GnRH-p53系列融合蛋白的表达及其抗肿瘤功能研究
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摘要
促性腺激素释放激素(gonadotrophin releasing hormone,GnRH)是下丘脑分泌的十肽激素,其生理功能是调节垂体前叶促性腺激素的分泌。近年来的研究发现在乳腺、卵巢、子宫内膜、前列腺、胰腺、结肠、肺、肝脏的肿瘤细胞上有GnRH受体分布。GnRH及其类似物可抑制这些肿瘤细胞生长,它们的抗肿瘤效应是通过垂体及垂体外的机制实现的。缺氧是实体肿瘤的特征性病理改变。氧依赖性降解区域(oxygen-dependent degradation domain,ODD)是缺氧诱导因子-1α(HIF-1α)上控制其稳定的关键区域,通过其核心脯氨酸的羟化,在常氧状态时使HIF-1α被蛋白酶降解。p53是细胞内重要的肿瘤抑制因子,它可以通过诱导肿瘤细胞凋亡和诱发肿瘤细胞发生细胞周期阻滞而产生抗肿瘤作用。研究表明人类50%以上的肿瘤的发生都与细胞内P53基因突变、缺失有关,因此p53一直是近年来抗肿瘤研究的热点。
本课题旨在以p53为肿瘤治疗的效应分子,通过GnRH与其受体特异结合介导蛋白进入相应的肿瘤细胞,应用ODD减轻p53对正常组织的损伤,增强蛋白对实体肿瘤的靶向作用,构建pET28a-GnRH-P53(pET28a-GP)、pET28a-GnRH-ODD-P53(pET28a-GOP)、pET28a-GnRHⅢ-P53(pET28a-G3P)、pET28a-GnRHⅢ-ODD-P53(pET28a-G3OP)、pET28a-GnRH-ODD557-574aa-P53 (pET28a-GSOP)、pET28a-GnRHⅢ-ODD557-574aa-P53(pET28a-G3SOP)原核表达载体,转化大肠杆菌BL21,诱导重组质粒表达相应的融合蛋白,将表达后菌体超声处理后离心,收集包涵体后进行变性、复性、纯化,从而得到具有生物学功能的融合蛋白。在细胞和动物水平研究融合蛋白的抗肿瘤作用。具体方法为:应用MTT法测定融合蛋白对肿瘤细胞生长的抑制作用;细胞免疫化学法、细胞免疫荧光法测定融合蛋白在细胞中的定位;应用流式细胞术测定融合蛋白诱导肿瘤细胞凋亡及其诱发肿瘤细胞周期阻滞;Western blot检测融合蛋白抗肿瘤的可能机制。以H1299细胞建立BALB/c裸鼠荷瘤模型,将实验动物随机分为C (正常对照组),TC (肿瘤对照组),GP(GnRH-p53),GOP(GnRH-ODD-p53),G3P (GnRHⅢ-p53),G3OP(GnRHⅢ-ODD- p53)六组,以5mg/kg/天的剂量腹腔给药(对照组动物给予等量的生理盐水),测定融合蛋白对模型鼠的抗肿瘤作用,同时对模型鼠血浆生化指标进行测定初步判断融合蛋白对动物的毒副作用。
本课题的实验结果:①构建了pET28a-GnRH-P53系列重组质粒载体,将其转入大肠杆菌BL21后成功表达了相应的融合蛋白。②经细胞免疫荧光法、细胞免疫化学法、Western blot检测表明融合蛋白可以进入GnRH-R表达阳性的H1299细胞、DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞。③经MTT法测定表明GP、GSOP、G3P、G3SOP在常氧和缺氧条件下均可以抑制H1299细胞的生长,并且这种作用具有剂量依赖性,即随着蛋白浓度的增加其抑制H1299细胞生长作用增强;GOP和G3OP在缺氧条件下对H1299细胞的生长抑制作用比其在常氧条件下的作用明显;融合蛋白对DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞也具有增殖抑制作用;融合蛋白对H1299细胞的增殖抑制作用强于相同剂量的GnRH类似物亮丙瑞林和G662。④流式细胞术测定融合蛋白诱导肿瘤细胞凋亡表明,融合蛋白在常氧状态下可以增加H1299细胞早期凋亡的百分率,其作用在48h最强,而与PBS对照相比,G3OP在给药24h时可显著诱导H1299细胞发生早期凋亡;在缺氧状态下,给药24h,与RPMI1640空白培养基对照相比较,各融合蛋白均可显著诱导H1299细胞发生早期凋亡; GOP和G3OP在缺氧状态下的作用比其在常氧状态下诱导细胞凋亡作用明显增强。⑤在常氧和缺氧状态下,各融合蛋白可以诱导H1299细胞G0/G1期细胞比例增加,而处于S期细胞的百分数减少,DNA合成受到抑制,即发生细胞G1期阻滞。⑥经Western blot检测,融合蛋白可以上调H1299细胞中的Caspase-3蛋白、p21蛋白表达水平,推测融合蛋白抑制H1299细胞生长的机制与活化Caspase-3蛋白诱导细胞凋亡及上调p21蛋白表达促进细胞发生G1期阻滞相关。⑦测量、计算各组实验动物瘤体体积发现,与TC组比较GP抑制瘤体生长作用十分显著(p<0.01),GOP、G3P、G3OP抑瘤作用较明显(p<0.05)。⑧免疫组化方法检测荷瘤模型小鼠肝脏和肿瘤切片,发现p53蛋白主要分布于肿瘤组织中,而且给药组动物肿瘤组织切片中Caspase-3蛋白、p21蛋白、PUMA蛋白的含量较对照组高,表明融合蛋白对荷瘤模型小鼠的抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。⑨动物血浆生化指标测定结果表明,融合蛋白对动物的肝、肾功能几乎没有影响。
由以上结果得出如下结论:在细胞和动物整体水平上,融合蛋白GP、GOP、G3P、G3OP具有抑制肿瘤生长作用,其抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。GP等融合蛋白有望开发成为新型的抗肿瘤药物。
As a 10-amino acid regulator of reproduction, gonadotrophin-releasing hormone (GnRH) is released by neurons in the hypothalamus, and transported via the hypothalamo-hypophyseal portal circulation to the anterior pituitary to trigger gonadotropin release for luteinizing hormone and follicle-stimulating hormone, which in turn stimulate the gonads for steroid production. In the past, GnRH receptor (GnRH-R) was found to be expressed in normal human reproductive tissues (e.g. breast, ovary, endometrial, prostate, pancreas, colon, lung and liver) and tumors derived from these tissues. Numerous studies have provided evidence for the suppress effect of GnRH and GnRH-a (GnRH analogue) in these tumor cells proliferation. The multiple actions of GnRH could be linked to the divergence of signaling pathways that are activated by GnRH-R. The oxygen-dependent degradation domain (ODD) of HIF-1αis a key on protein stability. In normal oxygen level, ODD can accelerate the degration of HIF-1αby the hydroxylation of its proline. p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain a mutation or deletion of p53. Therefore p53 has been an appealing target for new anticancer therapeutic strategies.
The aim of this project is to investigate the targeted tumor inhibition induced by p53 fusion proteins against tumors with p53 gene mutation or deficient. GnRH, as the ligand of GnRH-R, was used to deliver p53 into tumor cells. ODD was used to control the stability of proteins in tissue under different oxygen stress. GnRH, GnRHⅢ, ODD and p53 gene was constructed and cloned into the pET28a vector. The p53 fusion proteins were expressed and their targeted anti-tumor effects were determined. The viability of tumor cells treated with each protein was measured by the MTT method. Immunocytochemistry analysis and immunofluorescence analysis were also used to detect the location of p53 fusion proteins in tumor cells. The flow cytometry was employed to analyze the apoptosis and cell cycle arrest in the proteins treated H1299 cells. The possible mechanism of proliferation inhibition induced by p53 fusion proteins was measured by Western blot. The mice with xenograft tumor of H1299 cells were used to test the tumor proliferation inhibition induced by fusion proteins (GP, GOP, G3P and G3OP) in vivo, while saline buffer was used as control. The plasma biochemical parameters of mice were also determined.
A series of novel fusion proteins, GnRH-p53(GP), GnRH-ODD-p53(GOP),GnRHⅢ-p53 (G3P),GnRHⅢ-ODD-p53 (G3OP), GnRH-ODD557-574aa-p53(GSOP), GnRHⅢ-ODD557-574aa-p53(G3SOP), were expressed in E coli. BL21 (DE3). As the control protein, wild type p53 (p53), was also constructed. These proteins were used to investigate the targeted tumor inhibition induced by p53 fusion proteins. Immunocytochemistry analysis and immunofluorescence analysis showed that all of fusion proteins could permeate into the H1299 cells and located in the cytoplasm and nucleus. Cellular experiments demonstrated that GP, GSOP, G3P and G3SOP could obviously decrease the viability of H1299 cells, whereas GOP and G3OP did weaker anti-proliferation function under normoxia than hypoxia. The fusion proteins also had proliferation inhibition function in DU145 cells, T47D cells, MDA-MB-231 cell and SW480 cells. p53 fusion proteins had stronger cellular toxicity in tumor cells than the same molar concentration of GnRH analogues (Leuprorelin and G662) did. The results of flow cytometry showed that p53 fusion proteins could induce early apoptosis and G1 cell cycle arrest in H1299 cells. The fusion proteins fused with ODD domain exhibit mild apoptosis promoting action on the tumor cells under normoxia. The results of Western blot showed that the proteins in H1299 cells treated with fusion proteins could be well recognized by the mouse anti-p53 monoclonal antibody, rat anti-caspase-3 monoclonal antibody and mouse anti-p21 monoclonal antibody. This result of Western blot also indicated that the fusion proteins could increase the expression of caspase-3 proteins and p21 proteins in H1299 cells, which showed that the proliferation inhibition was related with cell apoptosis and cell cycle arrest induced by p53. Repeated intraperitoneal (i.p.) injection of fusion proteins at 5mg/kg/day resulted in the reduction in mass and volume of xenograft tumor, compared with saline buffer treatment control(GP, p<0.01; GOP, G3P, G3OP, p<0.05). The result of immunohistochemistry indicated that the fusion proteins could increase the expression of caspase-3, p21 and PUMA proteins in tumors. The plasma biochemical parameters of mice determined in this research were almost normal.
In conclusion, GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of cancer cells growth in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.
本课题旨在以p53为肿瘤治疗的效应分子,通过GnRH与其受体特异结合介导蛋白进入相应的肿瘤细胞,应用ODD减轻p53对正常组织的损伤,增强蛋白对实体肿瘤的靶向作用,构建pET28a-GnRH-P53(pET28a-GP)、pET28a-GnRH-ODD-P53(pET28a-GOP)、pET28a-GnRHⅢ-P53(pET28a-G3P)、pET28a-GnRHⅢ-ODD-P53(pET28a-G3OP)、pET28a-GnRH-ODD557-574aa-P53 (pET28a-GSOP)、pET28a-GnRHⅢ-ODD557-574aa-P53(pET28a-G3SOP)原核表达载体,转化大肠杆菌BL21,诱导重组质粒表达相应的融合蛋白,将表达后菌体超声处理后离心,收集包涵体后进行变性、复性、纯化,从而得到具有生物学功能的融合蛋白。在细胞和动物水平研究融合蛋白的抗肿瘤作用。具体方法为:应用MTT法测定融合蛋白对肿瘤细胞生长的抑制作用;细胞免疫化学法、细胞免疫荧光法测定融合蛋白在细胞中的定位;应用流式细胞术测定融合蛋白诱导肿瘤细胞凋亡及其诱发肿瘤细胞周期阻滞;Western blot检测融合蛋白抗肿瘤的可能机制。以H1299细胞建立BALB/c裸鼠荷瘤模型,将实验动物随机分为C (正常对照组),TC (肿瘤对照组),GP(GnRH-p53),GOP(GnRH-ODD-p53),G3P (GnRHⅢ-p53),G3OP(GnRHⅢ-ODD- p53)六组,以5mg/kg/天的剂量腹腔给药(对照组动物给予等量的生理盐水),测定融合蛋白对模型鼠的抗肿瘤作用,同时对模型鼠血浆生化指标进行测定初步判断融合蛋白对动物的毒副作用。
本课题的实验结果:①构建了pET28a-GnRH-P53系列重组质粒载体,将其转入大肠杆菌BL21后成功表达了相应的融合蛋白。②经细胞免疫荧光法、细胞免疫化学法、Western blot检测表明融合蛋白可以进入GnRH-R表达阳性的H1299细胞、DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞。③经MTT法测定表明GP、GSOP、G3P、G3SOP在常氧和缺氧条件下均可以抑制H1299细胞的生长,并且这种作用具有剂量依赖性,即随着蛋白浓度的增加其抑制H1299细胞生长作用增强;GOP和G3OP在缺氧条件下对H1299细胞的生长抑制作用比其在常氧条件下的作用明显;融合蛋白对DU145细胞、T47D细胞、MDA-MB-231细胞以及SW480细胞也具有增殖抑制作用;融合蛋白对H1299细胞的增殖抑制作用强于相同剂量的GnRH类似物亮丙瑞林和G662。④流式细胞术测定融合蛋白诱导肿瘤细胞凋亡表明,融合蛋白在常氧状态下可以增加H1299细胞早期凋亡的百分率,其作用在48h最强,而与PBS对照相比,G3OP在给药24h时可显著诱导H1299细胞发生早期凋亡;在缺氧状态下,给药24h,与RPMI1640空白培养基对照相比较,各融合蛋白均可显著诱导H1299细胞发生早期凋亡; GOP和G3OP在缺氧状态下的作用比其在常氧状态下诱导细胞凋亡作用明显增强。⑤在常氧和缺氧状态下,各融合蛋白可以诱导H1299细胞G0/G1期细胞比例增加,而处于S期细胞的百分数减少,DNA合成受到抑制,即发生细胞G1期阻滞。⑥经Western blot检测,融合蛋白可以上调H1299细胞中的Caspase-3蛋白、p21蛋白表达水平,推测融合蛋白抑制H1299细胞生长的机制与活化Caspase-3蛋白诱导细胞凋亡及上调p21蛋白表达促进细胞发生G1期阻滞相关。⑦测量、计算各组实验动物瘤体体积发现,与TC组比较GP抑制瘤体生长作用十分显著(p<0.01),GOP、G3P、G3OP抑瘤作用较明显(p<0.05)。⑧免疫组化方法检测荷瘤模型小鼠肝脏和肿瘤切片,发现p53蛋白主要分布于肿瘤组织中,而且给药组动物肿瘤组织切片中Caspase-3蛋白、p21蛋白、PUMA蛋白的含量较对照组高,表明融合蛋白对荷瘤模型小鼠的抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。⑨动物血浆生化指标测定结果表明,融合蛋白对动物的肝、肾功能几乎没有影响。
由以上结果得出如下结论:在细胞和动物整体水平上,融合蛋白GP、GOP、G3P、G3OP具有抑制肿瘤生长作用,其抗肿瘤作用主要是通过诱导肿瘤细胞凋亡和发生细胞周期阻滞而实现的。GP等融合蛋白有望开发成为新型的抗肿瘤药物。
As a 10-amino acid regulator of reproduction, gonadotrophin-releasing hormone (GnRH) is released by neurons in the hypothalamus, and transported via the hypothalamo-hypophyseal portal circulation to the anterior pituitary to trigger gonadotropin release for luteinizing hormone and follicle-stimulating hormone, which in turn stimulate the gonads for steroid production. In the past, GnRH receptor (GnRH-R) was found to be expressed in normal human reproductive tissues (e.g. breast, ovary, endometrial, prostate, pancreas, colon, lung and liver) and tumors derived from these tissues. Numerous studies have provided evidence for the suppress effect of GnRH and GnRH-a (GnRH analogue) in these tumor cells proliferation. The multiple actions of GnRH could be linked to the divergence of signaling pathways that are activated by GnRH-R. The oxygen-dependent degradation domain (ODD) of HIF-1αis a key on protein stability. In normal oxygen level, ODD can accelerate the degration of HIF-1αby the hydroxylation of its proline. p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain a mutation or deletion of p53. Therefore p53 has been an appealing target for new anticancer therapeutic strategies.
The aim of this project is to investigate the targeted tumor inhibition induced by p53 fusion proteins against tumors with p53 gene mutation or deficient. GnRH, as the ligand of GnRH-R, was used to deliver p53 into tumor cells. ODD was used to control the stability of proteins in tissue under different oxygen stress. GnRH, GnRHⅢ, ODD and p53 gene was constructed and cloned into the pET28a vector. The p53 fusion proteins were expressed and their targeted anti-tumor effects were determined. The viability of tumor cells treated with each protein was measured by the MTT method. Immunocytochemistry analysis and immunofluorescence analysis were also used to detect the location of p53 fusion proteins in tumor cells. The flow cytometry was employed to analyze the apoptosis and cell cycle arrest in the proteins treated H1299 cells. The possible mechanism of proliferation inhibition induced by p53 fusion proteins was measured by Western blot. The mice with xenograft tumor of H1299 cells were used to test the tumor proliferation inhibition induced by fusion proteins (GP, GOP, G3P and G3OP) in vivo, while saline buffer was used as control. The plasma biochemical parameters of mice were also determined.
A series of novel fusion proteins, GnRH-p53(GP), GnRH-ODD-p53(GOP),GnRHⅢ-p53 (G3P),GnRHⅢ-ODD-p53 (G3OP), GnRH-ODD557-574aa-p53(GSOP), GnRHⅢ-ODD557-574aa-p53(G3SOP), were expressed in E coli. BL21 (DE3). As the control protein, wild type p53 (p53), was also constructed. These proteins were used to investigate the targeted tumor inhibition induced by p53 fusion proteins. Immunocytochemistry analysis and immunofluorescence analysis showed that all of fusion proteins could permeate into the H1299 cells and located in the cytoplasm and nucleus. Cellular experiments demonstrated that GP, GSOP, G3P and G3SOP could obviously decrease the viability of H1299 cells, whereas GOP and G3OP did weaker anti-proliferation function under normoxia than hypoxia. The fusion proteins also had proliferation inhibition function in DU145 cells, T47D cells, MDA-MB-231 cell and SW480 cells. p53 fusion proteins had stronger cellular toxicity in tumor cells than the same molar concentration of GnRH analogues (Leuprorelin and G662) did. The results of flow cytometry showed that p53 fusion proteins could induce early apoptosis and G1 cell cycle arrest in H1299 cells. The fusion proteins fused with ODD domain exhibit mild apoptosis promoting action on the tumor cells under normoxia. The results of Western blot showed that the proteins in H1299 cells treated with fusion proteins could be well recognized by the mouse anti-p53 monoclonal antibody, rat anti-caspase-3 monoclonal antibody and mouse anti-p21 monoclonal antibody. This result of Western blot also indicated that the fusion proteins could increase the expression of caspase-3 proteins and p21 proteins in H1299 cells, which showed that the proliferation inhibition was related with cell apoptosis and cell cycle arrest induced by p53. Repeated intraperitoneal (i.p.) injection of fusion proteins at 5mg/kg/day resulted in the reduction in mass and volume of xenograft tumor, compared with saline buffer treatment control(GP, p<0.01; GOP, G3P, G3OP, p<0.05). The result of immunohistochemistry indicated that the fusion proteins could increase the expression of caspase-3, p21 and PUMA proteins in tumors. The plasma biochemical parameters of mice determined in this research were almost normal.
In conclusion, GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of cancer cells growth in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.
引文
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[10] Mangia A,Tommasi S,Reshkin SJ, et a1. Gonadotropin releasing hormone receptor in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses [J].Oncol Rep,2002, 9(5): 1127-1132.
[11] Nakatani T, Roy G, Fujimoto N, et al. Sex hormone dependency of diethylnitrosamine-induced liver tumors in mice and chemoprevention by leuprorelin [J]. Jpn J Cancer Res, 2000, 92 (3):249-256.
[12] Friess H, Buchler M,Kiesel I, et al. LH-RH receptors in the human pancreas. Basis for anti-hormonal treatment in ductal carcinoma of the pancreas [J]. Int J Pancreatol, 1991, 10(2): 151-159.
[13] Nagy A, Schally AV. Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers [J]. Biol Reprod, 2005, 73(5):851-859.
[14] Roberta M, moretti MM, Marina M, et al. Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on Melanoma cells [J]. J Clin Endocrinol Metab, 2002, 87(8): 3791-3797.
[15] Nechushtan Amotz, Yarkoni S, Marianovsky I, et al. Adenocacinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites [J]. J Biological Chemistry, 1997, 272(17): 11597-11603.
[16] Saad M, Garbuzenko OB, Ber E, et al. Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumor-specific treatment and imaging [J]? J Control Release, 2008, 130(2):107-14.
[17] Lane DP, Crawford LV. T antigene is bound to a host protein in SV40 transformation cells. Nature, 1979, 278:261-263.
[18] Haupt S, Berger M, Goldberg Z, et al. Apoptosis-the p53 network. J Cell Sci, 2003, 116:4077-4085.
[19] Adams JM and Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene,2007, 26:1324–1337.
[20] Lu X. p53: a heavily dictator of life and death. Cur Opin Genet Dev, 2005, 15:27-33.
[21] Beroud C, Soussi, T. The UMD-p53 database: new mutations and analysis tools. Hum Mutat,2003, 21:176-181.
[22] Lee JW, Bae SH, Jeong JW,et al. Hypoxia-inducible factor (HIF-1): its protein stability and biological functions. Experimental Mol Med, 2004, 36:1-12.
[23] Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF.Science, 2001,294: 1337-1340.
[24] Harada H, Hiraoka M, Kizaka-Kondoh1 S. Antitumor effect of tat-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells. Cancer Res, 2002, 62: 2013–2018.
[25] Sapan CV, Lundblad RL, Price NC. Colorimetric protein assay techniques. Biotechnol Appl Biochem, 1999,29 ( Pt 2):99-108.
[26] Sung C, Nardeli B,LaFleur DW, et al. An IFN-beta-albumin fusion protein that displays improved pharmacokenetic and pharmacodynamic properties in nonhuman primates. J Interferon Cytokine Res, 2003, 23(1):25-36.
[27] OlivierM, Eeles R, Hollstein M, et al. The IARC TP53 database: new online mutation analysis and recommendations to users.Hum M utat, 2002, 19 (6): 607 - 614.
[28] Hupp TR, SparksA, Lane DP. Small peptides activatethe latent sequence-specific DNA binding function of p53. Cell, 1995, 83 (2): 237 - 245.
[29] Bennett M, Macdonald K, Chan SW, et al. Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science, 1998, 282 (5387): 290-293.
[30] Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol JT - Current opinion in biotechnology, 1999, 10(5):411-21.
[31] Clark ED. Protein refolding for industrial processes. Curr Opin Biotechnol JT - Current opinion in biotechnology, 2001, 12(2):202-7.
[32] Gribskov M, Burgess RR. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene JT - Gene, 1983, 26(2-3):109-18.
[33] Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev JT - Microbiological reviews, 1996, 60(3):512-38.
[34] Clark EDB. Refolding of recombinant proteins. Curr Opin Biotechnol, 1998, 9(2):157-63.
[35] Hamaker KH, Liu J, Seely RJ, Ladisch CM, Ladisch MR. Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor. Biotechnol Prog, 1996, 12(2):184-9.
[36] Rudolph R, Lilie H. In vitro folding of inclusion body proteins. FASEB J, 1996, 10(1):49-56.
[37] Gho YS, Chae CB. Luteinizing hormone releasing hormone-RNase A conjugates specifically inhibit the proliferation of LHRH-receptor-positive human prostate and breast tumor cells [J].Mol Cells, 1999, 9(1):31-6.
[38] Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein [J]. Cell, 1988,55(6):1179-88.
[39] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse [J]. Science, 1999, 285(5433):1569-72.
[40] Ryu J, Lee HJ, Kim KA, et al. Intracellular delivery of p53 fused to the basic domain of HIV-1 Tat [J]. Mol Cells, 2004, 17(2):353-9.
[41] Yang Y, Ma J, Song Z, Wu M. HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors [J]. FEBS Lett, 2002, 532(1-2):36-44.
[42] Millar RP. GnRH II and type II GnRH receptors [J].Trends Endocrinol Metabo1, 2003(1), 14:35-43.
[43] Oh DY, Song JA. Membrane-Proximal region of the carboxyl terminus of the gonadotropin- releasing hormone receptor (GnRHR) confers differential signal transduction between mammalian and nonmammalian GnRHRs [J]. Mol Endocrinol, 2005, 19(3):722-731.
[44] Hazum E, Koch Y, Liscovitch M, Amsterdam A. Intracellular pathways of receptor-bound GnRH agonist in pituitary gonadotropes. Cell Tissue Res, 1985, 239(1):3-8.
[45] Steven J. Kuerbitz, Beverly S, et al. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Cell Biology. Proc. Natl. Acad. Sci. USA. 1992, 89:7491-7495.
[46] LaFevre-Bernt M, Wu SL and Lin XL. Recombinant, refolded tetrameric p53 and gonadotropin releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells. Mol Cancer Ther, 2008, 7(6):1420-1429.
[47] Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, Green DR.PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science,2005, 309(5741):1732-5.
[48] Wen-Shu Wu, Stefan Heinrichs, Dong Xu, Sean P. Garrison, Gerard P. Zambetti, Jerry M. Adams, A. Thomas Look.Slug Antagonizes p53-Mediated Apoptosis of Hematopoietic Progenitors by Repressing puma.18 November 2005, Cell,123(4) :641 - 653.
[49] Vousden KH. Apoptosis. p53 and PUMA: a deadly duo. Science, 2005, 309(5741):1685-1686.
1. Neill JD. GnRH and GnRH receptor genes in the human genome [J]. Endocrinology, 2002, 143(3):737-743.
2. Hiney JK, Sower SA, Yu WH, et al. Gonadotropin-releasing hormone neurons in the preoptichypothalamic region of the rat contain lamprey gonadotropin-releasing hormone-Ⅲ, mammalian luteinizing hormone-releasing hormone, or both peptides [J]. Proc Natl Acad Sci USA, 2001, 99(4):2386-2391.
3. Millar RP. GnRH II and type II GnRH receptors [J].Trends Endocrinol Metabo1, 2003(1), 14:35-43.
4. Naor ZF. Signaling by G-protein-coupled receptor (GPCR): Studies on the GnRH receptor [J]. Neuroendocrinol, 2009, 30(1):10-29.
5. Cheng CK. Molecular biology of gonadotropin-releasing hormone (GnRH)-1, GnRH-Ⅱ, and their receptors in humans [J]. Endocr Rev, 2005, 26(2):283-306.
6. Pawson AJ, Faccenda E, Maudsley S, et al. Mammalian type I gonadotropin-releasing hormone receptors undergo slow, constitutive, agonist-independent internalization [J]. Endocrinology, 2008, 149(3):1415-1422.
7. Silver MR, Nucci NV, Root AR, et al. Cloning and characterization of a functional type II gonadotropinreleasing hormone receptor with a lengthy carboxy-terminal tail from an ancestral vertebrate, the sea lamprey [J]. Endocrinology, 2005, 146(8):3351-3361.
8. Silver MR, Kawauchi H, Nozaki M, et al. Cloning and analysis of the lamprey GnRH-ⅢcDNA from eight species of lamprey representing the three families of Petromyzoniformes.General and Comparative [J]. Endocrinology, 2004, 139(1): 85-94.
9. Grundlker C, Gunthert AR, Millar RP, et al. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation [J]. J Clin Endocrinol Metab, 2002, 87(3): l427-l430.
10. Mangia A,Tommasi S,Reshkin SJ,et a1. Gonadotropin releasing hormone receptor in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses [J]. Oncol Rep, 2002, 9(5): 1127-1132.
11. Emons G, Gründker C, Günthert AR, et al. GnRH antagonists in the treatment of gynecological and breast cancers [J]. Endocr Relat Cancer, 2003(2), 10:291-299.
12. Franklin J, Hislop J, Flynn A, et al. Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus [J]. J Endocrinol, 2003, 176(2):275-84.
13. Ravenna I, Salvatori I, Morrone S, et al. Effects of triptorelin,a gonadotropin-releasing hormone agonist,on the human prostatic cell lines PC3 and lncap[J] . J Androl, 2000, 21(4): 549-557.
14. Montagnani Marelli M, Moretti RM, Mai S, et al. Type I Gonadotropin-Releasing Hormone Receptor (GnRH-R) Mediates the Antiproliferative Effects of GnRH-II on Prostate Cancer Cells[J]. J Clin Endocrinol Metab, 2009 Feb 3.
15. Nakatani T, Roy G, Fujimoto N, et al. Sex hormone dependency of diethylnitrosamine-induced liver tumors in mice and chemoprevention by leuprorelin [J]. Jpn J Cancer Res, 2000, 92 (3):249-256.
16. Friess H, Buchler M,Kiesel I, et al. LH-RH receptors in the human pancreas.Basis for anti-hormonal treatment in ductal carcinoma of the pancreas [J]. Int J Pancreatol, 1991, 10(2): 151-159.
17. Nagy A, Schally A.V. Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers [J]. Biol Reprod, 2005, 73(5):851-859.
18. Hered?i-Szabo ?K, Murphy RF and Lovas S. Different signal responses to lamprey GnRH-Ⅲin human cancer cells [J]. Intl J Pept Res Thera, 2006, 12(4): 359-364.
19. Roberta M, moretti MM, Marina M, et al. Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on Melanoma cells [J]. J Clin Endocrinol Metab, 2002, 87(8): 3791-3797.
20. Jakesz R. LHRH agonists [J]. European Jouran1 of Cancer, 2002, 38 (SuppB):S44.
21. Neill JD, Musgrove LC, Duck LW. Newly recognized GnRH receptors: function and relative role [J]. Trends Endocrinol Metab, 2004, 15(8): 383-392.
22. Yano T, Pinski J, Halmos G, et al. Inhibition of growth of OV-1063 human epithelial ovarian cancer xenografts in nude mice by treatm ent with luteinizing hormone releasing hormone antagonist SB-75 [J]. PNAS, l994, 91(15): 7090—7094.
23. Fister S, Günthert AR, Emons G, et al. Gonadotropin-releasing hormone type II antagonists induce apoptotic cell death in human endometrial and ovarian cancer cells in vitro and in vivo [J]. Cancer Res, 2007, 67(4):1750-1756.
24. Dondi D, Limonta P, Moretti RM, et al. Antiproliferative effects of luteinizing hormone-releasing hormone (LHRH) agonists on human androgen-independent prostate cancer cell line DU145: evidence for an autocrine-inhibitory LHRH loop [J]. Cancer Res, 1994, 54(15):4091-4095.
25. Koppán M, Nagy A, Schally AV, et al. Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits the growth of PC-82 human prostate cancer in nude mice [J]. Prostate, 1999, 38(2):151-8.
26. Limonta P, Moretti RM, Marelli MM, et al. The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway [J]. Endocrinology, 1999; 140(11):5250-6.
27. Teplan I. Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity [J]. Acta Biol Hung, 2000, 51 (1):1 - 29.
28. Günthert AR, Gründker C, Olota A, et al. Analogs of GnRH-I and GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen [J]. Eur J Endocrinol, 2005, 153(4):613-25.
29. Mez? G, Czajlik A, Manea M, et al. Structure, enzymatic stability and antitumor activity of sea lamprey GnRH-Ⅲand its dimer derivatives [J]. Peptides, 2007, (4):806-20.
30. Magdolna K, Borba′la V, Judit E, et al. Structure-activity study on the LH-and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-Ⅲanalogs [J]. Peptides, 2007, 28(4): 821– 829.
31. Szepeshazi K, Halmos G, Schally AV, et al. Growth inhibition of experimental pancreatic cancers and sustainedreduction in epidermal growth factor receptors during therapy with hormonal peptide analogs[J ]. J Cancer Res Clin Oncol, 1999, 125 (8 - 9): 444-452.
32. Szepeshazi K, Schally AV, Halmos G.LH-RH receptors in human colorectal cancers: unexpected molecular targets for experimental therapy [J]. Int J Oncol, 2007, 30(6):1485-1492.
33. Gho YS, Chae CB. Luteinizing hormone releasing hormone-RNase A conjugates specifically inhibit the proliferation of LHRH-receptor-positive human prostate and breast tumor cells [J]. Mol Cells, 1999, 9(1):31-6.
34. Nechushtan A , Yarkoni S, Marianovsky I, et al. Adenocacinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites [J]. J Biological Chemistry, 1997, 272(17): 11597-11603.
35. Saad M, Garbuzenko OB, Ber E, et al. Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumor-specific treatment and imaging [J]? J Control Release, 2008, 130(2):107-14.
36. Oh DY, Song JA. Membrane-Proximal region of the carboxyl terminus of the gonadotropin- releasing hormone receptor (GnRHR) confers differential signal transduction between mammalian and nonmammalian GnRHRs [J]. Mol Endocrinol, 2005, 19(3):722-731.
[2] Neill JD. GnRH and GnRH receptor genes in the human genome [J]. Endocrinology, 2002, 143(3):737-743.
[3] Hiney JK, Sower SA, Yu WH, et al. Gonadotropin-releasing hormone neurons in the preoptichypothalamic region of the rat contain lamprey gonadotropin-releasing hormone-Ⅲ, mammalian luteinizing hormone-releasing hormone, or both peptides [J]. Proc Natl Acad Sci USA, 2001, 99(4):2386-2391.
[4] Naor ZF. Signaling by G-protein-coupled receptor (GPCR): Studies on the GnRH receptor [J]. Neuroendocrinol, 2009, 30(1):10-29.
[5] Cheng CK. Molecular biology of gonadotropin-releasing hormone (GnRH)-1, GnRH-Ⅱ, and their receptors in humans [J]. Endocr Rev, 2005, 26(2):283-306.
[6] Pawson AJ, Faccenda E, Maudsley S, et al. Mammalian type I gonadotropin-releasing hormone receptors undergo slow, constitutive, agonist-independent internalization [J]. Endocrinology, 2008, 149(3):1415-1422.
[7] Silver MR, Nucci NV, Root AR, et al. Cloning and characterization of a functional type II gonadotropinreleasing hormone receptor with a lengthy carboxy-terminal tail from an ancestral vertebrate, the sea lamprey [J]. Endocrinology, 2005, 146(8):3351-3361.
[8] Kass SS, Grizzle W E, Neill JD. The nucleotide sequences of human GnRH receptors in breast and ovarin tumors are identical with that found in pituitary. Mol Cell Endocrenol, 1994, 106:l45-l49.
[9] Grundlker C, Gunthert AR, Millar RP, et al. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation [J]. J Clin Endocrinol Metab, 2002, 87(3): l427-l430.
[10] Mangia A,Tommasi S,Reshkin SJ, et a1. Gonadotropin releasing hormone receptor in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses [J].Oncol Rep,2002, 9(5): 1127-1132.
[11] Nakatani T, Roy G, Fujimoto N, et al. Sex hormone dependency of diethylnitrosamine-induced liver tumors in mice and chemoprevention by leuprorelin [J]. Jpn J Cancer Res, 2000, 92 (3):249-256.
[12] Friess H, Buchler M,Kiesel I, et al. LH-RH receptors in the human pancreas. Basis for anti-hormonal treatment in ductal carcinoma of the pancreas [J]. Int J Pancreatol, 1991, 10(2): 151-159.
[13] Nagy A, Schally AV. Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers [J]. Biol Reprod, 2005, 73(5):851-859.
[14] Roberta M, moretti MM, Marina M, et al. Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on Melanoma cells [J]. J Clin Endocrinol Metab, 2002, 87(8): 3791-3797.
[15] Nechushtan Amotz, Yarkoni S, Marianovsky I, et al. Adenocacinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites [J]. J Biological Chemistry, 1997, 272(17): 11597-11603.
[16] Saad M, Garbuzenko OB, Ber E, et al. Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumor-specific treatment and imaging [J]? J Control Release, 2008, 130(2):107-14.
[17] Lane DP, Crawford LV. T antigene is bound to a host protein in SV40 transformation cells. Nature, 1979, 278:261-263.
[18] Haupt S, Berger M, Goldberg Z, et al. Apoptosis-the p53 network. J Cell Sci, 2003, 116:4077-4085.
[19] Adams JM and Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene,2007, 26:1324–1337.
[20] Lu X. p53: a heavily dictator of life and death. Cur Opin Genet Dev, 2005, 15:27-33.
[21] Beroud C, Soussi, T. The UMD-p53 database: new mutations and analysis tools. Hum Mutat,2003, 21:176-181.
[22] Lee JW, Bae SH, Jeong JW,et al. Hypoxia-inducible factor (HIF-1): its protein stability and biological functions. Experimental Mol Med, 2004, 36:1-12.
[23] Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF.Science, 2001,294: 1337-1340.
[24] Harada H, Hiraoka M, Kizaka-Kondoh1 S. Antitumor effect of tat-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells. Cancer Res, 2002, 62: 2013–2018.
[25] Sapan CV, Lundblad RL, Price NC. Colorimetric protein assay techniques. Biotechnol Appl Biochem, 1999,29 ( Pt 2):99-108.
[26] Sung C, Nardeli B,LaFleur DW, et al. An IFN-beta-albumin fusion protein that displays improved pharmacokenetic and pharmacodynamic properties in nonhuman primates. J Interferon Cytokine Res, 2003, 23(1):25-36.
[27] OlivierM, Eeles R, Hollstein M, et al. The IARC TP53 database: new online mutation analysis and recommendations to users.Hum M utat, 2002, 19 (6): 607 - 614.
[28] Hupp TR, SparksA, Lane DP. Small peptides activatethe latent sequence-specific DNA binding function of p53. Cell, 1995, 83 (2): 237 - 245.
[29] Bennett M, Macdonald K, Chan SW, et al. Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Science, 1998, 282 (5387): 290-293.
[30] Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol JT - Current opinion in biotechnology, 1999, 10(5):411-21.
[31] Clark ED. Protein refolding for industrial processes. Curr Opin Biotechnol JT - Current opinion in biotechnology, 2001, 12(2):202-7.
[32] Gribskov M, Burgess RR. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene JT - Gene, 1983, 26(2-3):109-18.
[33] Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev JT - Microbiological reviews, 1996, 60(3):512-38.
[34] Clark EDB. Refolding of recombinant proteins. Curr Opin Biotechnol, 1998, 9(2):157-63.
[35] Hamaker KH, Liu J, Seely RJ, Ladisch CM, Ladisch MR. Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor. Biotechnol Prog, 1996, 12(2):184-9.
[36] Rudolph R, Lilie H. In vitro folding of inclusion body proteins. FASEB J, 1996, 10(1):49-56.
[37] Gho YS, Chae CB. Luteinizing hormone releasing hormone-RNase A conjugates specifically inhibit the proliferation of LHRH-receptor-positive human prostate and breast tumor cells [J].Mol Cells, 1999, 9(1):31-6.
[38] Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein [J]. Cell, 1988,55(6):1179-88.
[39] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse [J]. Science, 1999, 285(5433):1569-72.
[40] Ryu J, Lee HJ, Kim KA, et al. Intracellular delivery of p53 fused to the basic domain of HIV-1 Tat [J]. Mol Cells, 2004, 17(2):353-9.
[41] Yang Y, Ma J, Song Z, Wu M. HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors [J]. FEBS Lett, 2002, 532(1-2):36-44.
[42] Millar RP. GnRH II and type II GnRH receptors [J].Trends Endocrinol Metabo1, 2003(1), 14:35-43.
[43] Oh DY, Song JA. Membrane-Proximal region of the carboxyl terminus of the gonadotropin- releasing hormone receptor (GnRHR) confers differential signal transduction between mammalian and nonmammalian GnRHRs [J]. Mol Endocrinol, 2005, 19(3):722-731.
[44] Hazum E, Koch Y, Liscovitch M, Amsterdam A. Intracellular pathways of receptor-bound GnRH agonist in pituitary gonadotropes. Cell Tissue Res, 1985, 239(1):3-8.
[45] Steven J. Kuerbitz, Beverly S, et al. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Cell Biology. Proc. Natl. Acad. Sci. USA. 1992, 89:7491-7495.
[46] LaFevre-Bernt M, Wu SL and Lin XL. Recombinant, refolded tetrameric p53 and gonadotropin releasing hormone-p53 slow proliferation and induce apoptosis in p53-deficient cancer cells. Mol Cancer Ther, 2008, 7(6):1420-1429.
[47] Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, Green DR.PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science,2005, 309(5741):1732-5.
[48] Wen-Shu Wu, Stefan Heinrichs, Dong Xu, Sean P. Garrison, Gerard P. Zambetti, Jerry M. Adams, A. Thomas Look.Slug Antagonizes p53-Mediated Apoptosis of Hematopoietic Progenitors by Repressing puma.18 November 2005, Cell,123(4) :641 - 653.
[49] Vousden KH. Apoptosis. p53 and PUMA: a deadly duo. Science, 2005, 309(5741):1685-1686.
1. Neill JD. GnRH and GnRH receptor genes in the human genome [J]. Endocrinology, 2002, 143(3):737-743.
2. Hiney JK, Sower SA, Yu WH, et al. Gonadotropin-releasing hormone neurons in the preoptichypothalamic region of the rat contain lamprey gonadotropin-releasing hormone-Ⅲ, mammalian luteinizing hormone-releasing hormone, or both peptides [J]. Proc Natl Acad Sci USA, 2001, 99(4):2386-2391.
3. Millar RP. GnRH II and type II GnRH receptors [J].Trends Endocrinol Metabo1, 2003(1), 14:35-43.
4. Naor ZF. Signaling by G-protein-coupled receptor (GPCR): Studies on the GnRH receptor [J]. Neuroendocrinol, 2009, 30(1):10-29.
5. Cheng CK. Molecular biology of gonadotropin-releasing hormone (GnRH)-1, GnRH-Ⅱ, and their receptors in humans [J]. Endocr Rev, 2005, 26(2):283-306.
6. Pawson AJ, Faccenda E, Maudsley S, et al. Mammalian type I gonadotropin-releasing hormone receptors undergo slow, constitutive, agonist-independent internalization [J]. Endocrinology, 2008, 149(3):1415-1422.
7. Silver MR, Nucci NV, Root AR, et al. Cloning and characterization of a functional type II gonadotropinreleasing hormone receptor with a lengthy carboxy-terminal tail from an ancestral vertebrate, the sea lamprey [J]. Endocrinology, 2005, 146(8):3351-3361.
8. Silver MR, Kawauchi H, Nozaki M, et al. Cloning and analysis of the lamprey GnRH-ⅢcDNA from eight species of lamprey representing the three families of Petromyzoniformes.General and Comparative [J]. Endocrinology, 2004, 139(1): 85-94.
9. Grundlker C, Gunthert AR, Millar RP, et al. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation [J]. J Clin Endocrinol Metab, 2002, 87(3): l427-l430.
10. Mangia A,Tommasi S,Reshkin SJ,et a1. Gonadotropin releasing hormone receptor in primary breast cancer: comparison of immunohistochemical, radioligand and Western blot analyses [J]. Oncol Rep, 2002, 9(5): 1127-1132.
11. Emons G, Gründker C, Günthert AR, et al. GnRH antagonists in the treatment of gynecological and breast cancers [J]. Endocr Relat Cancer, 2003(2), 10:291-299.
12. Franklin J, Hislop J, Flynn A, et al. Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus [J]. J Endocrinol, 2003, 176(2):275-84.
13. Ravenna I, Salvatori I, Morrone S, et al. Effects of triptorelin,a gonadotropin-releasing hormone agonist,on the human prostatic cell lines PC3 and lncap[J] . J Androl, 2000, 21(4): 549-557.
14. Montagnani Marelli M, Moretti RM, Mai S, et al. Type I Gonadotropin-Releasing Hormone Receptor (GnRH-R) Mediates the Antiproliferative Effects of GnRH-II on Prostate Cancer Cells[J]. J Clin Endocrinol Metab, 2009 Feb 3.
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