and survival pathways in experimental glaucoma using the translimbal photocoagulation laser model. Glaucoma was induced unilaterally in 79 Wistar rats and all eyes developed elevated intraocular pressure. The involvement of caspase-3, p-AKT and members of the MAP kinase pathway was evaluated by immunohistochemistry and Western blotting. We found that protein levels of caspase-3 were elevated from day 15 to day 30 (p<0.05). All investigated members of the MAP kinase pathway were significantly activated. P-SAPK/JNK activation began on day 2, reaching a 6-fold elevation by day 30 (p<0.05). The p-P38 level was elevated on days 2 and 8 (p<0.05), followed by a decrease to baseline on day 15. The level of p-ATF-2, the substrate of P38, was significantly elevated at all time points tested, up to day 30 (p<0.05). P-ERK was detected early (p<0.05) on day 1, returning to normal on day 15. The pro-survival protein p-Akt, a member of the PI3-kinase survival pathway, was also detected early on day 1 (p<0.05) returning to baseline on day 8 and remaining unchanged up to 64days. We conclude that retinal ganglion cell death in glaucoma involves activation, at different time points, of multiple pro-apoptotic pathways (the MAP kinase pathway and the caspase family) and pro-survival (PI-3 Kinase/ Akt and p-ERK).     

甲状腺癌中p-Akt、c-Myc、E-cad及VEGF的表达及其临床病理意义

目的 :检测磷酸化蛋白激酶B(p-Akt)、c-Myc、上皮性钙黏附蛋白(E-cadherin,E-cad)及血管内皮生长因子(vascular endothelial growth factor,VEGF)在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)、甲状腺乳头状增生(thyroid papillary hyperplasia,TPH)、结节性甲状腺肿(nodular goiter,NG)、滤泡性腺瘤(follicular adenoma,FA)及正常甲状腺(normal thyroid,NT)组织中的表达,探讨各指标之间及其与PTC临床病理参数间的关系。方法:采用组织芯片免疫组织化学SP法检测136例PTC、80例TPH、57例NG、42例FA及50例NT组织中p-Akt、c-Myc、E-cad及VEGF的蛋白表达。结果 :1p-Akt、c-Myc及VEGF蛋白在PTC中的表达率明显高于TPH、NG、FA和NT组(P均<0.01);E-cad蛋白在PTC中的表达率明显低于TPH、NG、FA和NT组(P<0.01)。2PTC组织中p-Akt、c-Myc及VEGF的表达两两之间呈正相关(P<0.05)。3p-Akt、c-Myc及VEGF阳性高表达与PTC多灶性、滤泡亚型、甲状腺外浸润、淋巴结是否转移及临床病理分期密切相关(P均<0.05)。4E-cad在PTC中阳性低表达与年龄≥40岁、腺体外侵犯、淋巴结转移及临床病理分期相关((P均<0.05)。结论:PTC组织中p-Akt、c-Myc及VEGF的高表达和E-cad低表达可能促进PTC的发生、发展和肿瘤淋巴结转移,这可能与调控肿瘤新生血管有关,同时检测这4项指标对PTC的临床诊断和恶性程度及预后评估有一定临床意义。