前列腺癌细胞系LNCaP雄激素依赖性转化相关研究
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摘要
大多数前列腺癌开始治疗时为雄激素依赖型前列腺癌,并对抗雄激素治疗敏感,治疗后病情迅速好转,血清前列腺特异性抗原降低,但是,在临床上,经连续性药物抗雄激素治疗(平均12—18个月,最短3—6个月)后,前列腺癌细胞将失去对抗雄激素药物的敏感性,雄激素依赖型前列腺癌将逐渐转化为对抗雄激素药物耐受的雄激素非依赖型前列腺癌(androgen independeent prostate carcinoma,AIPC),甚至使抗雄激素药物转化为癌细胞的激动剂,并发生远处转移,成为抗雄激素治疗无效的中晚期前列腺癌,并导致患者最终死亡。
目的:
在本试验中,我们分别使用人工合成雄激素和雄激素受体拮抗剂氟他胺作用于前列腺癌雄激素反应性细胞系LNCaP,以研究雄激素和雄激素受体拮抗剂对前列腺癌细胞的确切作用及相关作用机理;并通过长期体外培养前列腺癌细胞系,建立了氟他胺耐受性前列腺癌细胞系LNCaP的亚系,LNCaP-flu。而后使用基因芯片技术,研究了在此过程中基因表达的变化,并以半定量逆转录聚合酶链反应技术验证力量基因芯片得出的结果。从而明确了雄激素和氟他胺对前列腺癌的具体作用及相关分子表达变化,并建立了氟他胺耐受性前列腺癌细胞系及氟他胺耐受细胞系的动物模型,明确了其药物耐受性的发生机理。从而为今后的氟他胺耐受性研究奠定了理论基础。
方法:
1.人工合成雄激素R1881对前列腺癌细胞的作用及其相关机理:
a) 应用MTT法研究R1881对LNCaP细胞的作用,并绘制生长曲线和细胞抑制率曲线。
b) 应用流式细胞仪研究在不同浓度R1881作用下细胞周期变化。
Although most prostate cancer began with androgen dependent phenotype, and they were sensitive to endocrinal therapy at first, they transit to androgen independent phenotype after 12-18 months of continuous anti-androgen treatment, which is insensitive to endocrinal therapy of any kind. Then the disease progresses, metastases accur, and patients die soon. Aim:In our research, we treated LNCaP cell with artificial androgen R1881 and androgen receptor antagonist flutamide respecitively to study their exact effect on prostate cancer cells and their correlated molecular mechanism. After continuous in vitro culturing, we succeeded in establishing a flutamide insensitive subline of LNCaP, LNCaP-flu. Then we studied the molecular changes of LNCaP-flu with cDNA microarray and reverse transcriptase-polymerase chain reaction. Thus clarified the exact effect of androgen and flutamide on prostate cancer cells, the molecular changes of flutamide insensitivity, and established flutamide insensitive prostate cancer cell line and animal model. Method: 1. Effect of artificial androgen R1881 on LNCaP cells and its correslatedmolecular mechanism:a) Studied the effect of R1881 on LNCaP with MTT assay, and drew
growth curve and inhibition rate curve.b) Studied cell cycle changes induced by R1881 of different concentrations with flowcytometer.c) Observe the morphological changes after R1881 treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of R1881's dual effect on LNCaP cells by gene chips, and confirmed the results by RT-PCR.2. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Studied the effect of flutamide on LNCaP with MTT assay, and drew growth curve and inhibition rate curve.b) Studied cell cycle changes induced by flutamide of different concentrations with flowcytometer.c) Observe the morphological changes after flutamide treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of flutamide's inhibitive effect on LNCaP cell by gene chips, and confirmed the results by RT-PCR.3. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-fiu, and study of its correlated molecular mechanism.a) Established a flutamide insensitive subline of LNCaP,LNCaP-flu, after continuous in vitro culture of LNCaP cells in presence of flutamide.b) Evaluated the expression of prostate specific antigen in the supernatant of cells with radio-immunity to clarify flutamide's effect on LNCaP cell PSA secretion.c) Studied the molecular mechanism of flutamide resistance of LNCaP-flu cell with gene chips, and confirmed the results by RT-PCR.d) Tried to find AR mutation by cloning and sequencing AR cDNA.4. Established LNCaP cell implant carcinoma in SCID mice with matrigel. And measured tumor volume and PSA expression.
Results:5. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) R1881 has a dual effect on LNCaP cell. 10~(-9)M and above could inhibit its proliferation, while 10~(-10)M and below could stimulate it. There is not noticeable morphological changes in this process.b) Microscope morphology: Flutamide exerted a prolific effect on LNCaP cells primarily. Then more than 15 days later, after around 5 generations, the effect reversed. The LNCaP cells were inhibited by flutamide. The cells turned thinner and lost satiety. R1881 doesn't have obvious effect on LNCaP cells morphologically.c) Molecular mechanisms of R1881's dual effect: cDNA microarray revealed that after LNCaP had been inhibited by 10~(-9)M R1881, there were 320 genes expressed differently. 170 were up-regulated, 150 were down-regulated. Among them, there are 2 genes may be classified as androgen receptor coactivator, viz. C4orfl and DDC. After LNCaP had been stimulated by 10~(-10)M R1881, there were 4608 genes expressed differently. 2046 were up-regulated, 2562 were down-regulated. Among them, 8 genes may be classified as androgen receptor coactivator, viz. FHL2, NCOR1, SVIL, GRIP1, PIAS3, ACTN2, TBLR1, NCOA2. RT-PCR further proved all these results.6. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Effect of flutamide on LNCaP cells: Flutamide may stimulate LNCaP cell proliferation at different concentrations. However, its effect could still not match with that of R1881's. After 5 generations of continuous in vitro culturing, its effect reversed. The inhibitive effect turns more obvious with the exposure time prolonging and the drug concentration rising. And a series of morphologic changes accompany all these.b) Molecular mechanisms of cell flutamide inhibition:cDNA microarray
revealed that after LNCaP had been inhibited by flutamide, there were 326 genes expressed differently. 97 were up-regulated, 219 were down-regulated. Among them, 8 genes may be correlated with cell cycle, viz. CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2. And RT-PCR confirmed the up-regulation of all these 8 genes.7. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a flutamide insensitive subline of LNCaP,LNCaP-flu, which could grow steadily in presence of 10~(-7)M flutamide.LNCaP-flu. And morphologic observation, cell growth test, PSA secretion, cell cycle assay and transwell in vitro invasive ability test proved that all cellular characters of LNCaP-flu are not susceptible to flutamide.b) Molecular mechanisms of flutamide insensitivity: cDNA microarray revealed that after LNCaP had been insensitive to flutamide, there were 2428 genes expressed differently. 1234 were up-regulated, 1194 were down-regulated. Among them, 5 genes may be correlated with androgen receptor function, viz. NCOR1,NCOA2,NCOA4 ,NC0A6 and DDC. And RT-PCR confirmed the up- and down-regulation of all these 5 genes.c) AR changes in flutamide resistance transition: In the process of flutamide resistance transition ,there is no AR expression changes. After cloning and sequencing , we found a new mutation of AR, ie. A3747G.8. Establishment of LNCaP cell Implant carcinoma in SCID mice: In the SCID mice, 75% (15/20) and 60% (12/20) of LNCaP and LNCaP-flu implants respecitively grew up to solid tumor at last. Immunohistochermstry revealed that there were androgen receptor expression in all the implant tumors, while there were not any PSA expression. There were no significant difference between LNCaP,
LNCaP-flu and LNCaP-flu/flu group cells in tumor weight and volume (P>0.05) . However, tumors from LNCaP/flu cells were much smaller (P<0.05) . Conclusion:1. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) The artificial androgen R1881 have a dual effect on LNCaP cell. 10~(-9)M and above could inhibit LNCaP cell proliferation, while 10~(-10)M and below could stimulate it.b) The down-regulation of androgen receptor coregulator DDC and C4orfl may play a role in the inhibition induced by 10~(-9)M R1881; while the down-regulation of ,FHL2,NCOR1,SVIL, GRIP1,PIAS3 and the up-regulation of ACTN2, TBLR1, NCOA2 may play a role in the prolific effect induced by 10~(-10)M R1881.2. Effect of artificial anti-androgen fiutamide on LNCaP cells and its correslated molecular mechanism:a) Fiutamide has dual effect on LNCaP cell. It could stimulate it at relative low concentration, or relative short time. But when the time prolonged or concentration rose, the effect was reversed.b) The inhibitive effect of fiutamide on LNCaP cells may be the up-regulation of CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2, which may affect downstream cell cycle related genes, and thus block the cell cycle at the G1/S checkpoint.3. Establishment of fiutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a fiutamide insensitive prostate cancer, LNCaP-flu.b) Androgen receptor related genes, such as NCOR1, NCOA2, NCOA4 , NCOA6 and DDC may be important in the transition of fiutamide sensitivity.
目的:
在本试验中,我们分别使用人工合成雄激素和雄激素受体拮抗剂氟他胺作用于前列腺癌雄激素反应性细胞系LNCaP,以研究雄激素和雄激素受体拮抗剂对前列腺癌细胞的确切作用及相关作用机理;并通过长期体外培养前列腺癌细胞系,建立了氟他胺耐受性前列腺癌细胞系LNCaP的亚系,LNCaP-flu。而后使用基因芯片技术,研究了在此过程中基因表达的变化,并以半定量逆转录聚合酶链反应技术验证力量基因芯片得出的结果。从而明确了雄激素和氟他胺对前列腺癌的具体作用及相关分子表达变化,并建立了氟他胺耐受性前列腺癌细胞系及氟他胺耐受细胞系的动物模型,明确了其药物耐受性的发生机理。从而为今后的氟他胺耐受性研究奠定了理论基础。
方法:
1.人工合成雄激素R1881对前列腺癌细胞的作用及其相关机理:
a) 应用MTT法研究R1881对LNCaP细胞的作用,并绘制生长曲线和细胞抑制率曲线。
b) 应用流式细胞仪研究在不同浓度R1881作用下细胞周期变化。
Although most prostate cancer began with androgen dependent phenotype, and they were sensitive to endocrinal therapy at first, they transit to androgen independent phenotype after 12-18 months of continuous anti-androgen treatment, which is insensitive to endocrinal therapy of any kind. Then the disease progresses, metastases accur, and patients die soon. Aim:In our research, we treated LNCaP cell with artificial androgen R1881 and androgen receptor antagonist flutamide respecitively to study their exact effect on prostate cancer cells and their correlated molecular mechanism. After continuous in vitro culturing, we succeeded in establishing a flutamide insensitive subline of LNCaP, LNCaP-flu. Then we studied the molecular changes of LNCaP-flu with cDNA microarray and reverse transcriptase-polymerase chain reaction. Thus clarified the exact effect of androgen and flutamide on prostate cancer cells, the molecular changes of flutamide insensitivity, and established flutamide insensitive prostate cancer cell line and animal model. Method: 1. Effect of artificial androgen R1881 on LNCaP cells and its correslatedmolecular mechanism:a) Studied the effect of R1881 on LNCaP with MTT assay, and drew
growth curve and inhibition rate curve.b) Studied cell cycle changes induced by R1881 of different concentrations with flowcytometer.c) Observe the morphological changes after R1881 treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of R1881's dual effect on LNCaP cells by gene chips, and confirmed the results by RT-PCR.2. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Studied the effect of flutamide on LNCaP with MTT assay, and drew growth curve and inhibition rate curve.b) Studied cell cycle changes induced by flutamide of different concentrations with flowcytometer.c) Observe the morphological changes after flutamide treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of flutamide's inhibitive effect on LNCaP cell by gene chips, and confirmed the results by RT-PCR.3. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-fiu, and study of its correlated molecular mechanism.a) Established a flutamide insensitive subline of LNCaP,LNCaP-flu, after continuous in vitro culture of LNCaP cells in presence of flutamide.b) Evaluated the expression of prostate specific antigen in the supernatant of cells with radio-immunity to clarify flutamide's effect on LNCaP cell PSA secretion.c) Studied the molecular mechanism of flutamide resistance of LNCaP-flu cell with gene chips, and confirmed the results by RT-PCR.d) Tried to find AR mutation by cloning and sequencing AR cDNA.4. Established LNCaP cell implant carcinoma in SCID mice with matrigel. And measured tumor volume and PSA expression.
Results:5. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) R1881 has a dual effect on LNCaP cell. 10~(-9)M and above could inhibit its proliferation, while 10~(-10)M and below could stimulate it. There is not noticeable morphological changes in this process.b) Microscope morphology: Flutamide exerted a prolific effect on LNCaP cells primarily. Then more than 15 days later, after around 5 generations, the effect reversed. The LNCaP cells were inhibited by flutamide. The cells turned thinner and lost satiety. R1881 doesn't have obvious effect on LNCaP cells morphologically.c) Molecular mechanisms of R1881's dual effect: cDNA microarray revealed that after LNCaP had been inhibited by 10~(-9)M R1881, there were 320 genes expressed differently. 170 were up-regulated, 150 were down-regulated. Among them, there are 2 genes may be classified as androgen receptor coactivator, viz. C4orfl and DDC. After LNCaP had been stimulated by 10~(-10)M R1881, there were 4608 genes expressed differently. 2046 were up-regulated, 2562 were down-regulated. Among them, 8 genes may be classified as androgen receptor coactivator, viz. FHL2, NCOR1, SVIL, GRIP1, PIAS3, ACTN2, TBLR1, NCOA2. RT-PCR further proved all these results.6. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Effect of flutamide on LNCaP cells: Flutamide may stimulate LNCaP cell proliferation at different concentrations. However, its effect could still not match with that of R1881's. After 5 generations of continuous in vitro culturing, its effect reversed. The inhibitive effect turns more obvious with the exposure time prolonging and the drug concentration rising. And a series of morphologic changes accompany all these.b) Molecular mechanisms of cell flutamide inhibition:cDNA microarray
revealed that after LNCaP had been inhibited by flutamide, there were 326 genes expressed differently. 97 were up-regulated, 219 were down-regulated. Among them, 8 genes may be correlated with cell cycle, viz. CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2. And RT-PCR confirmed the up-regulation of all these 8 genes.7. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a flutamide insensitive subline of LNCaP,LNCaP-flu, which could grow steadily in presence of 10~(-7)M flutamide.LNCaP-flu. And morphologic observation, cell growth test, PSA secretion, cell cycle assay and transwell in vitro invasive ability test proved that all cellular characters of LNCaP-flu are not susceptible to flutamide.b) Molecular mechanisms of flutamide insensitivity: cDNA microarray revealed that after LNCaP had been insensitive to flutamide, there were 2428 genes expressed differently. 1234 were up-regulated, 1194 were down-regulated. Among them, 5 genes may be correlated with androgen receptor function, viz. NCOR1,NCOA2,NCOA4 ,NC0A6 and DDC. And RT-PCR confirmed the up- and down-regulation of all these 5 genes.c) AR changes in flutamide resistance transition: In the process of flutamide resistance transition ,there is no AR expression changes. After cloning and sequencing , we found a new mutation of AR, ie. A3747G.8. Establishment of LNCaP cell Implant carcinoma in SCID mice: In the SCID mice, 75% (15/20) and 60% (12/20) of LNCaP and LNCaP-flu implants respecitively grew up to solid tumor at last. Immunohistochermstry revealed that there were androgen receptor expression in all the implant tumors, while there were not any PSA expression. There were no significant difference between LNCaP,
LNCaP-flu and LNCaP-flu/flu group cells in tumor weight and volume (P>0.05) . However, tumors from LNCaP/flu cells were much smaller (P<0.05) . Conclusion:1. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) The artificial androgen R1881 have a dual effect on LNCaP cell. 10~(-9)M and above could inhibit LNCaP cell proliferation, while 10~(-10)M and below could stimulate it.b) The down-regulation of androgen receptor coregulator DDC and C4orfl may play a role in the inhibition induced by 10~(-9)M R1881; while the down-regulation of ,FHL2,NCOR1,SVIL, GRIP1,PIAS3 and the up-regulation of ACTN2, TBLR1, NCOA2 may play a role in the prolific effect induced by 10~(-10)M R1881.2. Effect of artificial anti-androgen fiutamide on LNCaP cells and its correslated molecular mechanism:a) Fiutamide has dual effect on LNCaP cell. It could stimulate it at relative low concentration, or relative short time. But when the time prolonged or concentration rose, the effect was reversed.b) The inhibitive effect of fiutamide on LNCaP cells may be the up-regulation of CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2, which may affect downstream cell cycle related genes, and thus block the cell cycle at the G1/S checkpoint.3. Establishment of fiutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a fiutamide insensitive prostate cancer, LNCaP-flu.b) Androgen receptor related genes, such as NCOR1, NCOA2, NCOA4 , NCOA6 and DDC may be important in the transition of fiutamide sensitivity.
引文
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42. Craft N, Chhor C, Iran C, Belldegrun A, DeKernion J, Witte ON, Said J, Reiter RE, Sawyers CL. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res 1999,59:5030-5036.
43. Peets EA, Henson MF, Neri R.On the mechanism of the anti-androgenic action of flutamide (alpha-alpha-alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide) in the rat.Endocrinology. 1974 Feb;94(2):532-40.
44. Sogani PC, Whitmore WF Jr.Advanced prostatic carcinoma: flutamide therapy after conventional endocrine treatment.Urology. 1975 Aug;6(2): 164-6.
45. Brogden RN, Clissold SP.Flutamide. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in advanced prostatic cancer.Drugs. 1989 Aug;38(2): 185-203.
46. Silverio F, Tenaglia R, Bizzari M. Experience with flutamide in advancedprostatic cancer patients refractory to previous endocrine therapy. J DrugDevelopment. 1987, 1, (Suppl, 1): 10.
47.李炎唐.缓退瘤治疗前列腺癌:附32例报告.中华泌尿外科杂志.1995,16(7).416-417.
48. Librie F, Gomez JL, Cusan L. Neoadjuvant antiandrogen therapy forlocalized prostate cancer. Societe International D'Urologie 23rdCogress. Sep. 1994. Sydney, p 185.
49. Plail R, Hendry W, Demian J. Post-marketing surveillance of flutamide inUK hospitals. Societe International D'Urologie 23rd Cogress. Sep. 1994.Sydney, p 185.
50.邵竹平,周鹤同.睾丸实质切除联合缓退瘤治疗前列腺癌.中华男科学.2001,7(6):391-392.
51. Olea N, Sakabe K, Soto AM, Sonnenschein C. The proliferative effect of"anti-androgens" on the androgen-sensitive human prostate tumor cellline LNCaE Endocrinology. 1990; 126(3): 1457-63.
52. Wu HC, Hsieh JT, Gleave ME, et al. Derivation of androgen-independenthuman LNCaP prostatic cancer cell sublines: role of bone stromal cells.Int J Cancer 1994 May 1; 57(3): 406-12.
53. Kokontis JM, Hay N, Liao S. Progression of LNCaP prostate tumor cellsduring androgen deprivation: hormone-independent growth, repression ofproliferation by androgen, and role for p27Kip1 in androgen-induced cellcycle arrest. Mol Endocrinol 1998 Jul; 12(7): 941-53.
54. Hara T, Miyazaki J, Araki H, et al. Novel mutations of androgen receptor:a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res.2003 Jan 1; 63(1): 149-53.
55. Klugo RC, Farah RN, Cerny JC. Bilateral orchiectomy for carcinoma ofprostate. Response of serum testosterone and clinical response tosubsequent estrogen therapy. Urology. 1981 Jan; 17(1): 49-50.
56. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM,Mirand EA, Murphy GR LNCaP model of human prostatic carcinoma.Cancer Res. 1983 Apr;43(4): 1809-18.
57. Fowler JE Jr, Whitmore WF Jr. The response of metastatic adenocarcinoma of the prostate to exogenous testosterone.J Urol. 1981 Sep;126(3):372-5.
58. Kelly WK, Scher HI. Prostate specific antigen decline after antiandrogen withdrawal: the flutamide withdrawal syndrome. J Urol. 1993 Mar;149(3):607-9.
59. Fenton MA, Shuster TD, Fertig AM, Taplin ME, Kolvenbag G, Bubley GJ, Balk SP. Functional characterization of mutant androgen receptors from androgen-independent prostate cancer.Clin Cancer Res. 1997 Aug;3(8):1383-8.
60. Small EJ, Carroll PR. Prostate-specific antigen decline after casodex withdrawal: evidence for an antiandrogen withdrawal syndrome.Urology. 1994 Mar;43(3):408-10.
61. Blackledge GR, Lowery K. Role of prostate-specific antigen as a predictor of outcome in prostate cancer.Prostate Suppl. 1994;5:34-8.
62. Kantoff PW, Talcott JA. The radical prostatectomy series: apples are not oranges.J Clin Oncol. 1994 Nov;l2(11):2243-5.
63. Dawson NA. Treatment of progressive metastatic prostate cancer [published erratum of serious dosage error appears in Oncology (Huntingt) 1993 Jun;7(6):2]Oncology (Huntingt). 1993 May;7(5): 17-24, 27; discussion 27-9.
64. Rochlitz CF, Damon LE, Russi MB, Geddes A, Cadman EC. Cytotoxicity of ketoconazole in malignant cell lines.Cancer Chemother Pharmacol. 1988;21(4):319-22.
65. Small EJ.RE: Prostate specific antigen after gonadal androgen withdrawal and deferred flutamide treatment. J Urol. 1996 May;155(5):1704-5.
66. Mahler C, Verhelst J, Denis L. Ketoconazole and liarozole in the treatment of advanced prostatic cancer.Cancer. 1993 Feb 1;71(3 Suppl):1068-73.
67. Visakorpi T, Kallioniemi AH, Syvanen AC, Hyytinen ER, Karhu R, Tammela T, Isola JJ, Kallioniemi OP. Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization. Cancer Res. 1995 Jan 15;55(2):342-7.
68. Ferro MA, Gillatt D, Symes MO, Smith PJ. High-dose intravenous estrogen therapy in advanced prostatic carcinoma. Use of serum prostate-specific antigen to monitor response.Urology. 1989 Sep;34(3): 134-8.
69. Eisenberger MA, Simon R, O'Dwyer PJ, Wittes RE, Friedman MA. A reevaluation of nonhormonal cytotoxic chemotherapy in the treatment of prostatic carcinoma.J Clin Oncol. 1985 Jun;3(6):827-41.
70. Tannock IF, Osoba D, Stockler MR, Ernst DS, Neville AJ, Moore MJ, Armitage GR, Wilson JJ, Venner PM, Coppin CM, Murphy KC. Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer: a Canadian randomized trial with palliative end points.J Clin Oncol. 1996 Jun;14(6):1756-64.
71. Raghavan D, Cox K, Pearson BS, Coorey GJ, Rogers J, Watt WH, Coates AS, McNeil E, Grygiel JJ. Oral cyclophosphamide for the management of hormone-refractory prostate cancer.Br J Urol. 1993 Nov;72(5 Pt l):625-8.
72. Wafa LA, Cheng H, Rao MA, Nelson CC, Cox M, Hirst M, Sadowski I, Rennie PS. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system.Biochem J. 2003 Oct 15;375(Pt 2):373-83.
73. Sim DL, Chow VT.The novel human HUEL (C4orfl) gene maps to chromosome 4pl2-pl3 and encodes a nuclear protein containing the nuclear receptor interaction motif. Genomics. 1999 Jul 15;59(2):224-33.
74. Muller JM, Isele U, Metzger E, Rempel A, Moser M, Pscherer A, Breyer T, Holubarsch C, Buettner R, Schule R. FHL2, a novel tissue-specific coactivator of the androgen receptor.EMBO J. 2000 Feb 1;19(3):359-69.
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40. McDonnell TJ, Troncoso P, Brisbay SM,et al. Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 1992,52(24):6940-6944.
41. Liu AY, Corey E, Bladou F,et al. Prostatic cell lineage markers: emergence of BCL2~(+) cells of human prostate cancer xenograft LuCaP 23 following castration. Int J Cancer 1996,65:85-89.
42. Craft N, Chhor C, Iran C, Belldegrun A, DeKernion J, Witte ON, Said J, Reiter RE, Sawyers CL. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res 1999,59:5030-5036.
43. Peets EA, Henson MF, Neri R.On the mechanism of the anti-androgenic action of flutamide (alpha-alpha-alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide) in the rat.Endocrinology. 1974 Feb;94(2):532-40.
44. Sogani PC, Whitmore WF Jr.Advanced prostatic carcinoma: flutamide therapy after conventional endocrine treatment.Urology. 1975 Aug;6(2): 164-6.
45. Brogden RN, Clissold SP.Flutamide. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in advanced prostatic cancer.Drugs. 1989 Aug;38(2): 185-203.
46. Silverio F, Tenaglia R, Bizzari M. Experience with flutamide in advancedprostatic cancer patients refractory to previous endocrine therapy. J DrugDevelopment. 1987, 1, (Suppl, 1): 10.
47.李炎唐.缓退瘤治疗前列腺癌:附32例报告.中华泌尿外科杂志.1995,16(7).416-417.
48. Librie F, Gomez JL, Cusan L. Neoadjuvant antiandrogen therapy forlocalized prostate cancer. Societe International D'Urologie 23rdCogress. Sep. 1994. Sydney, p 185.
49. Plail R, Hendry W, Demian J. Post-marketing surveillance of flutamide inUK hospitals. Societe International D'Urologie 23rd Cogress. Sep. 1994.Sydney, p 185.
50.邵竹平,周鹤同.睾丸实质切除联合缓退瘤治疗前列腺癌.中华男科学.2001,7(6):391-392.
51. Olea N, Sakabe K, Soto AM, Sonnenschein C. The proliferative effect of"anti-androgens" on the androgen-sensitive human prostate tumor cellline LNCaE Endocrinology. 1990; 126(3): 1457-63.
52. Wu HC, Hsieh JT, Gleave ME, et al. Derivation of androgen-independenthuman LNCaP prostatic cancer cell sublines: role of bone stromal cells.Int J Cancer 1994 May 1; 57(3): 406-12.
53. Kokontis JM, Hay N, Liao S. Progression of LNCaP prostate tumor cellsduring androgen deprivation: hormone-independent growth, repression ofproliferation by androgen, and role for p27Kip1 in androgen-induced cellcycle arrest. Mol Endocrinol 1998 Jul; 12(7): 941-53.
54. Hara T, Miyazaki J, Araki H, et al. Novel mutations of androgen receptor:a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res.2003 Jan 1; 63(1): 149-53.
55. Klugo RC, Farah RN, Cerny JC. Bilateral orchiectomy for carcinoma ofprostate. Response of serum testosterone and clinical response tosubsequent estrogen therapy. Urology. 1981 Jan; 17(1): 49-50.
56. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM,Mirand EA, Murphy GR LNCaP model of human prostatic carcinoma.Cancer Res. 1983 Apr;43(4): 1809-18.
57. Fowler JE Jr, Whitmore WF Jr. The response of metastatic adenocarcinoma of the prostate to exogenous testosterone.J Urol. 1981 Sep;126(3):372-5.
58. Kelly WK, Scher HI. Prostate specific antigen decline after antiandrogen withdrawal: the flutamide withdrawal syndrome. J Urol. 1993 Mar;149(3):607-9.
59. Fenton MA, Shuster TD, Fertig AM, Taplin ME, Kolvenbag G, Bubley GJ, Balk SP. Functional characterization of mutant androgen receptors from androgen-independent prostate cancer.Clin Cancer Res. 1997 Aug;3(8):1383-8.
60. Small EJ, Carroll PR. Prostate-specific antigen decline after casodex withdrawal: evidence for an antiandrogen withdrawal syndrome.Urology. 1994 Mar;43(3):408-10.
61. Blackledge GR, Lowery K. Role of prostate-specific antigen as a predictor of outcome in prostate cancer.Prostate Suppl. 1994;5:34-8.
62. Kantoff PW, Talcott JA. The radical prostatectomy series: apples are not oranges.J Clin Oncol. 1994 Nov;l2(11):2243-5.
63. Dawson NA. Treatment of progressive metastatic prostate cancer [published erratum of serious dosage error appears in Oncology (Huntingt) 1993 Jun;7(6):2]Oncology (Huntingt). 1993 May;7(5): 17-24, 27; discussion 27-9.
64. Rochlitz CF, Damon LE, Russi MB, Geddes A, Cadman EC. Cytotoxicity of ketoconazole in malignant cell lines.Cancer Chemother Pharmacol. 1988;21(4):319-22.
65. Small EJ.RE: Prostate specific antigen after gonadal androgen withdrawal and deferred flutamide treatment. J Urol. 1996 May;155(5):1704-5.
66. Mahler C, Verhelst J, Denis L. Ketoconazole and liarozole in the treatment of advanced prostatic cancer.Cancer. 1993 Feb 1;71(3 Suppl):1068-73.
67. Visakorpi T, Kallioniemi AH, Syvanen AC, Hyytinen ER, Karhu R, Tammela T, Isola JJ, Kallioniemi OP. Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization. Cancer Res. 1995 Jan 15;55(2):342-7.
68. Ferro MA, Gillatt D, Symes MO, Smith PJ. High-dose intravenous estrogen therapy in advanced prostatic carcinoma. Use of serum prostate-specific antigen to monitor response.Urology. 1989 Sep;34(3): 134-8.
69. Eisenberger MA, Simon R, O'Dwyer PJ, Wittes RE, Friedman MA. A reevaluation of nonhormonal cytotoxic chemotherapy in the treatment of prostatic carcinoma.J Clin Oncol. 1985 Jun;3(6):827-41.
70. Tannock IF, Osoba D, Stockler MR, Ernst DS, Neville AJ, Moore MJ, Armitage GR, Wilson JJ, Venner PM, Coppin CM, Murphy KC. Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer: a Canadian randomized trial with palliative end points.J Clin Oncol. 1996 Jun;14(6):1756-64.
71. Raghavan D, Cox K, Pearson BS, Coorey GJ, Rogers J, Watt WH, Coates AS, McNeil E, Grygiel JJ. Oral cyclophosphamide for the management of hormone-refractory prostate cancer.Br J Urol. 1993 Nov;72(5 Pt l):625-8.
72. Wafa LA, Cheng H, Rao MA, Nelson CC, Cox M, Hirst M, Sadowski I, Rennie PS. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system.Biochem J. 2003 Oct 15;375(Pt 2):373-83.
73. Sim DL, Chow VT.The novel human HUEL (C4orfl) gene maps to chromosome 4pl2-pl3 and encodes a nuclear protein containing the nuclear receptor interaction motif. Genomics. 1999 Jul 15;59(2):224-33.
74. Muller JM, Isele U, Metzger E, Rempel A, Moser M, Pscherer A, Breyer T, Holubarsch C, Buettner R, Schule R. FHL2, a novel tissue-specific coactivator of the androgen receptor.EMBO J. 2000 Feb 1;19(3):359-69.
75. Berrevoets CA, Umar A, Trapman J, Brinkmann AO. Differential modulation of androgen receptor transcriptional activity by the nuclear receptor co-repressor (N-CoR). Biochem J. 2004 May l;379(Pt 3):731-8.
76. Ting HJ, Yeh S, Nishimura K, Chang C. Supervillin associates with androgen receptor and modulates its transcriptional activity. Proc Natl Acad Sci USA. 2002 Jan 22;99(2):661-6. Epub 2002 Jan 15.
77. Karvonen U, Janne OA, Palvimo JJ. Pure antiandrogens disrupt the recruitment of coactivator GRIP1 to colocalize with androgen receptor in nuclei. FEBS Lett. 2002 Jul 17;523(l-3):43-7.
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