CAPE在神经胶质瘤异常Wnt/β-catenin通路中的作用及机制研究
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摘要
咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)是一种非细胞毒性抗肿瘤物质,对肿瘤细胞具有选择性的抑制作用,而对正常细胞几乎没有毒性作用。CAPE最早由蜂胶中提炼出来,是蜂胶抗肿瘤作用的主要活性成分,现在可以通过人工合成的方法获得。研究发现其抗肿瘤作用可能在于抑制异常的Wnt/β-catenin信号通路,调节细胞凋亡相关基因转录和表达,诱导细胞凋亡和抑制核因子-κB(nuclear factor-κB,NF-κB)转录活性。
Wnt/β-catenin信号传导通路是胚胎发育所必须的信号系统,过高或过低的表达都将改变细胞的生存状态。目前已经证实该系统的减低表达是引起阿尔茨海默病的重要原因,而过度激活则与多种人类肿瘤的发生有密切的联系。Wnt/β-catenin信号途径可概括为:Wnt→Frz→Dsh→β-catenin的降解复合体解散→β-catenin积累,进入细胞核→TCF/LEF→基因转录(如c-myc、cyclinD1、VEGF)。β-catenin与Tcf结合成复合物并发生核转位是此通路被过度激活的使动因素,下游癌基因的激活则是肿瘤发生发展的直接推动者。
目前,在神经胶质瘤中已有关于干扰β-catenin表达的报道,但有关CAPE与神经胶质瘤中β-catenin表达的相关性目前还未见报道。我们考虑CAPE在神经胶质瘤中是作用于此信号通路的关键环节,也就是β-catenin的过表达与核转位。因为CAPE如果只是抑制了某个下游靶基因,则对上游基因影响很小。因此抑制上游关键基因,才会对抑制肿瘤起到明显的作用。
本研究的主要目的是:①研究CAPE对神经胶质瘤的抑制作用②检测CAPE体内、外对神经胶质瘤异常Wnt/β-catenin通路β-catenin、Tcf-4及靶基因c-myc、VEGF的影响,探讨CAPE作用于该通路的靶点。
我们以β-catenin为切入点,采用免疫荧光、实时荧光定量RT-PCR、流式细胞、Western blot等技术,观测CAPE在体内和体外对神经胶质瘤异常Wnt/β-catenin通路β-catenin、Tcf-4及靶基因c-myc、VEGF的影响,探讨CAPE作用于该通路的靶点,并构建重组腺病毒β-catenin及siRNA载体,探讨β-catenin在CAPE抑制肿瘤中的作用,从而研究CAPE对神经胶质瘤抑制作用的分子机制,以期为CAPE的研究提供理论基础。
本实验的第一部分成功构建Ad-β-catenin与Ad-β-catenin siRNA腺病毒载体,使我们可以加强或抑制β-catenin在神经胶质瘤U251细胞株及裸鼠移植瘤的表达。通过后面实验对细胞株及移植瘤β-catenin mRNA和蛋白的检测,两种病毒转染率高,表达稳定,可以很好地用于胶质瘤异常Wnt/β-catenin通路的研究。
本实验的第二部分通过流式细胞检测、激光共聚焦显微镜、实时荧光定量PCR(Real-Time Quantitative PCR)、Western blot等技术,我们观察到U251细胞株体外培养时给予CAPE干预后促进了细胞的凋亡,其基础表达、过表达、低表达的β-catenin均被进一步抑制,其下游的Tcf-4及靶基因c-myc、VEGF mRNA及蛋白均相应降低。实验结果提示:CAPE体外是通过抑制了U251细胞的β-catenin基因的表达与核转位,游靶基因的表达也相应下降,因此对神经胶质瘤起到一定的抑制作用。
本实验的第三部分通过实时荧光定量PCR、Western blot等技术,我们观察到U251细胞荷瘤鼠在给予CAPE干预后,其基础表达、过表达、低表达的β-catenin均被进一步抑制,CAPE对移植瘤的抑瘤率达到48.39%(>40%为有效)。实验结果提示:CAPE体内同样是抑制了移植瘤的异常Wnt/β-catenin通路,对胶质瘤的抑制效果明显。
综上所述,CAPE是通过抑制了神经胶质瘤U251细胞的异常Wnt/β-catenin通路中的β-catenin基因表达而对神经胶质瘤起到一定的抑制作用。
The coffeic acid benzene ethyl ester (caffeic acid phenethyl ester, CAPE) is a kind of non-cytotoxicity antitumorigenic substance, selectively inhibit the growth of tumour cells, while does not impact the growth of normal cells. CAPE was first refined from the bee glue, which is the main component accounting for its anti-tumor activity, and now, it can be synthesized artificially.
Previous studies have revealed that its anti-tumor function could be attributed to its ability to suppress the abnormal Wnt/β-catenin signaling pathway, modulate the expression of apoptosis-associated genes, induce cell apoptosis and inhibit the transcriptional activity of nuclear factor -κB (nuclear factor-κB, NF-κB).
As we know, Wnt/β-catenin signaling pathway is a developmental pathway that has been shown to play a role in embryonic development, whose dysfunction has great influence on the survival of cells. To date, it is quite clear that downregulation of Wnt-signaling pathway is the main cause of Alzheimers syndrome; besides, excessive activation of this pathway is closely related with the occurrence of many human tumors. The general Wnt/β-catenin signaling pathway could be schematically described as: dismission of the Wnt→Frz→Dsh→β-catenin degeneration complex→accumulation ofβ-catenin and translocation to the cell nucleus→β-catenin combination with TCF/LEF→gene expression(e.g. c-myc, cyclinD1, VEGF). Translocation ofβ-catenin-Tcf compound to nucleus could initiate this signaling pathway and cause signaling abnormality, which further activate the expression of downstream genes and thus promote cancer developmet.
It has been reported that CAPE could interference withβ-catenin in hepatoma and colon cancers, while it’s still unclear whether CAPE works in neuroglioma. So we hypothesized that the function of CAPE in neuroglioma could also be related withβ-catenin, since it is a key gene in the upstream of this pathway.
Here, the main purpose of research is to reveal the target of CAPE in this pathway in vivo and in vitro; and to detect whether CAPE could influenceβ-catenin, Tcf-4 and target gene c-myc in Wnt/β-catenin pathway of neuroglioma in vitro and in neuroglia mouse models (in vivo).
Immunity fluorescence, real-time RT-PCR and western blot methods were employed to analyze the levels ofβ-catenin, Tcf-4 and target gene c-myc, the VEGF in Wnt/β-catenin pathway in neuroglioma. These items were used to evaluate the efficacy of CAPE in neuroglioma, which will help to find the target of CAPE in this pathway. By constructing recombinant adenovirusβ-catenin and siRNA vectors, we investiged the role ofβ-catenin involved in the process of CAPE inhibiting tumor. This study will help to reveal the molecular mechanism of CAPE against neuroglioma, and finally provides the basic evidence for CAPE’s clinical use.
In the first part of our study, we have sucessfully constructed recombinant Ad-β-catenin and Ad-β-catenin siRNA vectors, which were subsequently used to enhance or suppress the expression ofβ-catenin in neuroglioma U251 cell line and transplanted tumor in athymic mice. The two recombinant vectors could be efficiently transfected to cell line and remain constant in cells and transplanted tumor by detecting mRNA and protein levels ofβ-catenin.
In the following part, we observed first that CAPE could induce cell apoptosis using FCM; moreover, overexpressedβ-catenin was suppressed after treatment with CAPE using real-time quantitative PCR; furthermore, we demonstrated that the expression of Tcf-4, c-myc and VEGF were decreased using western-blot assay. Taken together, these results indicated that CAPE could induce U251 cell apoptosis, and this function could attribute to its role againstβ-catenin. From the preliminary results, we may get the conclusion that CAPE could be therapeutically used to treat the neuroglioma.
In third part, in an attempt to further confirm that CAPE is also effective against tumor, U251 cell beared athymic mice was used to test the effect of CAPE. First, we observed that all forms ofβ-catenin could be surpressed by treatment with CAPE in U251 cell-beared mice. The expression ofβ-catenin downstream genes, such as, Tcf-4, c-myc and VEGF was also decreased. Moreove, 48.39% tumor inhibition rate can be achieved (>40% is thought to be effective). These in vivo results confirmed that CAPE could suppress the transplanted tumor load and was effective for the treatment of neuroglioma, which was through the inhibition of the abnormal Wnt/β-catenin pathway.
In a word, CAPE could induce U251 cell apoptosis, and from the results demonstrated here, this treatment effect could be attributed to its function of suppressing the expression ofβ-catenin.
Wnt/β-catenin信号传导通路是胚胎发育所必须的信号系统,过高或过低的表达都将改变细胞的生存状态。目前已经证实该系统的减低表达是引起阿尔茨海默病的重要原因,而过度激活则与多种人类肿瘤的发生有密切的联系。Wnt/β-catenin信号途径可概括为:Wnt→Frz→Dsh→β-catenin的降解复合体解散→β-catenin积累,进入细胞核→TCF/LEF→基因转录(如c-myc、cyclinD1、VEGF)。β-catenin与Tcf结合成复合物并发生核转位是此通路被过度激活的使动因素,下游癌基因的激活则是肿瘤发生发展的直接推动者。
目前,在神经胶质瘤中已有关于干扰β-catenin表达的报道,但有关CAPE与神经胶质瘤中β-catenin表达的相关性目前还未见报道。我们考虑CAPE在神经胶质瘤中是作用于此信号通路的关键环节,也就是β-catenin的过表达与核转位。因为CAPE如果只是抑制了某个下游靶基因,则对上游基因影响很小。因此抑制上游关键基因,才会对抑制肿瘤起到明显的作用。
本研究的主要目的是:①研究CAPE对神经胶质瘤的抑制作用②检测CAPE体内、外对神经胶质瘤异常Wnt/β-catenin通路β-catenin、Tcf-4及靶基因c-myc、VEGF的影响,探讨CAPE作用于该通路的靶点。
我们以β-catenin为切入点,采用免疫荧光、实时荧光定量RT-PCR、流式细胞、Western blot等技术,观测CAPE在体内和体外对神经胶质瘤异常Wnt/β-catenin通路β-catenin、Tcf-4及靶基因c-myc、VEGF的影响,探讨CAPE作用于该通路的靶点,并构建重组腺病毒β-catenin及siRNA载体,探讨β-catenin在CAPE抑制肿瘤中的作用,从而研究CAPE对神经胶质瘤抑制作用的分子机制,以期为CAPE的研究提供理论基础。
本实验的第一部分成功构建Ad-β-catenin与Ad-β-catenin siRNA腺病毒载体,使我们可以加强或抑制β-catenin在神经胶质瘤U251细胞株及裸鼠移植瘤的表达。通过后面实验对细胞株及移植瘤β-catenin mRNA和蛋白的检测,两种病毒转染率高,表达稳定,可以很好地用于胶质瘤异常Wnt/β-catenin通路的研究。
本实验的第二部分通过流式细胞检测、激光共聚焦显微镜、实时荧光定量PCR(Real-Time Quantitative PCR)、Western blot等技术,我们观察到U251细胞株体外培养时给予CAPE干预后促进了细胞的凋亡,其基础表达、过表达、低表达的β-catenin均被进一步抑制,其下游的Tcf-4及靶基因c-myc、VEGF mRNA及蛋白均相应降低。实验结果提示:CAPE体外是通过抑制了U251细胞的β-catenin基因的表达与核转位,游靶基因的表达也相应下降,因此对神经胶质瘤起到一定的抑制作用。
本实验的第三部分通过实时荧光定量PCR、Western blot等技术,我们观察到U251细胞荷瘤鼠在给予CAPE干预后,其基础表达、过表达、低表达的β-catenin均被进一步抑制,CAPE对移植瘤的抑瘤率达到48.39%(>40%为有效)。实验结果提示:CAPE体内同样是抑制了移植瘤的异常Wnt/β-catenin通路,对胶质瘤的抑制效果明显。
综上所述,CAPE是通过抑制了神经胶质瘤U251细胞的异常Wnt/β-catenin通路中的β-catenin基因表达而对神经胶质瘤起到一定的抑制作用。
The coffeic acid benzene ethyl ester (caffeic acid phenethyl ester, CAPE) is a kind of non-cytotoxicity antitumorigenic substance, selectively inhibit the growth of tumour cells, while does not impact the growth of normal cells. CAPE was first refined from the bee glue, which is the main component accounting for its anti-tumor activity, and now, it can be synthesized artificially.
Previous studies have revealed that its anti-tumor function could be attributed to its ability to suppress the abnormal Wnt/β-catenin signaling pathway, modulate the expression of apoptosis-associated genes, induce cell apoptosis and inhibit the transcriptional activity of nuclear factor -κB (nuclear factor-κB, NF-κB).
As we know, Wnt/β-catenin signaling pathway is a developmental pathway that has been shown to play a role in embryonic development, whose dysfunction has great influence on the survival of cells. To date, it is quite clear that downregulation of Wnt-signaling pathway is the main cause of Alzheimers syndrome; besides, excessive activation of this pathway is closely related with the occurrence of many human tumors. The general Wnt/β-catenin signaling pathway could be schematically described as: dismission of the Wnt→Frz→Dsh→β-catenin degeneration complex→accumulation ofβ-catenin and translocation to the cell nucleus→β-catenin combination with TCF/LEF→gene expression(e.g. c-myc, cyclinD1, VEGF). Translocation ofβ-catenin-Tcf compound to nucleus could initiate this signaling pathway and cause signaling abnormality, which further activate the expression of downstream genes and thus promote cancer developmet.
It has been reported that CAPE could interference withβ-catenin in hepatoma and colon cancers, while it’s still unclear whether CAPE works in neuroglioma. So we hypothesized that the function of CAPE in neuroglioma could also be related withβ-catenin, since it is a key gene in the upstream of this pathway.
Here, the main purpose of research is to reveal the target of CAPE in this pathway in vivo and in vitro; and to detect whether CAPE could influenceβ-catenin, Tcf-4 and target gene c-myc in Wnt/β-catenin pathway of neuroglioma in vitro and in neuroglia mouse models (in vivo).
Immunity fluorescence, real-time RT-PCR and western blot methods were employed to analyze the levels ofβ-catenin, Tcf-4 and target gene c-myc, the VEGF in Wnt/β-catenin pathway in neuroglioma. These items were used to evaluate the efficacy of CAPE in neuroglioma, which will help to find the target of CAPE in this pathway. By constructing recombinant adenovirusβ-catenin and siRNA vectors, we investiged the role ofβ-catenin involved in the process of CAPE inhibiting tumor. This study will help to reveal the molecular mechanism of CAPE against neuroglioma, and finally provides the basic evidence for CAPE’s clinical use.
In the first part of our study, we have sucessfully constructed recombinant Ad-β-catenin and Ad-β-catenin siRNA vectors, which were subsequently used to enhance or suppress the expression ofβ-catenin in neuroglioma U251 cell line and transplanted tumor in athymic mice. The two recombinant vectors could be efficiently transfected to cell line and remain constant in cells and transplanted tumor by detecting mRNA and protein levels ofβ-catenin.
In the following part, we observed first that CAPE could induce cell apoptosis using FCM; moreover, overexpressedβ-catenin was suppressed after treatment with CAPE using real-time quantitative PCR; furthermore, we demonstrated that the expression of Tcf-4, c-myc and VEGF were decreased using western-blot assay. Taken together, these results indicated that CAPE could induce U251 cell apoptosis, and this function could attribute to its role againstβ-catenin. From the preliminary results, we may get the conclusion that CAPE could be therapeutically used to treat the neuroglioma.
In third part, in an attempt to further confirm that CAPE is also effective against tumor, U251 cell beared athymic mice was used to test the effect of CAPE. First, we observed that all forms ofβ-catenin could be surpressed by treatment with CAPE in U251 cell-beared mice. The expression ofβ-catenin downstream genes, such as, Tcf-4, c-myc and VEGF was also decreased. Moreove, 48.39% tumor inhibition rate can be achieved (>40% is thought to be effective). These in vivo results confirmed that CAPE could suppress the transplanted tumor load and was effective for the treatment of neuroglioma, which was through the inhibition of the abnormal Wnt/β-catenin pathway.
In a word, CAPE could induce U251 cell apoptosis, and from the results demonstrated here, this treatment effect could be attributed to its function of suppressing the expression ofβ-catenin.
引文
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[9]. Miyagishi M, Sumimoto H, Miyoshi H, et al. Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells[J]. J Gene Med, 2004,6:715-23.
[10]. Li T, Zhang Y, Fu L, et al. siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. Gene Ther, 2005,12:751-61. ublishing
[11]. Miyake K, Flygare J, Kiefer T, et al. Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond-blackfan anemia using inducible expression of siRNA against RPS19[J]. Mol Ther, 2005,11:627-37.
[1]. Takemoto K, Kuranaga E, Tonoki A, et al. Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo[J]. Proc Natl Acad Sci U S A, 2007,104:13367-72.
[2]. Bayline RJ, Dean DM,Booker R. Inhibitors of ubiquitin-dependent proteolysis can delay programmed cell death of adult intersegmental muscles in the moth Manduca sexta[J]. Dev Dyn, 2005,233:445-55.
[3]. Kuenzler KA, Arthur LG,Schwartz MZ. A possible mechanism for prevention of intestinal programmed cell death after ischemia-reperfusion injury by hepatocyte growth factor pretreatment[J]. J Pediatr Surg, 2002,37:1696-9.
[4]. Hacker G, The morphology of apoptosis[J].Cell Tissue Res 2000;301 (1):5-17.
[5]. Fadok VA, Voelker DRk Campbell PA, et al.Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[J]. J Immunol, 1992;148 (7):2207-2216.
[6]. Kerr J F R, Wyllie A H, Curie A R. Apoptosis :a base biological phenomenon wit h winderanging implications in tissue kinet2ics[J].Bj J Cancer ,1972;26:238-257.
[7]. Tkaczyk ER, Tkaczyk AH, Katnik S, et al. Extended cavity laser enhanced two-photon flow cytometry[J]. Biomed Opt, 2008,13:041319.
[8]. Matzdorff A. Platelet function tests and flow cytometry to monitor antiplatelet therapy[J]. Semin Thromb Hemost, 2005,31:393-9.
[9]. Assuncao P, Rosales RS, Rifatbegovic M, et al. Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry[J]. Front Biosci, 2006,11:492-7.
[10]. Vermes I, Haanen C, Steffens2Nakken H, et al.A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluoresce in labeled AnnexinⅤ[ J]. Immunol Methods, 1995;184 (1):39-51.
[11]. Hibi K, Ushio H, Fukuda H, et al. Immunomagnetic separation using carbonyl iron powder and flow cytometry for rapid detection of Flavobacterium psychrophilum[J]. Anal Bioanal Chem, 2008,391:1147-52.
[12]. Maloum K, Charlotte F, Divine M, et al. A comparison of the sensitivity of flow cytometry and bone marrow biopsy in the detection of minimal residual disease in chronic lymphocytic leukemia. [J] Haematologica, 2006,91:860-1.
[13]. Hewitt CJ, Nebe-Von-Caron G. An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations[J]. Cytometry, 2001,44:179-87.
[1]. Shtutman M, Zhurinsky J, Simcha I, et al.The cyclin D1 gene is a target of the ?-catenin/LEF-1 pathway[J]. Proc Natl Acad Sci,1999,96:5522–5527.
[2]. Brabletz T, Herrmann K, Jung A, et al. Expression of nuclearβ-catenin and c-myc is correlated with tumor size but not with proliferative activity of colorectal adenomas[J]. Am J Pathol ,2000,156:865–870.
[3]. He TC, Chan TA, Vogelstein B. PPAR is an APC-regulated target of nonsteroidal anti-inflammatory drugs[J]. Cell,1999, 99:335–345.
[4]. Giaginis C, Theocharis S,Tsantili-Kakoulidou A. Quantitative structure-activity relationships for PPAR-gamma binding and gene transactivation of tyrosine-based agonists using multivariate statistics[J]. Chem Biol Drug Des, 2008,72:257-64.
[5]. Jennifer LS. ICAT is a multipotent inhibitor ofβ-catenin. Focus on "Role for ICAT inβ-catenin-dependent nuclear signaling and cadherin functions"[J]. Am JPhysiol Cell Physiol,2004,286: C745-746.
[6]. Valenta T, Lukas J, Doubravska L, et al. HIC1 attenuates Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies[J]. EMBO J, 2006,25:2326-37.
[7]. Lutterbach B, Hiebert SW. Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation[J]. Gene,2000,245:223-5.
[8]. Crawford HC, Fingleton BM, Rudolph-Owen LA, et al. The metalloproteinase matrilysin is a target of ?-catenin transactivation in intestinal tumors[J]. Oncogene,1999,18:2883–2891.
[9]. Mikami T, Saegusa M, Mitomi H, et al. Significant correlations of E-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas[J]. Am J Clin Pathol,2001,116:369–376.
[10]. Na HK, Wilson MR, Kang KS, et al. Restoration of gap junctional intercellular communication by caffeic acid phenethyl ester (CAPE) in a ras-transformed rat liver epithelial cell line[J]. Cancer Lett, 2000,157:31-8.
[11]. Xiang D, Wang D, He Y, Xie J, Zhong Z, Li Z, Xie J. Caffeic acid phenethyl ester induces growth arrest and apoptosis of colon cancer cells via the beta-catenin/T-cell factor signaling[J]. Anticancer Drugs. 2006 Aug;17(7):753-762.
[1]. Ghosh MK, Mitra AK. Carboxylic ester hydrolase activity in hairless and athymic nude mouse skin[J]. Pharm Res, 1990,7:251-5.
[2]. Castigli E, Sciaccaluga M, Schiavoni G, et al. GL15 and U251 glioblastoma-derived human cell lines are peculiarly susceptible to induction of mitotic death by very low concentrations of okadaic acid[J]. Oncol Rep, 2006,15:463-70.
[3]. Wang SJ, Wang JH, Zhang YW, et al. [Effects of small interfering RNA targeting basic fibroblast growth factor on proliferation and apoptosis of glioma cell line U251[J]. Ai Zheng, 2008,27:905-9
[4]. Martinez-Murillo R, Martinez A. Standardization of an orthotopic mouse brain tumor model following transplantation of CT-2A astrocytoma cells[J]. Histol Histopathol, 2007,22:1309-26.
[5]. Ohsawa I, Uemoto S, Kobayashi E, et al. Control of cyclosporine A-induced tumor progression using 15-deoxyspergualin for rat cardiac transplantation[J]. Transplantation, 2007,84:424-8.
[6]. De Giorgi U, Rosti G, Papiani G, et al. The status of high-dose chemotherapy with hematopoietic stem cell transplantation in germ cell tumor patients[J]. Haematologica, 2002,87:95-104.
[7]. Densem CG, Hutchinson IV, Yonan N, et al. Influence of tumor necrosisfactor-alpha gene-308 polymorphism on the development of coronary vasculopathy after cardiac transplantation[J]. Heart Lung Transplant, 2001,20:1265-73.
[8]. Sharp JG, Kessinger A, Mann S, et al. Outcome of high-dose therapy and autologous transplantation in non-Hodgkin's lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest[J]. Clin Oncol, 1996,14:214-9.
[1]. Garg AK, Buchholz TA, Aggarwal BB. Chemosensitization and radiosensitization of tumors by plant polyphenols[J]. Antioxid Redox Signal, 2005, 7(11-12): 1630-1647.
[2]. Wang D, Xiang DB, He YJ, Li ZP, Wu XH, Mou JH, Xiao HL, Zhang QH. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro[J]. World J Gastroenterol, 2005, 11(26): 4008-4012.
[3]. Xiang D, Wang D, He Y, Xie J, Zhong Z, Li Z, Xie J. Caffeic acid phenethyl ester induces growth arrest and apoptosis of colon cancer cells via the beta-catenin/T-cell factor signaling[J]. Anticancer Drugs. 2006 Aug;17(7):753-762.
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[3]. Nagaoka T, Banskota AH, Tezuka Y, Saiki I, Kadota S. Selective antiproliferative activity of caffeic acid phenethyl ester analogues on highly liver-metastatic murine colon 26-L5 carcinoma cell line[J]. Bioorg Med Chem, 2002, 10(10): 3351-3359
[4]. Xiang DB, Wang D, He Y, Xie J, Zhong Z, Li Z, Xie J. Caffeic acid phenethyl ester induces growth arrest and apoptosis of colon cancer cells via the beta-catenin/T-cell factor signaling. [J] Anticancer Drugs. 2006 Aug;17(7):753-762.
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[8]. En YJ, Shiao MS, Hsu ML, Tsai TH, Wang SY. Effect of caffeic acid phenethyl ester, an antioxidant from propolis, on inducing apoptosis in human leukemic HL-60 cells[J]. J Agric Food Chem, 2001, 49(11): 5615-5619
[9]. Rose F, Dahlem G, Guthmann B, Grimminger F, Maus U, Hanze J, Duemmer N, Grandel U, Seeger W, Ghofrani HA. Mediator generation and signaling events in alveolar epithelial cells attacked by S. aureus alpha-toxin[J]. Am J Physiol Lung Cell Mol Physiol, 2002, 282(2): L207-214
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[1]. Kalantidis K, Tsagris M,Tabler M. Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing[J]. Plant J, 2006,45:1006-16.
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[9]. Miyagishi M, Sumimoto H, Miyoshi H, et al. Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells[J]. J Gene Med, 2004,6:715-23.
[10]. Li T, Zhang Y, Fu L, et al. siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. Gene Ther, 2005,12:751-61. ublishing
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[1]. Takemoto K, Kuranaga E, Tonoki A, et al. Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo[J]. Proc Natl Acad Sci U S A, 2007,104:13367-72.
[2]. Bayline RJ, Dean DM,Booker R. Inhibitors of ubiquitin-dependent proteolysis can delay programmed cell death of adult intersegmental muscles in the moth Manduca sexta[J]. Dev Dyn, 2005,233:445-55.
[3]. Kuenzler KA, Arthur LG,Schwartz MZ. A possible mechanism for prevention of intestinal programmed cell death after ischemia-reperfusion injury by hepatocyte growth factor pretreatment[J]. J Pediatr Surg, 2002,37:1696-9.
[4]. Hacker G, The morphology of apoptosis[J].Cell Tissue Res 2000;301 (1):5-17.
[5]. Fadok VA, Voelker DRk Campbell PA, et al.Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages[J]. J Immunol, 1992;148 (7):2207-2216.
[6]. Kerr J F R, Wyllie A H, Curie A R. Apoptosis :a base biological phenomenon wit h winderanging implications in tissue kinet2ics[J].Bj J Cancer ,1972;26:238-257.
[7]. Tkaczyk ER, Tkaczyk AH, Katnik S, et al. Extended cavity laser enhanced two-photon flow cytometry[J]. Biomed Opt, 2008,13:041319.
[8]. Matzdorff A. Platelet function tests and flow cytometry to monitor antiplatelet therapy[J]. Semin Thromb Hemost, 2005,31:393-9.
[9]. Assuncao P, Rosales RS, Rifatbegovic M, et al. Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry[J]. Front Biosci, 2006,11:492-7.
[10]. Vermes I, Haanen C, Steffens2Nakken H, et al.A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluoresce in labeled AnnexinⅤ[ J]. Immunol Methods, 1995;184 (1):39-51.
[11]. Hibi K, Ushio H, Fukuda H, et al. Immunomagnetic separation using carbonyl iron powder and flow cytometry for rapid detection of Flavobacterium psychrophilum[J]. Anal Bioanal Chem, 2008,391:1147-52.
[12]. Maloum K, Charlotte F, Divine M, et al. A comparison of the sensitivity of flow cytometry and bone marrow biopsy in the detection of minimal residual disease in chronic lymphocytic leukemia. [J] Haematologica, 2006,91:860-1.
[13]. Hewitt CJ, Nebe-Von-Caron G. An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations[J]. Cytometry, 2001,44:179-87.
[1]. Shtutman M, Zhurinsky J, Simcha I, et al.The cyclin D1 gene is a target of the ?-catenin/LEF-1 pathway[J]. Proc Natl Acad Sci,1999,96:5522–5527.
[2]. Brabletz T, Herrmann K, Jung A, et al. Expression of nuclearβ-catenin and c-myc is correlated with tumor size but not with proliferative activity of colorectal adenomas[J]. Am J Pathol ,2000,156:865–870.
[3]. He TC, Chan TA, Vogelstein B. PPAR is an APC-regulated target of nonsteroidal anti-inflammatory drugs[J]. Cell,1999, 99:335–345.
[4]. Giaginis C, Theocharis S,Tsantili-Kakoulidou A. Quantitative structure-activity relationships for PPAR-gamma binding and gene transactivation of tyrosine-based agonists using multivariate statistics[J]. Chem Biol Drug Des, 2008,72:257-64.
[5]. Jennifer LS. ICAT is a multipotent inhibitor ofβ-catenin. Focus on "Role for ICAT inβ-catenin-dependent nuclear signaling and cadherin functions"[J]. Am JPhysiol Cell Physiol,2004,286: C745-746.
[6]. Valenta T, Lukas J, Doubravska L, et al. HIC1 attenuates Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies[J]. EMBO J, 2006,25:2326-37.
[7]. Lutterbach B, Hiebert SW. Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation[J]. Gene,2000,245:223-5.
[8]. Crawford HC, Fingleton BM, Rudolph-Owen LA, et al. The metalloproteinase matrilysin is a target of ?-catenin transactivation in intestinal tumors[J]. Oncogene,1999,18:2883–2891.
[9]. Mikami T, Saegusa M, Mitomi H, et al. Significant correlations of E-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas[J]. Am J Clin Pathol,2001,116:369–376.
[10]. Na HK, Wilson MR, Kang KS, et al. Restoration of gap junctional intercellular communication by caffeic acid phenethyl ester (CAPE) in a ras-transformed rat liver epithelial cell line[J]. Cancer Lett, 2000,157:31-8.
[11]. Xiang D, Wang D, He Y, Xie J, Zhong Z, Li Z, Xie J. Caffeic acid phenethyl ester induces growth arrest and apoptosis of colon cancer cells via the beta-catenin/T-cell factor signaling[J]. Anticancer Drugs. 2006 Aug;17(7):753-762.
[1]. Ghosh MK, Mitra AK. Carboxylic ester hydrolase activity in hairless and athymic nude mouse skin[J]. Pharm Res, 1990,7:251-5.
[2]. Castigli E, Sciaccaluga M, Schiavoni G, et al. GL15 and U251 glioblastoma-derived human cell lines are peculiarly susceptible to induction of mitotic death by very low concentrations of okadaic acid[J]. Oncol Rep, 2006,15:463-70.
[3]. Wang SJ, Wang JH, Zhang YW, et al. [Effects of small interfering RNA targeting basic fibroblast growth factor on proliferation and apoptosis of glioma cell line U251[J]. Ai Zheng, 2008,27:905-9
[4]. Martinez-Murillo R, Martinez A. Standardization of an orthotopic mouse brain tumor model following transplantation of CT-2A astrocytoma cells[J]. Histol Histopathol, 2007,22:1309-26.
[5]. Ohsawa I, Uemoto S, Kobayashi E, et al. Control of cyclosporine A-induced tumor progression using 15-deoxyspergualin for rat cardiac transplantation[J]. Transplantation, 2007,84:424-8.
[6]. De Giorgi U, Rosti G, Papiani G, et al. The status of high-dose chemotherapy with hematopoietic stem cell transplantation in germ cell tumor patients[J]. Haematologica, 2002,87:95-104.
[7]. Densem CG, Hutchinson IV, Yonan N, et al. Influence of tumor necrosisfactor-alpha gene-308 polymorphism on the development of coronary vasculopathy after cardiac transplantation[J]. Heart Lung Transplant, 2001,20:1265-73.
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