摘要
胰腺癌是一种消化系统恶性肿瘤,据2002年全球癌症统计,胰腺癌的发病率占第11位,而死亡率却已经上升至第8位。死亡率高的主要原因与胰腺癌恶性度高,诊断时大多数患者已经到了晚期,肿瘤过大(直径大于4 cm)、浸润到周围脏器或者已经发生了淋巴转移。胰腺癌复发率高,即使进行了手术也预后极差。因此,寻找与胰腺癌发生发展密切相关的特异基因或蛋白具有重要意义。
目前研究表明,胰腺癌的组织和细胞已有许多基因发生了突变,比如VEGF、EGF、Notch、IGF、MUC、K-Ras、P13K-AKT和sonic hedgehog等。这些突变因子主要归属于3条重要信号转导通路:Ras-MAPK信号转导通路、PI3K-AKT-mTOR信号转导通路和Hedgehog信号转导通路。K-ras基因在胰腺癌患者中的突变率高达90%以上,突变的k-ras基因导致Ras蛋白的持续激活,激活的Ras蛋白自身磷酸化并激活其下游蛋白,包括Ras-MAPK信号转导通路中的一个重要结点——MEKK1也被激活。
MEKK1是MAP/ERK KK1激酶的缩写,属于MAPKKK家族的成员,在MAPK信号通路中处于结点位置,也是MAPK通路与其他信号通路的结点,包括IKK-NFκB通路、JAK-STAT通路和GSK3β通路。MEKK1具有重要的生物学功能,如影响细胞的存活和凋亡,参与肿瘤细胞的运动和迁移,据文献报道与胃癌、乳腺癌和卵巢癌的侵袭密切相关。全长的MEKK1是196kD的蛋白,可与许多蛋白都发生相互作用,如骨架蛋白、丝氨酸/苏氨酸激酶和粘着斑相关的酪氨酸激酶(FAK)等。活化的MEKK1又可激活其下游的JNK通路、P38通路和ERK信号转导通路。
为探索MEKK1在胰腺癌发生发展中的作用,本研究首先采用RT-PCR方法对不同组织来源的肿瘤细胞株及正常细胞株中MEKK1的mRNA表达水平进行了检测,结果2株胰腺癌细胞BxPC3和Capan2均呈高表达。随后,将胰腺癌细胞系增加到4株(BxPC3、Capan2、SW1990和PANC1),并运用多种方法对MEKK1的表达水平进行了检测。如运用RT-PCR检测了mRNA水平的表达差异,采用WesternBlot和免疫荧光方法检测了蛋白水平的表达差异。结果MEKK1在BxPC3细胞株中表达水平最高,Capan2的表达水平其次,SW1990和PANC1表达水平均较低。同时也采用RT-PCR方法检测了4株胰腺癌MEKK2、MEKK3和MEKK4的mRNA表达水平,结果MEKK4在4株胰腺癌细胞系的表达模式与MEKK1相似。
为探讨MEKK1与胰腺癌细胞生物行为之间的关系,对4株胰腺癌细胞株的生物学行为进行了检测。采用软琼脂集落方法检测其恶性度,划痕愈合实验检测其运动能力,粘附实验检测其异质粘附特性,Transwell方法检测其侵袭能力。结果表明,4株胰腺癌细胞中BxPC3细胞的粘附、侵袭、转移能力和恶性度都为最高,粘附、侵袭和转移能力的变化趋势与MEKK1的表达水平相一致。因此推测胰腺癌细胞的生物学特性可能与MEKK1和/或MEKK4的高表达有关。
为进一步探讨MEKK1是否与胰腺癌的侵袭转移能力呈正相关,我们以MEKK1表达水平最高和侵袭转移能力最强的BxPC3细胞作为研究对象,采用RNA干扰技术对MEKK1基因进行沉默,检测沉默后该细胞的运动、粘附、侵袭、转移以及分泌MMP的能力。结果表明,MEKK1基因沉默后,BxPC3细胞的运动、粘附、侵袭、转移以及分泌MMP_2的能力均低于未转染和转染阴性对照siRNA的BxPC3细胞(P<0.05)。
MEKK1的下游信号转导通路主要有ERK通路、JNK通路和P38通路。MEKK1的哪条下游通路在胰腺癌的侵袭转移中起主要作用尚不清楚,为此我们检测了MEKK1沉默后其下游蛋白ERK、JNK和P38磷酸化与非磷酸化水平。结果只有磷酸化ERK的表达水平明显下降,而磷酸化的JNK和p38只为轻度抑制。提示MEKK1参与胰腺癌的侵袭转移主要通过ERK信号转导通路实现。
为进一步探讨MEKK1在人胰腺癌发生发展中的作用,与哈尔滨医科大学附属医院合作,对2001年到2006年的41例胰腺癌患者的组织标本进行了回顾性分析。采用组织化学方法检测了组织标本中的MEKK1蛋白表达,结合患者病理特征进行了分析。结果表明,MEKK1阳性例数有32例(占78.1%),MEKK1表达与淋巴转移、TNM分级和肿瘤分化程度高度相关(P<0.01),而与患者的年龄、性别、肿瘤大小和肿瘤发生部位等不相关(P>0.05)。MEKK1的蛋白表达主要位于胞浆,但随着分化程度的不同,其细胞定位有所改变,高分化癌的细胞核中几乎没有表达,中分化癌细胞核中表达增加,而低分化癌细胞核中全部都有表达。ERK的蛋白表达也与MEKK1呈正相关。提示MEKK1蛋白在细胞核中的表达与分化程度密切相关,且在胰腺癌的侵袭转移过程中起一定的作用,并且与ERK高度相关。
综上所述,MEKK1的蛋白高表达与胰腺癌的侵袭转移有关,并且与其下游基因ERK高度相关,MEKK1很可能成为治疗胰腺癌的潜在分子靶点。
Icogenin是由中国医学科学院药物研究所化学合成室雷平生研究员课题组从龙血树(Dracaena draco)中提取的一种甾体皂苷,具有一定的细胞毒作用。前期MTT筛选实验研究发现,Icogenin对胰腺癌PANC1细胞和人鳞状上皮癌A431细胞的生长抑制作用最强。目前临床上治疗胰腺癌最理想的办法仍是外科根治手术加药物化疗,对于不能手术或进行姑息性手术的患者,化疗则更为重要,而细胞毒性药物在目前临床实施的化疗方案中仍占主导地位,因此研究Icogenin的抗胰腺癌作用及其作用机制则具有重要意义。
本研究采用人胰腺癌细胞株BxPC3作为体外细胞研究对象,MTT方法测得Icogenin的IC_(50)为0.84±0.10μmol/L,但斜率较陡。5μmol/L Icogenin可明显地抑制BxPC3细胞生长和集落形成,使BxPC3细胞阻滞在G2/M期,Icogenin作用6h即可诱导BxPC3细胞出现凋亡,细胞内核小体DNA断裂出现典型的梯形条带。同时流式细胞术、AO/EB染色等多种方法均证实了细胞凋亡的存在。Western blot结果表明,Icogenin诱导BxPC3细胞凋亡主要与抑制线粒体通路的bcl-2家族有关(Bax、bcl-xl、Bak、bcl-2等均有变化),而与P53无关。诱导凋亡剂量对于MAPK信号通路的影响则是磷酸化的ERK和P38蛋白表达水平降低,而磷酸化的JNK蛋白表达水平增加。
体外实验还发现在低于凋亡剂量(1μmol/L小于48h的IC_(10))的Icogenin还具有抗胰腺癌侵袭转移作用。研究中分别采用穿膜方法、粘附实验和单层细胞划痕实验探讨Icogenin对胰腺癌BxPC3细胞侵袭、粘附和运动能力的影响。Western blot方法检测Icogenin对MAPK信号传导通路蛋白表达水平的影响,明胶酶谱法分析Icogenin对MMP2分泌的影响。结果表明,Icogenin在体外可显著抑制人胰腺癌BxPC3细胞的侵袭、运动以及粘附能力(P<0.05),并呈较好的剂量依赖关系,ERK抑制剂PD98059和U0126也可抑制BxPC3细胞的侵袭和转移能力。Icogenin可抑制MMP2的分泌,其作用机制与抑制MAPK信号传导通路中的磷酸化ERK有关。
说明Icogenin对胰腺癌细胞具有一定的抗侵袭转移作用,大剂量可通过线粒体通路诱导细胞出现凋亡而起到抗胰腺癌作用。
Pancreatic cancer is one of the gastrointestinal malignancies with extremely poor prognosis.Although the incidence rate remains relatively low,pancreatic cancer contributes the eighth leading cause of cancer death.To date,few therapeutic options are available for patients with this metastatic disease.The main problem of this cancer is the late diagnosis as its cancer-specific symptoms occur only at an advanced stage when the tumor size is too large(above 4 cm in diameter) with local spread or lymph node metastasis.
Many factors have been identified to be closely related to the carcinogenesis and progression of pancreatic cancer.The factors include those involved in VEGF,EGF, Notch,IGF,MUC,K-Ras,PI3K-AKT,and sonic hedgehog signaling pathways. Importantly,the factors in the Ras-MAPK,PI3K-AKT-mTOR and Hedgehog pathways were reported to be the most important ones that contribute to invasion and metastasis of pancreatic cancer.
Ras/Raf/MAPK pathway plays an important role in most of tumor invasion and metastasis.In pancreatic cancer,activating mutation of K-Ras is frequently found.Ras activation leads to activation of downstream signaling proteins including MEKK1 and MEK.MEKK1 is a Ser/Thr protein kinase,with 196 KD in mass,belonging to the MEKK/STE11 subgroup of the MAPKKK family,and is a hub of the MAPK signal pathway.MEKK1 functions as to interact with many proteins such as scaffold proteins, serine-threonine kinase,and a focal adhesion-associated kinase(FAK).MEKK1 is able to activate its cascade proteins including ERK1/2,JNK and P38 pathways,and it has also been shown to regulate both IKK-NFκB and JAK-STAT pathways.MEKK1 plays a critical role in cell growth,cell differentiation as well as invasion and metastasis.To date, MEKK1 has been reported to mediate tumor metastasis of gastric carcinomas,mammary and ovarian cancers.However,its role in pancreatic cancer has not been well documented.
To observe whether the MEKK1 expression is related to pancreatic cancer in vitro,we examined the MEKK1 mRNA and protein levels in different pancreatic cancer cell lines (BxPC3,Capan2,SW1990 and PANC1).The data showed that MEKK1 mRNA level remained highly abundant in BxPC3 and Capan2 cell lines while it had weak expression in the SW1990 and PANC1 cell lines.A Western blot analysis and immunofluence assay further confirmed the MEKK1 protein expression in a similar pattern in the four observed pancreatic cell lines.
To clarify whether the MEKK1 expression level is related to the malignancy of pancreatic cancer cells,softagar assay,woudhealing assay,adhesion assay and a tumor invasion assay were performed by use of the four human pancreatic cancer cell lines.The data revealed that BxPC3 cells had stronger abilities of adhesion,molity and invasion. These results suggest that MEKK1 expression is positively associated with metastatic potential of the human pancreatic cancer cells.
To examine whether the highly expressed MEKK1 is the cause for the strong invasive ability,we depleted MEKK1 by using the RNA interference method in BxPC3 cells.We selected and synthesized three siRNAs against MEKK1.The efficiency of the selected siRNAs was examined by transfection in the BxPC3 cells.RT-PCR analysis revealed that siRNAl and siRNA3 have a significant inhibitory effect on the endogenous MEKK1 expression while the siRNA2 showed no effect.The effects of the selected siRNAs were further confirmed by a Western blot analysis and the results consistently showed that siRNA3 was the strongest one to inhibit MEKK1 expression.Based on these results we determined to use siRNA3 for the following experiments.
To study the role of MEKK1 on the cell invasion,motility and adhesion,which are the major characteristics of the metastasis,we used siRNA3,the most powerful one we selected,to deplete the endogenous expression of MEKK1 in the BxPC3 cells.In an invasion assay,we calculated the number of cells that migrated to the bottom side of the membrane on a chamber where the cells were seeded.The data showed that wild type BxPC3 cells had more numbers of the migrated cells with or without transfection of a control siRNA.However,when siRNA3 targeting MEKK1 was transfected into the cells, the number of the migrated cells decreased dramatically.These results suggested that depletion of MEKK1 significantly suppressed the migration ability of BxPC3 cells.
To examine whether depletion of MEKK1 has any effect on the motive ability of the cells, we performed a wound healing experiment using BxPC3 cells transfected with or without control-siRNA and siRNA3.The data showed that the wild type BxPC3 cells had no difference in the relative wound closure with or without transfection of the control siRNA,but a significant difference was observed for the cells transfected with or without siRNA3 targeting MEKK1 both for 15 and 24 hours.
The highly expressed MEKK1 correlated to the pancreatic tumor metastasis in patients with the tumor reminded us of examining whether MEKK1 has an effect on cell adhesion to ECM(extracellular matrix).For this purpose,we performed a cell adhesive assay by using siRNA3 targeting MEKK1 in the BxPC3 cells.The results showed that the cells, when transfected with siRNA3,obtained low adhesion ability,compared with the wild type cells.The results were in consistence during different times as we observed in 30,60 and 120 min,respectively.These results indicated that MEKK1 might help the tumor cells adhere to the ECM.Taking together,our data suggest that MEKK1 exerts its effects on metastasis by promoting invasion,migration and adhesion in BxPC3 cells.
Matrix-metalloproteinases(MMPs) have been reported to play pivotal roles in tumor invasion through degradation of basement membrane and ECM.To reveal the mechanisms of the inhibition role of siRNA3 on pancreatic cell metastasis,the activity of MMP2 protein was investigated by a Gelatin zymography assay.The data showed that the activity of MMP2 can be observed in the gelatin zymography assay in HT1080 cells, a canonical cell line for the MMP2 activity measurement.When wild type BxPC3 cells were used,we observed high activities of MMP2 in the wild type and mock cells but a significant low activity of MMP2 in the cells transfected with siRNA3 targeting MEKK1. We did not observed the activity of MMP9 in BxPC3 cells although we examined that in HT1080 cells.This result indicated that MEEK1 contributed to the activation of MMP2 in the BxPC3 cells.
MEKK1 functions as a hub in three signal pathways including ERK1/2,P38 and JNK.To identify which downstream pathways are critical in the metastasis of pancreatic tumor cells,we investigated the phosphorylation of ERK1/2,P38 and JNK in the MEKK-depleted BxPC3 cells.The results showed that the phosphorylation levels of ERK1/2 were significantly inhibited,while the levels of phosphorylated p38 and JNK were weakly affected by siRNA3 targeting MEKK1.These data suggest that ERK1/2 play a major role in the activation of the cascade of MEKK1 in the pancreatic tumor cells.
To identify the detailed roles of MEKK1 in the regulation of malignant characteristics of human pancreatic adenocarcinoma in vivo.In this study,we analyzed MEKK1 expression in patient's samples by immunohistochemistry assay.The results demonstrated that MEKK1 positive staining(Figure 24) was noted in 32 samples among the 41 pancreatic adenocarcinomas(78.1%).All these results suggested that the MEKK1 expression was significantly associated with differentiation,TNM staning and lymph node metastasis in the observed pancreatic cancer patients(Table 8;P<0.05).The MEKK1 staining did not show significantly different among the patients of different gender,age,tumor location and tumor size(table 8;P>0.05).In addition,there is a mainly founding that MEKK1 cell location verified with cancer differentiation grade.The amounts of nuclear staining is 100%in poorly differentiated cancers,while the amount of nuclear staining in moderate and well differentiated cancers were 72.2%and 22.2%,respectively.
In conclusion,this study demonstrates that MEKK1 plays a major role in the metastasis of pancreatic cancer,possibly through activating specifically ERK to regulate expression of MMP2.Our findings suggest that MEKK1 may be a potential molecule target for pancreatic cancer therapy.
Icogenin is one of steroidal saponins extracted from plant named Dracaena Draco. Icogenin has been reported to have certain cytotoxicity in HL60 cells.It has been found that Icogenin prefers inhibiting the cancer cell growth of PANC1 and A431 among eight cancer cell lines by MTT assay.At present,it is still the best way to treat pancreatic cancer combining radical surgical resection with chemotherapy drugs.Chemotherapy is even more important for patients with unresected and advanced pancreatic cancer. Cytotoxic chemotherapy drugs lie in the dominated position.Therefore,it is of great significance to explore the activity of anti-pancreatic cancer and its mechanisms by Icogenin.
In this study,we choose a pancreatic cancer cell line BxPC3 as cell model in vitro. Icogenin inhibited the abilities of cell proliferation and colony formation of BxPC3 cells, the IC_(50) is 0.84±0.02μmol/L.Cell apoptosis was found at 6 hour by 5μmol/L Icogenin. At the same time,the results by flow cytometry,AO/EB staining and other methods have confirmed the existence of apoptosis.The DNA ladder was also found.BxPC3 cell cycle was arrested at G2/M phase.Western blot analysis results showed that BxPC3 cell apoptosis mainly related to inhibite the mitochondrial pathway bc1-2 family(Bax,Bak, bc1-2 and bcl-x1 were changed).The P53 level was not varied.The phosphorylation ERK and P38 were decreased and the phosphorylated JNK was increased in MAPK signal pathway by apoptosic dose Icogenin.
We also found that Icogenin has the role against invasion and metastasis in pancreatic cancer at lower doses(1μmol/L is less than IC_(10) at 48h point).To investigate the activities of Icogenin on anti-cancer and anti-metastasis and their mechanisms in pancreatic cancer cell line BxPC3 in vitro,using transwell assay,the effects of Icogenin on the invasion of BxPC3 cells were measured.The abilities of cell motility and adhesion in BxPC3 cells were detected by adhesion assay and wound healing assay respectively. The MAPK signal pathway proteins were analysed by Western blotting.At the same time, the activity of MMP2 was observed by zymography assay.Icogenin inhibited the abilities of motility,adhesion and invasion of pancreatic cancer BxPC3 cells in vitro,in dose-depended manner,and inhibited the secretion of MMP2 and phosphorylation of ERK.PD98059 and U0126 which were ERK inhibitors could suppress the abilities of invasion and metastasis of pancreatic cancer cells BxPC3(P<0.05).
In summary,mechanism of Icogenin against pancreatic cancer may relate to inhibite the phosphoration of ERK and P38.On the other side,Icogenin induces pancreatic cancer cell apoptosis by activing the JNK proteins which induced Bcl-2 dependent apoptosis in mitochondria.
引文
[1].Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55:74-108.
[2].Radinsky R Growth factors and their receptors in metastasis [J].Semin Cancer Biol,1991,2:169-177.
[3].Seo Y,Baba H,Fukuda T,et al.High expression of vascular endothelial growth factor is associated with liver metastasis and a poor prognosis for patients with ductal pancreatic adenocarcinoma[J].Cancer,2000,88:2239-2245.
[4].Bloomston M,Bhardwaj A,Ellison EC,et al.Epidermal growth factor receptor expression in pancreatic carcinoma using tissue microarray technique[J].Dig Surg,2006,23:74-79.
[5].Doucas H,Mann CD,Sutton CD,et al.Expression of nuclear Notch3 in pancreatic adenocarcinomas is associated with adverse clinical features,and correlates with the expression of STAT3 and phosphorylated Akt[J].J Surg Oncol,2008,97:63-68.
[6].Hakam A,Fang Q,Karl R,et al.Coexpression of IGF-1R and c-Src proteins in human pancreatic ductal adenocarcinoma[J].Dig Dis Sci,2003,48:1972-1978.
[7].Yamasaki H,Ikeda S,Okajima M,et al.Expression and localization of MUC1,MUC2,MUC5AC and small intestinal mucin antigen in pancreatic tumors[J].Int J Oncol,2004,24:107-113.
[8].Almoguera C,Shibata D,Forrester K,et al.Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes[J].Cell,1988,53:549-554.
[9].Asano T,Yao Y,Zhu J,et al.The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells[J].Oncogene,2004,23:8571-8580.
[10].Thayer SP,di Magliano MP,Heiser PW,et al.Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis[J].Nature,2003,425:851-856.
[11].Furukawa T Molecular targeting therapy for pancreatic cancer:current knowledge and perspectives from bench to bedside[J].J Gastroenterol,2008,43:905-911.
[12].Campbell PM,Groehler AL,Lee KM,et al.K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling[J].Cancer Res,2007,67:2098-2106.
[13].Motoshima K,Kohara N,Shiogama T,et al.[Immunohistological study on ras and myc oncogene products in pancreatic cancer][J].Nippon Geka Gakkai Zasshi,1990,91:123-129.
[14].Ding Y,Huang D & Chen XG Development of an enzyme-linked immunosorbent assay (ELISA)for the specific detection of MEKK1 expression in cells[J].J Pharmacol Toxicol Methods,2005,51:159-167.
[15].Lange-Carter CA,Pleiman CM,Gardner AM,et al.A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf[J].Science,1993,260:315-319.
[16].Cuevas BD,Abell AN & Johnson GL Role of mitogen-activated protein kinase kinase kinases in signal integration[J].Oncogene,2007,26:3159-3171.
[17].Uhlik MT,Abell AN,Cuevas BD,et al.Wiring diagrams of MAPK regulation by MEKK1,2,and 3[J].Biochem Cell Biol,2004,82:658-663.
[18].Xia Y & Kao WW The signaling pathways in tissue morphogenesis:a lesson from mice with eye-open at birth phenotype[J].Biochem Pharmacol,2004,68:997-1001.
[19].Zhang L,Wang W,Hayashi Y,et al.A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure[J].Embo J,2003,22:4443-4454.
[20].Xia Y,Makris C,Su B,et al.MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration[J].Proc Natl Acad Sci USA,2000,97:5243-5248.
[21].Chen Z & Cobb MH Activation of MEKK1 by Rho GTPases[J].Methods Enzymol,2006,406:468-478.
[22].Yujiri T,Nawata R,Takahashi T,et al.MEK kinase 1 interacts with focal adhesion kinase and regulates insulin receptor substrate-1 expression[J].J Biol Chem,2003,278:3846-3851.
[23].Cuevas BD,Abell AN,Witowsky JA,et al.MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts [J].Embo J,2003,22:3346-3355.
[24].Ricono JM,Huang M,Barnes LA,et al.Specific cross-talk between epidermal growth factor receptor and integrin alphavbeta5 promotes carcinoma cell invasion and metastasis[J].Cancer Res,2009,69:1383-1391.
[25].Murata T,Naomoto Y,Yamatsuji T,et al.Localization of FAK is related with colorectal carcinogenesis [J].Int J Oncol,2008,32:791-796.
[26].Huang D,Zhang Y & Chen X Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography [J].J Chromatogr B Analyt Technol Biomed Life Sci,2003,784:101-109.
[27].Allema JH,Reinders ME,van Gulik TM,et al.Prognostic factors for survival after pancreaticoduodenectomy for patients with carcinoma of the pancreatic head region[J].Cancer,1995,75:2069-2076.
[28].Moll UM,Youngleib GL,Rosinski KB,et al.Tumor promoter-stimulated Mr 92,000 gelatinase secreted by normal and malignant human cells:isolation and characterization of the enzyme from HT1080 tumor cells[J].Cancer Res,1990,50:6162-6170.
[29].Novina CD,Murray MF,Dykxhoorn DM,et al.siRNA-directed inhibition of HIV-l infection[J].Nat Med,2002,8:681-686.
[30].Giering JC,Grimm D,Storm TA,et al.Expression of shRNA from a tissue-specific pol Ⅱ promoter is an effective and safe RNAi therapeutic[J].Mol Ther,2008,16:1630-1636.
[31].Ni B,Shi X,Li Y,et al.Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA[J].Antivir Ther,2005,10:527-533.
[32].Orlacchio A,Bernardi G,Orlacchio A,et al.RNA interference as a tool for Alzheimer's disease therapy[J].Mini Rev Med Chem,2007,7:1166-1176.
[33].Grunweller A&Hartmann RK RNA interfefence as a gene-specific approach for molecular medicine[J].Curr Med Chem,2005,12:3143-3161.
[34].Judge AD,Robbins M,Tavakoli I,et al.Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice[J].J Clin Invest,2009.
[35].李玉军,张伟 胰腺癌MTl-MMP和MMP-2表达与神经浸润、转移及预后关系[J].临床与实验病理学杂志,2008,24:5.
[36].张翼,徐泽宽,张国新,等.骨桥蛋白和基质金属蛋白酶-2在胰腺癌中的表达及其临床意义[J].南京医科大学学报(自然科学版),2008,28:4.
[37].Sen T,Moulik S,Dutta A,et al.Multifunctional efiect of epigallocatechin-3-gallate(EGCG)in downregulation of gelatinase-A(MMP-2)in human breast cancer cell line MCF-7[J].Life Sci,2009,84:l94-204.
[38].Kim JY,Kim YJ,Lee S,et al.The critical role of ERK in death resistance and invasiveness of hypoxia-selected glioblastoma cells[J].BMC Cancer,2009,9:27.
[39].Kim S,Choi JH,Kim JB,et al.Berberine suppresses TNF-alpha-induced MMP-9 and cell invasion through inhibition of AP-l activity in MDA-MB-231 human breast cancer cells[J].Molecules,2008,13:2975-2985.
[40].Stahle M,Veit C,Bachfischer U,et al.Mechanisms in LPA-induced tumor cell migration:critical role of phosphorylated ERK[J].J Cell Sci,2003,116:3835-3846.
[41].Suh SJ,Kim JR,Jin UH,et al.Deoxypodophyllotoxin,flavolignan,from Anthriscus sylvestris Hoffm.inhibits migration and MMP-9 via MAPK pathways in TNF-alpha-induced HASMC[J],Vascul Pharmacol,2008.
[42].Rangaswami H,Bulbule A & Kundu GC JNK1 differentially regulates osteopontin-induced nuclear factor-inducing kinase/MEKKl-dependent activating protein-1-mediated promatrix metalloproteinase-9 activation [J].J Biol Chem,2005,280:19381-19392.
[43].Hou S,Xu P,Zhou L,et al.Synthesis and antitumor activity of icogenin and its analogue[J].Bioorg Med Chem Lett,2006,16:2454-2458.
[44].Hernandez JC,Leon F,Quintana J,et al.Icogenin,a new cytotoxic steroidal saponin isolated from Dracaena draco[J].Bioorg Med Chem,2004,12:4423-4429.
[45].Li D,Xie K,Wolff R,et al.Pancreatic cancer [J].Lancet,2004,363:1049-1057.
[46].Burris HA,3rd,Moore MJ,Andersen J,et al.Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer:a randomized trial [J].J Clin Oncol,1997,15:2403-2413.
[47].Danovi SA,Wong HH & Lemoine NR Targeted therapies for pancreatic cancer[J].Br Med Bull,2008,87:97-130.
[48].Reni M,Cereda S,Mazza E,et al.PEFG (cisplatin,epirubicin,5-fluorouracil,gemcitabine)regimen as second-line therapy in patients with progressive or recurrent pancreatic cancer after gemcitabine-containing chemotherapy [J].Am J Clin Oncol,2008,31:145-150.
[49].Chauffert B,Mornex F,Bonnetain F,et al.Phase Ⅲ trial comparing intensive induction chemoradiotherapy (60 Gy,infusional 5-FU and intermittent cisplatin)followed by maintenance gemcitabine with gemcitabine alone for locally advanced unresectable pancreatic cancer.Definitive results of the 2000-01 FFCD/SFRO study [J].Ann Oncol,2008,19:1592-1599.
[50].Lipton A,Harvey H,Witters L,et al.Gemcitabine/Irinotecan/celecoxib in pancreatic cancer[J].Oncology (Williston Park),2004,18:43-45.
[51].Baize N,Abu Shalaa A,Berthier F,et al.Gemcitabine combined with oxaliplatin is safe and effective in patients with previously untreated advanced pancreatic adenocarcinoma[J].Gastroenterol Clin Biol,2005,29:1006-1009.
[52].Demols A,Peeters M,Polus M,et al.Gemcitabine and oxaliplatin (GEMOX)in gemcitabine refractory advanced pancreatic adenocarcinoma:a phase Ⅱ study [J].Br J Cancer,2006,94:481-485.
[53].Oettle H,Richards D,Ramanathan RK,et al.A phase Ⅲ trial of pemetrexed plus gemcitabine versus gemcitabine in patients with unresectable-or metastatic pancreatic cancer[J].Ann Oncol,2005,16:1639-1645.
[54].Morgan MA,Parsels LA,Kollar LE,et al.The combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation in pancreatic cancer[J].Clin Cancer Res,2008,14:5142-5149.
[55].Bramhall SR,Schulz J,Nemunaitis J,et al.A double-blind placebo-controlled,randomised study comparing gemcitabine and marimastat with gemcitabine and placebo as first line therapy in patients with advanced pancreatic cancer[J].Br J Cancer,2002,87:161-167.
[56].Van Cutsem E,van de Velde H,Karasek P,et al.Phase Ⅲ trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer[J].J Clin Oncol,2004,22:1430-1438.
[57].Moore MJ,Goldstein D,Hamm J,et al.Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer:a phase Ⅲ trial of the National Cancer Institute of Canada Clinical Trials Group[J].J Clin Oncol,2007,25:1960-1966.
[58].Van Laar ES,Roth S,Weitman S,et al.Activity of irofulven against human pancreatic carcinoma cell lines in vitro and in vivo[J].Anticancer Res,2004,24:59-65.
[59].Takhar AS,Gilliam AD,Watson SA,et al.The effect of jaundice on the generation of anti-gastrin antibodies in G17DT immunized patients with advanced pancreatic cancer[J].Eur J Surg Oncol,2006,32:197-200.
[60].Chau I,Cunningham D,Russell C,et al.Gastrazole (JB95008),a novel CCK2/gastrin receptor antagonist,in the treatment of advanced pancreatic cancer:results from two randomised controlled trials[J].Br J Cancer,2006,94:1107-1115.
[61].Li J,Zhu J,Melvin WS,et al.A structurally optimized celecoxib derivative inhibits human pancreatic cancer cell growth[J].J Gastrointest Surg,2006,10:207-214.
[62].Debatin KM & Krammer PH Death receptors in chemotherapy and cancer[J].Oncogene,2004,23:2950-2966.
[63].Bold RJ,Chandra J & McConkey DJ Gemcitabine-induced programmed cell death (apoptosis)of human pancreatic carcinoma is determined by Bcl-2 content[J].Ann Surg Oncol,1999,6:279-285.
[64].Gupta S Molecular signaling in death receptor and mitochondrial pathways of apoptosis (Review)[JJ.Int J Oncol,2003,22:15-20.
[65].Desagher S & Martinou JC Mitochondria as the central control point of apoptosis[J].Trends Cell Biol,2000,10:369-377.
[66].Koizumi K,Tanno S,Nakano Y,et al.Activation of p38 mitogen-activated protein kinase is necessary for gemcitabine-induced cytotoxicity in human pancreatic cancer cells[J].Anticancer Res,2005,25:3347-3353.
[67].Habiro A,Tanno S,Koizumi K,et al.Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells[J].Biochem Biophys Res Commun,2004,316:71-77.
[68].Song IH [Cancer metastasis and metastasis suppressors][J].Korean J Gastroenterol,2004,43:1-7.
[69].Sun Y,Liu M,Yang B,et al.Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma[J].Eur Arch Otorhinolaryngol,2008.
[70].Liu J & Lin A Wiring the cell signaling circuitry by the NF-kappa B and JNK1 crosstalk and its applications in human diseases[J].Oncogene,2007,26:3267-3278.
[71].Zhuang ZH,Zhou Y,Yu MC,et al.Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli [J].Cell Signal,2006,18:441-448.
[72].Cheng YJ,Lee CH,Lin YP,et al.Caspase-3 enhances lung metastasis and cell migration in a protease-independent mechanism through the ERK pathway [J].Int J Cancer,2008,123:1278-1285.
[73].Xia Y,Wang J,Xu S,et al.MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress[J].Mol Cell Biol,2007,27:510-517.
[74].Zebrowski DC,Alcendor RR,Kirshenbaum LA,et al.Caspase-3 mediated cleavage of MEKK1 promotes p53 transcriptional activity [J].J Mol Cell Cardiol,2006,40:605-618.
[75].Coulombe P & Meloche S Atypical mitogen-activated protein kinases:structure,regulation and functions[J].Biochim Biophys Acta,2007,1773:1376-1387.
[76].Johnson GL,Dohlman HG & Graves LM MAPK kinase kinases (MKKKs)as a target class for small-molecule inhibition to modulate signaling networks and gene expression[J].Curr Opin Chem Biol,2005,9:325-331.
[77].Meloche S & Pouyssegur J The ERK 1/2 mitogen-activated protein kinase pathway as a master regulator of the G1-to S-phase transition[J].Oncogene,2007,26:3227-3239.
[78].Roberts PJ & Der CJ Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer[J].Oncogene,2007,26:3291-3310.
[79].Bode AM&Dong Z The functional Contrariety of JNK[J].Mol Carcinog,2007,46:591-598.
[80].Cuevas BD,Uhlik MT.Garrington TP,et al.MEKKl regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability[J].Oncogene,2005.24:801-809.
[81].Matsumoto T,Turesson I,Book M,et al.p38 MAP kinase negatively regulates endothelial cell survival,proliferation,and differentiation in FGF-2-stimulated angiogenesis[J].J Cell Biol,2002,156:149-160.
[82].Baud V,Liu ZG,Bennett B,et al.Signaling by proinnflammatory cytokines:oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain[J].Genes Dev,1999,13:1297-1308.
[83].Lim CP & Cao X Regulation of Stat3 activation by MEK kinase 1[J].J Biol Chem,2001,276:21004-21011.
[84].Nakamura Y,Yujiri T,Nawata R,et al.MEK kinase 1 is essemial for Bcr-Abl-induced STAT3 and self-renewal activity in embryonic stem cells[J].Oncogene,2005.24:7592-7598.
[85].Cuevas BD,Winter-Vann AM,Johnson NL,et al.MEKKl controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer[J].Oncogene,2006,25:4998-5010.
[86].Li Y,Minamino T,Tsukamoto O,et al.Ablation of MEK kinase l suppresses intimal hyperplasia by impairing smooth muscle cell migration and urokinase plasminogen activator expression in a mouse blood-now cessation model[J].Circulation,2005.111:1672-1678.
[87].Yujiri T,Ware M,Widmann C,et al.MEK kinase l gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B aetivation[J].Proe Natl Acad Sci U S A,2000,97:7272-7277.
[88].Dhillon AS,Hagan S,Rath O,et al.MAP kinase signalling pathways in cancer[J].Oncogene,2007,26:3279-3290.
[89].Sebolt-Leopold JS,Herrera R & Ohren JF The mitogen-activated protein kinase pathway for molecular-targeted cancer treatment[J].Recent Results Cancer Res,2007,172:155-167.
[90].Sussman MA Cause of death:a“broken” MEKK?[J].J Mol Cell Cardiol,2006,40:593-596