鞘脂激活肽NP上调AR基因表达及功能影响前列腺癌细胞增殖的研究
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摘要
鞘脂激活蛋白原(Prosaposin)是一种高度保守的热稳定糖蛋白(65-72kDa,527氨基酸),其基因位于10号染色体长臂(q21-22),全长17kb,有14个外显子。鞘脂激活蛋白原经水解后产生4种小的热稳定糖蛋白--鞘脂激活蛋白(Saposin)A、B、C、D,他们在溶酶体酶解鞘脂的过程中发挥重要作用。研究表明,鞘脂激活蛋白原还可作为一种分泌蛋白具有神经营养作用,能够促进神经胶质源性细胞突触增长、细胞增殖以及增加其抗凋亡的能力,其神经营养活性位于鞘脂激活蛋白C(Saposin C)的N端氨基酸序列(LIDNNRTEELLY)。Prosaposin、Saposin C以及包括这一功能区域的一些鞘脂激活肽(如:ProsaptideTX14A)都能够相似的神经营养作用,其作用方式是通过结合细胞膜上的G蛋白偶联受体(GPCR)发挥的。
新的研究发现,Prosaposin基因失活可导致前列腺组织发育不良,Koochekpour S等人研究证实鞘脂激活蛋白原基因在前列腺癌组织中扩增并且高表达;外源性的saposin C以及Prosaptide TX14A可以通过多种信号途径促进前列腺癌细胞的生长、增殖、转移和侵蚀,并且抑制癌细胞的凋亡,包括MAPK途径、PI3K/AKT途径等。
前列腺癌是一种死亡率很高的恶性肿瘤,其复发后往往转变为雄激素非依赖性前列腺癌(AIPC),目前缺乏有效的治疗手段。尽管人们尚不清楚前列腺癌发生雄激素非依赖性转化的分子机制,但是研究证实雄激素受体AR可能在AIPC的发生发展过程中起决定性作用,因为它是唯一在AIPC发生发展过程中持续性高表达的分子。AR通过与雄激素结合后被磷酸化激活进入细胞核内,与DNA的雄激素受体应答元件(AREs)结合,从而激活与细胞生长和存活相关的基因转录。在AIPC发生发展过程中由AR介导的信号途径涉及AR受体基因的突变和扩增;生长因子或细胞因子的异常调节;共激活分子的改变等。大量的研究表明,AIPC的发生与雄激素受体(AR)的反常激活即在雄激素缺乏的情况下发生雄激素非依赖性激活有关。
该研究旨在探讨鞘脂激活蛋白原功能结构域--鞘脂激活肽NP(Neurotrophic Peptide,NP)对AR的调节以及对前列腺癌细胞增殖的影响。本实验利用基因克隆方法构建含有鞘脂激活肽NP基因序列的真核表达载体以及含有NP和TAT-PTD蛋白转导结构域基因序列的融合蛋白表达载体。脂质体转染法转染雄激素依赖性前列腺癌细胞(LNCaP)和雄激素非依赖性前列腺癌细胞(PC-3),通过RT-PCR、Western Blot、荧光素酶分析、流式细胞术检测等方法分析NP对AR基因表达和转录激活活性的调节以及NP对前列腺癌细胞周期和生长的影响,进一步探讨NP在AIPC发生发展过程中的作用。
结果显示: (1)成功构建真核表达载体pcDNA-TAT-NP和pcDNA-NP,通过脂质体转染法证实NP及TAT-NP能够在前列腺癌细胞中持续表达。(2)MTT及流式细胞术检测证实NP及TAT-NP都能够促使细胞进入S和G2/M期,刺激细胞增殖。(3)RT-PCR、Western Blot实验证实在mRNA和蛋白水平,NP及TAT-NP都具有增加AR基因表达的作用。(4)通过荧光素酶分析证实NP及TAT-NP都能够增加AR的转录激活活性。
总之,本研究结果表明,细胞内异源性表达的鞘脂激活肽NP能够增加AR的基因表达并增强AR的转录激活活性,同时促使前列腺癌细胞进入S和G2/M期,刺激前列腺癌细胞增殖。这一研究结果为解释AIPC的发生发展提供了新的分子机理,同时该研究对AIPC发生发展的分子机理的积累有可能为治愈AIPC提供可行的基因治疗途径。
Prosaposin is a highly conserved glycoprotein (65-72kDa, 527 amino acids), the gene was localized to the long arm of chromosome 10 (q21-22) and encompasses 17 kb of genomic sequence with 14 exons. Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins (saposins A, B, C and D, which are required for the enzymatic hydrolysis of sphingolipids in lysosomes). In addition to its intracellular presence and function, Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro. As a secretory protein, Prosaposin could promote survival, prevent apoptosis and stimulate synapse growth of neuroglial-derived cells. Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH_2-terminal end of the saposin C domain (LIDNNRTEELLY). Several synthetic peptides (14-22 residues, e.g. TX14A) derived from this region are equally as bioactive as Prosaposin. Prosaposin, saposin C, and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor (GPCR).
Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland. Koochekpour S. et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells, and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth, metastasis and invasion via activated MAPK, PI_3K/AKT et al singling pathways in prostate cancer.
Prostate cancer is the highly strong lethal disease, once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present, this is no effective treatment options for the patients. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor (AR) has been' shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC. The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily. Binding androgen, AR can be activated by phosphorylation. The active AR enters into the nuclear and binds to the androgen-responsive element (ARE) of the target DNA in order to activate the target gene transcription and promote cells growth. During the development of AIPC the singling pathways AR mediating involved in AR gene mutation, AR gene amplification, the aberrant regulation of AR by growth factors and cytokines, and AR cofactors change. Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.
The present study is aimed to determine effect of the function domain of saposin C (neurotrophic peptide, NP) on androgen receptor (AR) expression and transcriptional activity and cells proliferation in prostate cancer. We constructed DNA vectors that can express NP or a chimeric peptide of a viral TAT transduction domain and NP by gene cloning technology. The effect of ectopic expression of NP with or without the TAT transduction domain on cell growth was examined by MTT assay and flow cytometry. Then reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and transient transfection experiments were used to determine the effect of NP on AR expression and activation.
The Results showed that the eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed and confirmed by sequencing, and they can normally express in prostate cancer cells. MTT and flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation, and promoted the cells to enter S and G_2/M phase. The RT-PCR and Western blot analyses displayed that TAT-NP and NP induced AR gene expression. Dual-luciferace experiment results demonstrated that TAT-NP and NP activated transactivation of the AR.
Collectively, our study showed that NP, as a growth stimulating factor to PCa cells, exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner. Further investigation focused on how NP regulates the AR will give more insight into its role in PCa progression as well as a new cancer therapeutic target.
新的研究发现,Prosaposin基因失活可导致前列腺组织发育不良,Koochekpour S等人研究证实鞘脂激活蛋白原基因在前列腺癌组织中扩增并且高表达;外源性的saposin C以及Prosaptide TX14A可以通过多种信号途径促进前列腺癌细胞的生长、增殖、转移和侵蚀,并且抑制癌细胞的凋亡,包括MAPK途径、PI3K/AKT途径等。
前列腺癌是一种死亡率很高的恶性肿瘤,其复发后往往转变为雄激素非依赖性前列腺癌(AIPC),目前缺乏有效的治疗手段。尽管人们尚不清楚前列腺癌发生雄激素非依赖性转化的分子机制,但是研究证实雄激素受体AR可能在AIPC的发生发展过程中起决定性作用,因为它是唯一在AIPC发生发展过程中持续性高表达的分子。AR通过与雄激素结合后被磷酸化激活进入细胞核内,与DNA的雄激素受体应答元件(AREs)结合,从而激活与细胞生长和存活相关的基因转录。在AIPC发生发展过程中由AR介导的信号途径涉及AR受体基因的突变和扩增;生长因子或细胞因子的异常调节;共激活分子的改变等。大量的研究表明,AIPC的发生与雄激素受体(AR)的反常激活即在雄激素缺乏的情况下发生雄激素非依赖性激活有关。
该研究旨在探讨鞘脂激活蛋白原功能结构域--鞘脂激活肽NP(Neurotrophic Peptide,NP)对AR的调节以及对前列腺癌细胞增殖的影响。本实验利用基因克隆方法构建含有鞘脂激活肽NP基因序列的真核表达载体以及含有NP和TAT-PTD蛋白转导结构域基因序列的融合蛋白表达载体。脂质体转染法转染雄激素依赖性前列腺癌细胞(LNCaP)和雄激素非依赖性前列腺癌细胞(PC-3),通过RT-PCR、Western Blot、荧光素酶分析、流式细胞术检测等方法分析NP对AR基因表达和转录激活活性的调节以及NP对前列腺癌细胞周期和生长的影响,进一步探讨NP在AIPC发生发展过程中的作用。
结果显示: (1)成功构建真核表达载体pcDNA-TAT-NP和pcDNA-NP,通过脂质体转染法证实NP及TAT-NP能够在前列腺癌细胞中持续表达。(2)MTT及流式细胞术检测证实NP及TAT-NP都能够促使细胞进入S和G2/M期,刺激细胞增殖。(3)RT-PCR、Western Blot实验证实在mRNA和蛋白水平,NP及TAT-NP都具有增加AR基因表达的作用。(4)通过荧光素酶分析证实NP及TAT-NP都能够增加AR的转录激活活性。
总之,本研究结果表明,细胞内异源性表达的鞘脂激活肽NP能够增加AR的基因表达并增强AR的转录激活活性,同时促使前列腺癌细胞进入S和G2/M期,刺激前列腺癌细胞增殖。这一研究结果为解释AIPC的发生发展提供了新的分子机理,同时该研究对AIPC发生发展的分子机理的积累有可能为治愈AIPC提供可行的基因治疗途径。
Prosaposin is a highly conserved glycoprotein (65-72kDa, 527 amino acids), the gene was localized to the long arm of chromosome 10 (q21-22) and encompasses 17 kb of genomic sequence with 14 exons. Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins (saposins A, B, C and D, which are required for the enzymatic hydrolysis of sphingolipids in lysosomes). In addition to its intracellular presence and function, Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro. As a secretory protein, Prosaposin could promote survival, prevent apoptosis and stimulate synapse growth of neuroglial-derived cells. Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH_2-terminal end of the saposin C domain (LIDNNRTEELLY). Several synthetic peptides (14-22 residues, e.g. TX14A) derived from this region are equally as bioactive as Prosaposin. Prosaposin, saposin C, and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor (GPCR).
Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland. Koochekpour S. et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells, and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth, metastasis and invasion via activated MAPK, PI_3K/AKT et al singling pathways in prostate cancer.
Prostate cancer is the highly strong lethal disease, once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present, this is no effective treatment options for the patients. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor (AR) has been' shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC. The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily. Binding androgen, AR can be activated by phosphorylation. The active AR enters into the nuclear and binds to the androgen-responsive element (ARE) of the target DNA in order to activate the target gene transcription and promote cells growth. During the development of AIPC the singling pathways AR mediating involved in AR gene mutation, AR gene amplification, the aberrant regulation of AR by growth factors and cytokines, and AR cofactors change. Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.
The present study is aimed to determine effect of the function domain of saposin C (neurotrophic peptide, NP) on androgen receptor (AR) expression and transcriptional activity and cells proliferation in prostate cancer. We constructed DNA vectors that can express NP or a chimeric peptide of a viral TAT transduction domain and NP by gene cloning technology. The effect of ectopic expression of NP with or without the TAT transduction domain on cell growth was examined by MTT assay and flow cytometry. Then reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and transient transfection experiments were used to determine the effect of NP on AR expression and activation.
The Results showed that the eukaryotic expression vectors pcDNA-TAT-NP and pcDNA-NP were constructed and confirmed by sequencing, and they can normally express in prostate cancer cells. MTT and flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation, and promoted the cells to enter S and G_2/M phase. The RT-PCR and Western blot analyses displayed that TAT-NP and NP induced AR gene expression. Dual-luciferace experiment results demonstrated that TAT-NP and NP activated transactivation of the AR.
Collectively, our study showed that NP, as a growth stimulating factor to PCa cells, exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner. Further investigation focused on how NP regulates the AR will give more insight into its role in PCa progression as well as a new cancer therapeutic target.
引文
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4．贺松琴，曹文广。雄激素非依赖性前列腺癌发生的分子机制。国外医学泌尿系统分册 2005：25：811—815
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2. Trachtenberg J. Looking to the future. Advence in the management of hormone-refractory prostate cancer. Eur Urol Suppl 2002; 1: 44-53
3. Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med, 2004, 10: 33
4．贺松琴，曹文广。雄激素非依赖性前列腺癌发生的分子机制。国外医学泌尿系统分册 2005：25：811—815
5. Eder IE, Haag P, Bartsch G, Klocker H. Targeting the androgen receptor in hormone-refractory prostate cancer—new concepts. Future Oncol. 2005 Feb; 1(1): 93-101.
6. Edwards J, Bartlett JM. The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 1: Modifications to the androgen receptor. BJU Int. 2005 Jun; 95(9): 1320-6
7. Edwards J, Bartlett JM. The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 2: Androgen-receptor cofactors and bypass pathways. BJU Int. 2005; 95(9): 1327-35.
8. O'Brien JS, Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders. FASEB J. 1991; 5(3): 301-8.
9. Kishimoto Y, Hiraiwa M, O'Brien JS. Saposins: structure, function, distribution, and molecular genetics. J Lipid Res. 1992; 33(9): 1255-67.
10. Campana WM, O'Brien JS, Hiraiwa M, Patton S: Secretion of prosaposin, a multifunctional protein, by breast cancer cells. Biochim Biophys Acta 1999, 1427: 392-400.
11. Morimoto S, Yamamoto Y, O'Brien JS, Kishimoto Y: Determination of saposin proteins (sphingolipid activator proteins) in human tissues. Anal Biochem 1990, 190: 154-157.
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