ERK信号转导通路与乳腺癌关系的研究进展

乳腺癌严重的危害着女性的生命健康,近年来其发病率呈逐年上升的趋势,在西方国家和我国发达大城市已位居女性恶性肿瘤发病率之首.正常乳腺细胞的增殖、分化、代谢、凋亡受到体内细胞信号转导通路的精细调节,细胞在致癌因素的刺激下逐渐癌变的过程中,可能涉及到调控细胞的信号转导通路异常改变.     

MAPK/ERK通路影响人胃癌细胞对奥沙利铂敏感性的机制研究

目的:探讨MAPK/ERK通路对人胃癌细胞奥沙利铂敏感性的影响,并进一步探讨其机制.方法:采用MTT法测定奥沙利铂对人胃癌BGC823和SGC7901细胞的增殖抑制影响;MAPK/ERK特异性抑制剂PD98059预处理上述两种细胞后,测定奥沙利铂的药物敏感性.Western blot方法检测胃癌细胞中p-ERK、ERK及谷胱甘肽-S-转移酶π(glutathione S-transferases π,GST-π)蛋白表达.应用SPSS 13.0软件包进行统计学分析.结果:1mg/L-100mg/L奥沙利铂分别作用BGC823和SGC7901细胞,48h的IC50分别为24.26mg/L和18.44 mg/L;20μmol/L的PD98059预处理BGC823和SGC7901细胞,再加入奥沙利铂继续培养48h,IC50分别降至12.42mg/L和7.97mg/L,表明对奥沙利铂的敏感性显著提高.进一步检测发现PD98059处理BGC823和SGC7901细胞24h后,p-ERK蛋白表达明显下调,GST-π蛋白亦显著下调.结论:PD98059通过抑制MAPK/ERK通路,下调GST-π蛋白表达,显著提高人胃癌细胞对奥沙利铂的药物敏感性.     

长链非编码RNA RAD51-AS1通过ERK信号通路对乳腺癌细胞迁移侵袭的影响

目的 探讨RAD51反义RNA 1(RAD51-AS1)通过抑制细胞外信号调节激酶(ERK)信号调控乳腺癌细胞增殖、迁移和侵袭等生物学行为的潜在机制。方法 采用实时荧光定量PCR(qPCR)检测55对乳腺癌组织和癌旁正常乳腺组织以及乳腺癌细胞(BT549、MCF7、SK-BR-3、BT474、MDA-MB-231)和正常乳腺上皮细胞(MCF-10A)中RAD51-AS1的表达情况。培养MDA-MB-231细胞并分为NC组(转染pcDNA3.1)、RAD51-AS1组(转染pcDNA3.1-RAD51-AS1以过表达RAD51-AS1)和RAD51-AS1+ERK激动剂组(同时转染pcDNA3.1-RAD51-AS1和ERK激动剂以过表达RAD51-AS1并激活ERK信号)。通过CCK-8法、划痕实验和Transwell实验分别评估细胞的增殖、迁移和侵袭能力。利用qPCR和Western blot检测细胞中ERK、p-ERK和基质金属蛋白酶9(MMP-9)的表达情况。结果 乳腺癌组织的RAD51-AS1表达量低于癌旁组织(0.419±0.039 vs. 1.000±0.063,P<...     

顺铂调控三阴性乳腺癌细胞的PD-L1表达及机制的研究

目的 探讨顺铂对三阴性乳腺癌(TNBC)细胞程序性死亡蛋白配体1(PD-L1)表达的调控作用及可能机制.方法 分别用浓度为2.5、5、10、20、50、100μmol/L的顺铂处理MDA-MB-231细胞,0.625、1.25、2.5、5、10、20μmol/L的顺铂处理SUM1315细胞,48 h后通过活细胞计数(C...     

JNK信号通路介导ghrelin对乳腺癌MDA-MB-231细胞耐药蛋白表达的影响

目的探讨ghrelin调控c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号转导通路对乳腺癌细胞P-糖蛋白表达的影响。方法培养人乳腺癌MDA-MB-231细胞,分别加入生长激素释放肽(ghrelin,100 nmol/L,干预72小时)、多柔比星(0.8 mg/L,干预72小时)、ghrelin联合多柔比星(ghrelin 100 nmol/L,多柔比星0.8 mg/L,干预72小时)。采用MTT方法检测细胞增殖,Western blot法检测细胞JNK、P-糖蛋白(P-glycoprotein,P-gp)的表达及p-JNK水平。结果 MTT检测显示,人乳腺癌MDA-MB-231细胞在ghrelin的干预下增殖(P<0.01),存活率在多柔比星的干预下降低(P<0.01),ghrelin联合多柔比星能够抑制多柔比星对MDA-MB-231细胞的杀伤作用(P<0.01)。ghrelin组JNK表达及p-JNK水平上升(均P<0.01),且P-gp蛋白表达上升(P<0.05);多柔比星组JNK表达及p-JNK水平下降(均P<0.01);ghrelin联合多柔比星较多柔比星组JNK表达及p-JNK水平增加,且P-gp表达明显上升(均P<0.05)。结论 ghrelin激活JNK信号通路干预乳腺癌细胞P-gp表达增加从而抑制多柔比星诱导乳腺癌细胞凋亡。     

食管鳞癌ECA109细胞中Survivin通过ERK信号通路调控c-myc基因表达的机制

目的：探讨在食管鳞癌ECA109细胞中Survivin对c-myc基因的调控作用。方法：将食管鳞癌ECA109细胞分为Survivin shRNA干扰组、阴性质粒对照组（Control shRNA）和空白细胞株（未加任何处理的食管癌ECA109细胞）,将2μg Survivin shRNA质粒、2μg Control shRNA质粒分别转染ECA109细胞,48 h后收集各组细胞,采用RT-PCR法检测各组Survivin、c-myc mRNA的表达,Western blot法检测Survivin、c-myc蛋白及p-ERK蛋白的表达;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号传导通路的特异性抑制剂分别阻断ECA109细胞中关键激酶的表达,RT-PCR法检测c-myc mRNA的表达。结果：与阴性质粒对照组和空白细胞组比较,Survivin shRNA组Survivin表达下调,c-myc mRNA及蛋白的表达降低,差异均具有统计学意义（P均 < 0.05）;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号通路抑制剂分别作用于食管癌ECA109细胞,PD98059（ERK信号通路阻断剂）组c-myc mRNA及蛋白表达降低（P < 0.05）;Survivin shRNA干扰沉默Survivin基因后ERK磷酸化蛋白表达降低（P < 0.05）。结论：Survivin对c-myc具有正向调控作用,可能通过ERK信号传导通路调控c-myc的表达。     

苦参碱和MAPK/ERK信号通路在抑制肺癌细胞诱导人脐静脉内皮细胞增殖和迁移中的作用

背景与目的 苦参碱是中国传统中药槐根的主要生物碱成分之一,具有抗炎、抑制肿瘤细胞增殖、诱导肿瘤细胞分化和凋亡等作用.丝裂原活化蛋白激酶/细胞外调节蛋白激酶(Motigen-activated proteinkinase/extraceUuar regulated protein kinase,MAPK/ERK)级联通路是血管内皮细胞信号转导的重要途径.本研究目的 是探讨苦参碱(Matrine,Mat)和MAPK/ERK信号转导通路在抑制肺腺癌A549细胞条件培养基(A549 human lung cancer cellsconditioned medium,A549CM)诱导的人脐静脉内皮细胞(human umbilical veins endothelial cens,HUVECs)增殖和迁移中的作用.方法 应用Mat与MAPK/ERK特异性抑制剂PD98059(PD)处理A549CM培养的HUVECs,采用细胞计数、划痕损伤实验和Western Blot技术,观察Mat、PD98059对A549CM诱导的人脐静脉内皮细胞增殖、迁移及磷酸化ERK(p-ERK)表达的影响.结果 干预后24h,与对照组相比,A549CM显著促进HUVECs增殖、迁移及P-ERK的表达.与A549CM组相比,Mat能明显抑制A549CM诱导的HUVECs增殖和迁移及p-ERK的表达;PD98059可以抑制HUVECs增殖及P-ETK表达,而对迁移没有作用.结论 Mat和PD98059均能有效抑制A549CM诱导的HUVECs增殖和P-ERK(表达,Mat还能抑制A549CM诱导的HUVECs迁移,而PD98059对A549CM诱导的HUVECs迁移没有影响,提示抑制MAPK/ERK(信号传导通路可能是苦参碱抗肺癌血管生成的部分机制.     

ERK/MAPK信号通路活性表达与鼻咽癌细胞增殖凋亡的关系

目的:探讨鼻咽癌细胞中ERK/MAPK信号传导通路异常激活与细胞增殖凋亡的关系.方法:SP法检测36例鼻咽癌、42例对照鼻咽黏膜上皮中p-ERK,Cyelin D1 和Ki-67蛋白的表达.用不同浓度阻滞刺PD98059处理CNE-2Z细胞,Hoechst33342/PI荧光双染法观察细胞凋亡,流式细胞仪检测凋亡,蛋白质印迹法检测Cyclin D1蛋白表达,免疫细胞化学法检测tyERK、Cyclin D1 和Ki-67蛋白的表达.结果:鼻咽癌组织中p-ERK、Cyclin D1和Ki-67的阳性表达率分别为69.44%(25/36)、80.56%(29/36)和80.56%(29/36),鼻咽黏膜上皮组织中则分别为23.80%(10/42)、19.05%(8/42)和9.52%(4/42),P<0.01.鼻咽癌组织中p-ERK-与Cyelin D1、Ki-67蛋白的表达呈正相关,r分别为0.604和0.668,P均<0.01.不同浓度PD98059作用于CNE-2Z细胞后,可观察到凋亡细胞,检测到凋亡峰,Cyclin D1蛋白表达的减少呈剂量依赖性,p-ERK、Cyclin D1和Ki-67蛋白的阳性表达细胞数减少.结论:鼻咽癌组织及CNE-2Z细胞中存在p-ERK蛋白的高表达和活化异常,p-ERK可能通过上调Cyclin D1和Ki-67的表达促进鼻咽癌细胞增殖并抑制凋亡.ERK信号通路有可能成为鼻咽癌治疗的新靶点.     

哈萨克族食管癌患者组织中细胞外信号调节激酶/丝裂原活化蛋白激酶信号通路的表达

目的 探讨细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)信号通路在哈萨克族食管鳞癌患者组织中的表达变化及意义.方法 采用Western blot技术检测在血清饥饿条件下、U0126梯度浓度处理下,食管癌细胞系EC9706中磷酸化ERK1(p-ERK1)和磷酸化ERK2(p-ERK2)的表达变化.采用实时荧光定量PCR法检测25例哈萨克族食管癌患者肿瘤组织和癌旁正常组织中总ERK1(t-ERK1)和总ERK2(t-ERK2)mRNA的表达变化.采用Western blot技术检测25例哈萨克族食管癌患者肿瘤组织、癌旁正常组织以及5例哈萨克族非食管癌者正常食管组织中t-ERK1、t-ERK2、p-ERK1和p-ERK2蛋白的表达变化.采用免疫组化染色法,在126例石蜡包埋标本(正常食管黏膜19例,食管原位癌55例,食管浸润癌52例)中验证p-ERK1和p-ERK2蛋白的表达变化.结果 在食管癌EC9706细胞中,ERK/MAPK信号通路呈高度活化状态.血清瞬时刺激10 min后,p-ERK1和p-ERK2的表达达到峰值.EC9706细胞在50 μmol/L的U0126处理下,p-ERK1和p-ERK2的表达几乎完全被抑制.t-ERK1 mRNA在25例哈萨克族食管癌患者肿瘤组织中的表达量为1.92±3.49,明显低于癌旁组织(3.67±7.47,P<0.05);t-ERK2 mRNA在食管癌组织和癌旁组织中的表达量差异无统计学意义(P>0.05).t-ERK1和t-ERK2蛋白在食管癌组织、相应癌旁组织和正常食管黏膜组织中的表达差异无统计学意义(P>0.05);但p-ERK1和p-ERK2蛋白在食管癌组织中的表达量(分别为0.87±0.14和0.79±0.10)均明显低于癌旁组织(分别为1.10±0.13和1.32±0.12,P<0.05)和正常食管黏膜组织(分别为1.50±0.22和1.64±0.18,P<0.05).免疫组化染色验证的结果 显示,p-ERK1和p-ERK2蛋白在浸润性食管癌组织中的阳性表达率均为7.7%(4/52),在癌旁正常食管黏膜组织中的阳性表达率均为31.6%(6/19),在食管原位癌组织中的阳性表达率均为85.5%(47/55),不同组织间的表达差异有统计学意义(P<0.05).结论 在食管癌细胞中,ERK/MAPK信号通路呈活化状态.ERK/MAPK信号通路活化水平的改变可能参与了哈萨克族食管癌患者肿瘤的早期发生.

Abstract：

Objective To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients. Methods The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas. Results ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 μmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92±3.49 in the ESCC tissues and 3.67±7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P<0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P>0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87±0.14 and 0.79±0.10 in the ESCC tissues, and 1.10±0.13 and 1.32±0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P<0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P>0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P<0.05). Conclusions ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.     

磷脂酶A2和ERK2与乳腺癌发生发展的关系

目的磷脂酶A2（CPLA2）作为活化的细胞外信号调节激酶（P—ERK2）的底物,与乳腺癌发生发展具有密切关系。本研究旨在阐明CPLA2、ERK2在乳腺癌发生发展中的作用及其相互之间存在的关系。方法应用免疫组织化学二步法,分别检测48例乳腺癌、21例乳腺纤维腺瘤和21例乳腺腺病组织中CPLA2和ERK2的表达水平。结果乳腺癌组织中CPLA2和ERK2的表达明显高于乳腺纤维腺瘤和乳腺腺病组织,且乳腺癌组织中CPLA2和ERK2的表达与TNM分期呈正相关,CPLA2与乳腺癌组织肿瘤直径、术后生存时间、淋巴结转移个数均成正相关,且CPLA2和ERK2之间呈显著正相关关系。结论ERK2可以通过促进CPLA2的表达促进乳腺癌的发生发展。     

Overexpression of Snail accelerates adriamycin induction of multidrug resistance in breast cancer cells

In addition to several molecular and morphologic changes, epithelial-mesenchymal transition (EMT) cells also show variation in sensitivity to chemotherapeutics agents. The aim of this study was to investigate whether overexpression of Snail in MCF-7 cells is associated with facilitated acquisition of P-gp mediated multidrug resistance (MDR). The results demonstrated that over-expression of Snail indeed resulted in slight enhancement of adriamycin-induced MDR in MCF-7/Snail cells without detectable increase of P-gp. However, in the longer term, MCF-7 cells overexpressing Snail were prone to be resistant to adriamycin, in this case with increased expression of P-gp. These results provide evidence that a strategy involving Snail inhibition may be a useful and promising therapeutic aspect in modulating MDR.     

Effects of inhibiting JAK on invasion and metastasis of the human breast cancer cells through ERK signaling transduction pathway

Objective: To explore the effects of Janus activated kinase (JAK) inhibitor AG490 on the phosphorylation of extracellular signal regulated protein kinase (ERK) in human breast cancer cells MDA-MB-231 and the roles of JAK in the invasion and metastasis of the human breast cancer cells through ERK signaling transduction pathways.Methods: MDA-MB-231 cells were treated with 20 (mol/L, 40 (mol/L, 80 (mol/L Janus kinase inhibitor AG490 for 24, 48 and 72 h. Proliferation and adhesion of MDA-MB-231 cells to matrigel were measured with MTT assay. When treated with 40 (mol/L AG490 for 24 h, the expressions of P-ERK and MMP-9 of cells were detected by Western-blot and invasion and metastasis of MDA-MB-231 cells were evaluated with transwell chamber.Results: After being treated with 20 (mol/L, 40 (mol/L, 80 (mol/L AG490 for 24, 48 and 72 h, the proliferation of MDA-MB-231 cells was inhibited in a dose-and time-dependent manner. MDA-MB-231 cells treated with 40 (mol/L AG490 for 30, 60, 90 and 120 min resulted in the increasing adhesion of cells to Matrigel in a time-dependent manner. However, capacity of adhesion in the group treated with AG490 was significantly decreased in comparison with the control group (P<0.01). The expression level of P-ERK and MMP-9 were decreased when treated with AG490. After treatment with 40 (mol/L AG490, in invasion assay, the number of cells in AG490 treated group to migrate to filter coated with Matrigel was reduced compared with control group (P<0.05). Meanwhile, in migration assay, the number of cells in AG490 treated group to migrate to filter was also decreased compared with control group (P<0.05).Conclusion: Our study indicates that JAK kinase could affect the activity of ERK signal transduction pathway through the phosphorylation of ERK. The inhibitory effects of JAK kinase on MMP-9 expression and invasion of breast cancer cells were associated with the down-regulation of the ERK signaling pathway.     

Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway

Multidrug resistance (MDR) is one of the major obstacles to the efficiency of cancer chemotherapy, which often results from the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). In the present study, we determined the effect of dasatinib which was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph⁺) acute lymphoblastic leukemia (ALL) treatment on P-gp-mediated MDR. Our results showed that dasatinib significantly increased the sensitivity of P-gp-overexpressing MCF-7/Adr cells to doxorubicin in MTT assays; thus lead to an enhanced cytotoxicity of doxorubicin in MCF-7/Adr cells. Additionally, dasatinib increased the intracellular accumulation, inhibited the efflux of doxorubicin in MCF-7/Adr cells, and significantly enhanced doxorubicin-induced apoptosis in MCF-7/Adr cells. Further studies showed that dasatinib altered the expression levels of mRNA, protein levels of P-gp, and the phosphorylation of signal–regulated kinase (ERK) both in time-dependent (before 24 h) and dose-dependent manners at concentrations that produced MDR reversals. In conclusion, dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression, which may be partly attributed to the inhibition of ERK pathway. Dasatinib may play an important role in circumventing MDR when combined with other conventional antineoplastic drugs.     

ERK信号通路参与RANKL诱导的乳腺癌细胞迁移

目的：探讨细胞外调节蛋白激酶（ERK）信号通路在RANKL诱导的乳腺癌细胞迁移中的作用.方法：流式细胞术检测MDA-MB-231细胞表面RANK蛋白的表达;western-blot检测RANKL刺激后磷酸化ERK（P-ERK）及ERK的表达;Transwell法测定RANKL刺激后细胞迁移能力的改变.结果：MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强.RANKL刺激后MDA-MB-231细胞P-ERK表达升高,PD98059抑制RANKL诱导的细胞迁移.结论：ERK信号通路参与RANKL诱导的乳腺癌细胞迁移.     

RNAi‐mediated downregulation of uPAR synergizes with targeting of HER2 through the ERK pathway in breast cancer cells

Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB‐2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2‐amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.     

Therapeutic potential of ERK5 targeting in triple negative breast cancer

Triple negative breast cancers (TNBCs) account for 15% of all breast cancers, and represent one of the most aggressive forms of the disease, exhibiting short relapse-free survival. In contrast to other breast cancer subtypes, the absence of knowledge about the etiopathogenic alterations that cause TNBCs force the use of chemotherapeutics to treat these tumors. Because of this, efforts have been devoted with the aim of incorporating novel therapies into the clinical setting. Kinases play important roles in the pathophysiology of several tumors, including TNBC. Since expression of the MAP kinase ERK5 has been linked to patient outcome in breast cancer, we analyzed the potential value of its targeting in TNBC. ERK5 was frequently overexpressed and active in samples from patients with TNBC, as well as in explants from mice carrying genetically-defined TNBC tumors. Moreover, expression of ERK5 was linked to a worse prognosis in TNBC patients. Knockdown experiments demonstrated that ERK5 supported proliferation of TNBC cells. Pharmacological inhibition of ERK5 with TG02, a clinical stage inhibitor which targets ERK5 and other kinases, inhibited cell proliferation by blocking passage of cells through G₁ and G₂, and also triggered apoptosis in certain TNBC cell lines. TG02 had significant antitumor activity in a TNBC xenograft model in vivo, and also augmented the activity of chemotherapeutic agents commonly used to treat TNBC. Together, these data indicate that ERK5 targeting may represent a valid strategy against TNBC, and support the development of trials aimed at evaluating the clinical effectiveness of drugs that block this kinase.     

PD98059抑制MAPK/ERK信号通路对胃癌细胞生物学功能的影响

目的：研究MAPK/ERK信号通路中关键信号分子MEK和ERK在胃癌SGC-7901细胞中的表达及PD98059抑制MAPK/ERK通路对胃癌细胞生物学功能的影响。方法：体外培养胃癌细胞株SGC-7901,不同浓度（0、25、50、100、200、300和400 mmol/L）PD98059处理24 h后CCK-8法检测细胞增殖率变化;再用0、25、50和100 μmol/L PD98059处理24 h后采用实时荧光定量PCR（qPCR）检测MEK和ERK mRNA的表达量;Western blot检测MEK和ERK蛋白的表达;流式细胞术检测细胞周期和凋亡变化。同时设正常胃黏膜上皮GES-1细胞为对照。结果：与正常胃黏膜上皮GES-1细胞相比,胃癌SGC-7901细胞中MEK和ERK mRNA的表达升高,差异具有统计学意义（P < 0.05）;p-MEK、p-ERK蛋白的表达亦显著升高,差异具有统计学意义（P < 0.05）。0~200 μmol/L PD98059处理SGC-7901细胞后,细胞增殖率随着抑制剂浓度的升高而降低（P < 0.05）。当PD98059浓度处于200~400 μmol/L时抑制作用逐渐趋于平稳。0~100 μmol/L PD98059作用后MEK、ERK mRNA的表达量低于对照组（P < 0.05）,随着PD98059浓度升高,ERK mRNA表达量逐渐降低（P < 0.05）。Western blot检测结果显示50和100 μmol/L PD98059作用后p-MEK1/2、p-ERK1/2蛋白表达降低（P < 0.05）。且抑制剂PD98059使胃癌SGC-7901细胞发生G0/G1期阻滞,可诱导细胞凋亡。结论：MAPK/ERK信号通路在胃癌细胞中激活,PD98059通过抑制MAPK/ERK信号通路的活性可影响胃癌细胞的生物学功能。     

赖氨匹林抑制乳腺癌细胞增殖的机制初探

背景与目的:有研究证实非类固醇类抗炎药(nonsteroidal anti-inflammatory drugs,NSAIDs)通过抑制环氧合酶(cyclooxygenase,COX)-2干扰致癌作用,但并不是唯一机制。本实验探讨非选择性环氧合酶-2抑制剂赖氨匹林(aspisol)对体外培养的雌激素受体(ER)阴性(MDA-MB-231)和阳性(MCF-7)人乳腺癌细胞增殖的影响及其ERK(extracellular sigal-regulated kinase,细胞外信号调节蛋白激酶)信号通路的影响。方法:应用MTT比色法分析细胞生长抑制作用,流式细胞技术测定凋亡率,Western blot方法检测赖氨匹林对两种乳腺癌细胞外信号调节蛋白激酶(ERK)/磷酸化(P-ERK)的表达的影响。结果:1~10 mmol/L赖氨匹林可抑制MDA-MB-231、MCF-7细胞的生长,且其作用具有剂量和时间依赖性,药物浓度越大,时间越长,抑制作用越明显,差异有显著性(P<0.01)。赖氨匹林(1,5,10 mmol/L)作用24 h后诱导MDA-MB-231细胞的早期凋亡率分别为(5.76±1.02)%、(10.28±1.95)%、(15.41±2.14)%,与对照组(0.27±0.17)%比较,差异有显著性(P<0.01),并呈浓度依赖性。赖氨匹林(1,5,10 mmol/L)作用24 h后诱导MCF-7细胞的早期凋亡率分别为(4.62±1.05)%、(6.86±2.51)%、(11.40±3.56)%,与对照组(0.21±0.11)%相比,差异有显著性(P<0.01),并呈浓度依赖性。赖氨匹林可不同程度下调两种乳腺癌细胞P-ERK的表达水平(P<0.01),但不影响总ERK表达(P>0.05)。结论:赖氨匹林对ER阳性和ER阴性乳腺癌细胞均有抑制增生,诱导凋亡的作用,其作用机制与抑制乳腺癌ERK信号通路有关。