摘要
多胺通过改变DNA结构和调节信号传导途径等方式调节基因的表达,从而在细胞生长和分化过程中发挥着重要的作用。研究发现,在肿瘤细胞和组织中多胺的含量和生物合成明显增高。鸟氨酸脱羧酶(ODC)是多胺生物合成途径中第一个限速酶,可催化L-鸟氨酸脱羧生成腐胺。ODC可被化学致癌物以及多种癌基因,如v-src、neu、ras等激活,在多种肿瘤中表达增高,并与肿瘤复发有关。S—腺苷甲硫氨酸脱羧酶(AdoMetDC)是多胺合成的第二个限速酶,主要是催化S-腺苷甲硫氨酸(SAM)脱羧基形成脱羧的SAM(dcSAM),为精脒和精胺的合成提供丙氨基。抑制ODC和AdoMetDC的活性可以防治肿瘤的形成和转移。因此ODC和AdoMetDC是肿瘤治疗的重要靶标。
在西方,前列腺癌的发病率甚高,在我国也有升高趋势。我国前列腺癌在男性泌尿、生殖系统恶性肿瘤中发病率居第三位,目前一线治疗手段主要是手术和化疗,但五年生存率只能达到40%。因此寻找一种新型非手术干预的治疗方法对于前列腺癌的预防和治疗具有非常重要意义。我们曾构建针对ODC的反义腺病毒表达载体,并证明其在前列腺癌细胞中具有明显的增殖抑制作用。但因其缺乏组织特异性,推广应用受到限制,故研制组织特异,特别是肿瘤细胞特异的反义RNA表达载体,是当前极待解决的问题。
前列腺特异抗原主要在前列腺细胞中表达,在前列腺肿瘤细胞中表达增强。Sang-jin Lee将PSA的启动子进行了改造,获得了高效的PSA的启动子(PSES)并证明其确实具有前列腺组织特异性。以该改造的PSES启动子作为反义RNA表达载体的表达调控元件,可以起到组织导向作用,达到靶向治疗的目的。因为ODC和AdoMetDC是性激素下游调控物质,抑制其表达不会影响性激素的其它生理功能,可避免抗雄激素疗法所致之副作用。又因为PSES启动子所调控的基因表达在前列腺肿瘤细胞中表达增强,其介导的ODC和AdoMetDC双反义RNA表达载体可以在前列腺肿瘤细胞中表达而抑制肿瘤细胞增殖、恶变和转移,虽然在正常前列腺细胞中也可能有表达并抑制正常细胞增殖,但因前列腺是非必需器官,切除前列腺对患者生命质量影响不大,故仍然认为PSES启动子不失为一种较好的前列腺肿瘤靶向基因治疗的启动子。所以参考上述国内外研究进展,根据我们以往研究的结果而设计,在已构建的ODC单反义腺病毒和可以同时表达ODC和AdoMetDC反义RNA双反义腺病毒的基础上,我们又构建起以ODC和AdoMetDC为靶标,分别以经过改造的非雄激素依赖和雄激素依赖的PSES启动子为表达调控元件的前列腺特异的ODC和AdoMetDC双反义RNA表达载体,并验证它们对前列腺癌细胞增殖和侵袭的抑制作用,从而为前列腺癌的基因治疗研究提供了新的方法和途径。
第一章前列腺非雄激素依赖启动子介导的ODC、AdoMetDC双反义RNA表达载体的构建
研究目的:构建前列腺非雄激素依赖启动子介导的同时表达ODC和AdoMetDC双反义RNA腺病毒载体,为非雄激素依赖的前列腺癌靶向治疗研究提供新工具和手段。
研究方法:
1.将ODC,AdoMetDC两种PCR产物与pMD19-T simple进行连接、转化,经筛选、鉴定获得TA—AdoMetDC—ODC。
2.将TA—AdoMetDC—ODC经酶切后回收目的基因片段,反向插入经同样酶切的穿梭质粒pAdTrack中,构建成质粒pAdTrack—AdoMetDC—ODC,并将改造的前列腺非雄激素依赖启动子(PPA8)插入调控部位,建立前列腺特异的重组穿梭质粒pAdTrack-PPA8-AdoMetDC—ODC。
3.PCR法从pAdTrack-PPA8-ODC-AdoMetDC扩增目的基因PPA8—AdoMetDC—ODC,将目的基因克隆到穿梭载体pShuttle-EGFP-Basic,得到pShuttle-EGFP-Basic-PPA8-AdoMetDC-ODC
4.PCR法从pShuttle-CMV上扩增polyA,将PCR产物与pShuttle-EGFP-Basic-PPA8-AdoMetDC-ODC进行连接得到pShuttle-EGFP-Basic-PPA8-AdoMetDC-ODC—polyA,测序鉴定。
5.将目的基因PPA8-AdoMetDC-ODC—polyA转移到pAdxsi载体,得到pADxsi-PPA8-AdoMetDC-ODC-PolyA,转染293细胞包装和扩增出腺病毒颗粒Ad-PPA8-ODC-AdoMetDCas。荧光显微镜和PCR的方法对重组腺病毒Ad-PPA8-ODC-AdoMetDCas进行鉴定。
实验结果:
1.TA—AdoMetDC—ODC经酶切鉴定和测序分析,目的片断基因序列正确。
2.TA—AdoMetDC—ODC经酶切后反向插入到pAdTrack中,并将PPA8插入调控部位,经酶切鉴定和测序分析,目的片断基因序列及插入方向正确。表明已成功构建出前列腺特异的重组穿梭质粒pAdTrack-PPA8—AdoMetDC—ODC。
3.pShuttle-EGFP-Basic-PPA8-AdoMetDC-ODC—polyA经酶切鉴定和测序分析,目的片断基因序列及插入方向正确。
4.重组质粒pADxsi-PPA8-AdoMetDC-ODC-PolyA转染293细胞进行包装扩增,可见明显病毒空斑形成,荧光显微镜下可见绿色荧光蛋白在293细胞中表达。扩增后腺病毒滴度为5×10~(10)pfu/ml,PCR证实基因组中含有目的基因PPA8,ODC和AdoMetDC。
实验结论:成功构建并扩增出前列腺非雄激素依赖启动子介导的ODC和AdoMetDC双反义RNA腺病毒载体,为非雄激素依赖的前列腺癌的基因治疗和预防研究提供了必要的工具。
第二部分前列腺雄激素依赖启动子介导的ODC、AdoMetDC双反义RNA表达载体的构建
实验目的:构建前列腺雄激素依赖启动子介导的同时表达ODC和AdoMetDC双反义RNA腺病毒载体,为雄激素依赖的前列腺癌靶向治疗研究提供新工具和手段。
实验方法:
1.将pAdTrack-PPA8—AdoMetDC—ODC经酶切后回收目的基因片段AdoMetDC—ODC,插入经同样酶切的穿梭质粒pShuttle-Basic中,构建成质粒pShuttle-Basic-AdoMetDC-ODC,并将改造的前列腺雄激素依赖启动子(P61)插入调控部位,建立前列腺特异的重组穿梭质粒pShuttle-Basic-P61-AdoMetDC-ODC
2.PCR法从pShuttle-CMV上扩增polyA,将PCR产物与pShuttle-EGFP-Basic-P61-AdoMetDC-ODC进行连接得到pShuttle-EGFP-Basic-P61-AdoMetDC-ODC—polyA,测序鉴定。
3.将目的基因P61-AdoMetDC-ODC—polyA转移到pAdxsi载体,得到pADxsi-P61-AdoMetDC-ODC-PolyA,转染293细胞包装和扩增出腺病毒颗粒Ad-P61-ODC-AdoMetDCas。荧光显微镜和PCR的方法对重组腺病毒Ad-P61-ODC-AdoMetDCas进行鉴定。
实验结果:
1.pAdTrack-PPA8—AdoMetDC—ODC经酶切后,目的基因片段AdoMetDC—ODC插入到pShuttle-Basic中,并将P61插入调控部位,经酶切鉴定和测序分析,目的片断基因序列及插入方向正确。表明已成功构建出前列腺特异的重组穿梭质粒pShuttle-Basic-P61-AdoMetDC-ODC。
2.pShuttle-EGFP-Basic-P61-AdoMetDC-ODC—polyA经酶切鉴定和测序分析,目的片断基因序列及插入方向正确。
3.重组质粒pADxsi-P61-AdoMetDC-ODC-PolyA转染293细胞进行包装扩增,可见明显病毒空斑形成,荧光显微镜下可见绿色荧光蛋白在293细胞中表达。扩增后腺病毒滴度为5×10~(10)pfu/ml,PCR证实基因组中含有目的基因P61,ODC和AdoMetDC。
实验结论:成功构建并扩增出前列腺雄激素依赖启动子介导的ODC和AdoMetDC双反义RNA腺病毒载体,为雄激素依赖的前列腺癌的基因防治研究提供了必要的工具。
第三部分重组腺病毒抑癌作用的体外研究
实验目的:研究前列腺特异性启动子介导的ODC,AdoMetDC双反义RNA的重组腺病毒Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas的组织特异性,以及它们对前列腺癌细胞的多胺合成,细胞增殖以及侵袭的抑制作用。
实验方法:
1.Ad-GFP分别感染前列腺癌雄激素依赖细胞株LNCap,前列腺癌非雄激素依赖细胞株Du145,结直肠癌细胞株HT-29,肺癌细胞株H1299,肝癌细胞株HepG2。以不同的MOI值病毒感染24小时以后观察细胞状态,并观察荧光强度。
2.采用RT-PCR法从分子水平检测雄激素依赖的和非雄激素依赖的两种腺病毒的反义RNA对LNCap,Du145,HT-29,H1299,HepG2细胞中ODC和AdoMetDC基因表达的干扰效果。
3.采用Western印迹的方法分别检测两种腺病毒对LNCap,Du145,HT-29,H1299,HepG2细胞中P21,ODC和AdoMetDC蛋白表达,以验证重组载体对ODC和AdoMetDC表达的抑制作用及其细胞特异性,以及与CDK抑制蛋白p21WAF1/CIP1/SDI1表达的关系。
4.采用HPLC的方法分别检测两种腺病毒对LNCap,Du145细胞内多胺含量的影响。
5.采用MTT法检测Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对LNCap,Du145,HT-29,H1299,HepG2细胞增殖的影响,分别测定各组细胞的生长曲线。
6.采用流式细胞术分别检测Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对LNCap,Du145,HT-29,H1299,HepG2细胞周期分布的影响以及与CDK抑制蛋白p21 WAF1/CIP1/SDI1表达的关系,以进一步验证上述处理对前列腺癌的治疗作用及其机制。
7.采用Matrigel侵袭实验分析两种腺病毒对LNCap,Du145,HT-29,H1299,HepG2细胞侵袭活性的改变。
实验结果:
1.基因转染效率实验结果显示:各种实验细胞系在感染90 MOI的Ad-GFP,24小时后呈现GFP阳性最多,荧光最强。在MOI值为90时候为最佳转染浓度值。以下所有实验腺病毒的转染体系均以此次转染体系为准。
2.RT-PCR结果显示,感染实验细胞系的腺病毒的反义RNA对于H1299,HT29以及HepG2三种细胞株中的ODC和AdoMetDCas基因没有明显的干扰效果。对于DU145以及LNCap中的ODC和AdoMetDCas两个基因的分子水平存在一定的干扰影响。Ad-P61-ODC-AdoMetDCas表现出更高的高干扰效果。
3.western blot结果显示,重组腺病毒Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对于HepG2,H1299,HT29三种细胞系中ODC,AdoMetDCas以及P21三种基因的蛋白表达水平没有显著的影响;对于DU145和LNCap,在蛋白水平上表现出对于ODC和AdoMetDCas的抑制,Ad-P61-ODC-AdoMetDCas抑制率更高。P21蛋白表达水平显著增强。
4.HPLC结果显示,前列腺癌细胞株DU145和LNCap感染Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas后细胞内三种多胺含量都明显降低,且抑制作用不被外源性的腐胺恢复。
5.流式细胞术结果显示感染了Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas的前列腺癌细胞周期阻滞在G1期,且伴随有CDK抑制蛋白p21表达升高,而对于其他实验细胞系则没有影响。
6.重组腺病毒Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对于HT29,H1299以及HepG2细胞株的细胞增殖没有显著的影响,而感染重组腺病毒以后的DU145和LNCap与对照组NS和Ad-GFP相比较,生长明显缓慢。说明重组腺病毒对于前列腺细胞的生长有着明显的抑制作用。
7.Matrigel侵袭实验显示,Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas可显著降低前列腺癌细胞株细胞的体外侵袭能力,Ad-P61-ODC-AdoMetDCas较Ad-PPA8-ODC-AdoMetDCas相比可以更明显抑制前列腺细胞的侵袭能力。
实验结论:Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas具有显著抑制前列腺癌增殖和侵袭的作用,而且对于前列癌的防治研究具有重要的意义。Ad-P61-ODC-AdoMetDCas较Ad-PPA8-ODC-AdoMetDCas相比抑制作用更明显。
第四部分重组腺病毒抑癌作用的体内研究
实验目的:裸鼠皮下种植前列腺癌细胞,制备前列腺癌动物模型,体内观察Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas抑制前列腺癌的作用及其组织特异性。
实验方法:
1.检测Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas预防肿瘤形成的作用:收集体外感染重组腺病毒的前列腺癌细胞,按2×10~6/100μl接种于裸鼠协腹皮下,肿瘤体积每星期测量两次,测量到注射后第40天。
5.2.检测Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对已形成肿瘤的作用:收集前列腺癌细胞,调节细胞浓度为5×10~6个/100μl,将100μl细胞悬液注射入裸鼠皮下,当肿瘤直径达到5～7mm时,被随机分组,瘤内注射病毒3×10~9pfu,每三天注射一次,共三次,肿瘤体积每周测量一次。
实验结果:
1.ODC和AdoMetDCas反义RNA的表达可以抑制前列腺癌细胞在体内的成瘤,接受Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas处理细胞的实验组裸鼠在6周的观察期中所生成的肿瘤的体积和生长速率和较无病毒处理组和Ad-GFP处理组的肿瘤显著降低。
2.重组腺病毒Ad-P61-ODC-AdoMetDCas和Ad-PPA8-ODC-AdoMetDCas对已形成的裸鼠前列腺癌移植瘤能够减缓其生长,增高苛瘤鼠的存活率,30天Ad-GFP组裸鼠存活率为50%,而重组腺病毒组存活率为100%。
实验结论:
通过研究重组腺病毒对裸鼠移植瘤模型的作用,结果显示ODC和AdoMetDCas反义RNA对前列腺肿瘤有预防作用,即抑制肿瘤的形成;对已形成的裸鼠皮下前列腺肿瘤可减缓生长,延长存活时间。
Ornithine decarboxylase,ODC is the first rate-limiting enzyme in the process of polyamine synthesis,which can catalyze L-ornithine to decarboxylate and have other biological functions.ODC can be activated by chemical inducers and various oncogenes such as v-src,neu,ras etc.It can be expressed in many cancer tissues. S-adenosylmethionine decarboxylase,AdoMetDC is another key enzyme,is also a rate-limiting enzyme in the process of polyamino synthesis.It can catalyze the decarboxylation of S-adenosylmethionine followed the production of SAM(dcSAM), providing propylamino for the synthesis of spermidine and amine.Therefore,both are important in the prostatic carcinoma prevention,and some efforts have been given in inhibiting the activity of ODC and AdoMetDC.Several people have suggested that ODC and AdoMetDc are important targets for tumor therapy.However the lack of ODC and AdoMetDc tissue specificity has partially limited their clinical application. Prostate cancer is one of the most common malignancies in the Western world.It is also the third leading cause of cancer mortality in the US.With changes in the profiles of diet the morbidity of disease in China become higher.First-line therapy is radical surgery with adjuvant chemotherapy.But overall 5 year survival rate for this disease is around 40%.Therefore,it is important to find a novel non-surgical intervention for treating Prostate cancer.Previous studies have demonstrated the correlation between Prostate cancer and aberrant polyamine biosynthesis pathway.Therefore,ODC and AdoMetDC become important targets for treating prostate cancer.We had constructed an adenovirus expressing antisense ODC RNA and proved its inhibitory effects on prostate cancer growth.Prostate-specific antigen(PSA) normally expressed in prostatic cells and strongly expressed in prostatic carcinomar cells.Sang-jin Lee has modified a promoter for the expression of PSA gene,enhancing the PSA expression efficiency and tissue specificity.The modified PSA gene promoter can be a regulatory element for tissue targeting.Notably,given the ubiquitous expression of ODC and AdoMetDCas in both normal and tumor cells,general disruption of both genes might suffer from the serious adverse effects.Thus the specific gene disruption of ODC and AdoMetDCas expression in prostate cells is a desirable strategy in prostate gene therapy.In this study,ODC and AdoMetDC were used as targets,and a modified prostatic Prostate-specific antigen promoter-based double anti-sense RNA expression vector was produced.The vector was used for its ability of prevention and therapy of prostatic carcinoma.
Part 1 Construction of a prostatic androgen-independent promoter mediated ODC and AdoMetDC antisense RNA-expressing adenovirus
Aim:To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase(ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) antisenses specifically in prostate cancer cells.
Methods:Fragments of ODC and AdoMetDC cDNAs were inserted into pMD19-T simple vector followed by ligation with PGL-PPA8 fragment,and ligated into shuttle vector pAdTrack named pAdTrack-PPA8-AdoMetDC-ODC.Primers were designed using pAdTrack- PPA8-AdoMetDC-ODC as the template,The PCR product was then subject to gel electrophoresis.Next,the 0.9kb fragment was recovered and treated with KpnⅠ,and then gel electrophoresis and subsequent purification were carried out. The shuttle vector pShuttle-Basic was digested with KpnⅠand dephosphorylated with CIP,and after gel electrophoresis the 4.2kb fragment was recovered and ligated to the above inserting fragment,resulting in vector pShuttle-EGFP-Basic-PPA8-AdoMetDC -ODC.PolyA was PCR amplified using pShuttle-CMV as the template,with primers added with PolyA-F and PolyA-R adaptors with EcoRI.The above PCR product was subject to gel electrophoresis,and subsequently cut with EcoR I.The resulting product was subject to gel electrophoresis and recovered,pShuttle-Basic-PPA8 -AdoMetDC-odc vector was treated with MfeI and dephosphorylated with CIP,and the 8.5kb vector segment was recovered and then ligated to the inserting fragment. The target gene was transferred to pAdxsi vector,resulting in the pADxsi-PPA8-AdoMetDC-ODC-PolyA vector,which was subsequently treated by I-CeuI and I-SceI endonucleases and dephosphorylated with CIP,then inactivated in 10%SDS for 10min at 75℃,and recovered with ethanol precipitation. pShuttle-EGFP-Basic-PPA8-AdoMetDC-ODC-PolyA vector was treated with I-CeuI and I-SceI endonucleases.After electrophoresis,the 2.8kb fragment was recovered and then ligated to the above treated vector fragment,resulting in pADxsi-PPA8-AdoMetDC-ODC-PolyA. After package in 293 cells,the recombinant adenovirus pADxsi-PPA8-AdoMetDC-ODC-PolyA was obtained.
Results:
To identify whether ODC and AdoMetDC had been inserted into the constructed shuttle vector,pAdTrack-PPA8-AdoMetDC-ODC plasmids extracted from three candidate clones were digested with KpnⅠ/EcoRⅤand product fragments were separated in agarose gel electrophoresis.The results indicated that all the three clones are positive ones and thus applied in the following experiments.The recombinant vector pShuttle-Basic-PPA8-AdoMetDC-ODC and pADxsi-PPA8-AdoMetDC-ODC -PolyA were successfully confirmed by sequencing analysis.During the processure of viral package and amplification in 293 cells,lots of obvious viral plagues and GFP expression were detected with the light microscopy and fluorescent microscopy.The appearance of target gene in adenovirus genome was confirmed by PCR technique.
Conclusion:The biantisense recombinant adenoviruses Ad-PPA8-AdoMetDC -ODCas were successfully constructed and amplified.
Part2.Construction of a prostatic androgen-dependent promoter mediated ODC and AdoMetDC antisense RNA-expressing adenovirus
Aim:To construct a prostatic androgen-dependent promoter mediated ODC and AdoMetDC antisense RNA-expressing adenovirus
Methods:PShuttle-Basic vector was treated with HindⅢand KpnⅠ,after electrophoresis,the 2.4kb vector fragment was recovered,pAdTrack-PPA8-AdoMetDC-ODC vector was also treated with HindⅢand KpnⅠ,and the 0.3kb fragment was recovered and then ligated to the 2.4kb vector fragment,resulting in pShuttle-Basic-AdoMetDC-ODC,which was confirmed by HindⅢand KpnⅠdigestion,pShuttle-Basic-AdoMetDC-ODC was digested with HindⅢ,and dephosphorylated with CIP,after electrophoresis,the 2.7kb vector fragment was recovered.PGL3-P61 construct was also treated with HindⅢ,and the 5.8kb fragment was recovered and then ligated to the treated vector fragment,resulting in pShuttle-Basic-P61-AdoMetDC-ODC,which was confirmed by KpnⅠdigestion The construction procedures of pShuttle-Basic-P61-AdoMetDC-ODC-PolyA and pADxsi-P61-AdoMetDC-ODC-PolyA was the same as those of pADxsi-PPA8-AdoMetDC-ODC-PolyA and pADxsi-PPA8-AdoMetDC-ODC-PolyA.After package in 293 cells,we got the recombinant adenovirus pAdxsi-P61-AdoMetDC-ODC -PolyA.
Results:The sequence and derection of inserted genes were confirmed by sequencing. Several positive recombinant clones were identified.During the processure of viral package and amplification in 293 cells,lots of obvious viral plagues and GFP expression were detected with the light microscopy and fluorescent microscopy.The appearance of target gene in adenovirus genome was confirmed by PCR technique.
Conclusion:The biantisense recombinant adenoviruses Ad-P61-AdoMetDC-ODCas were successfully constructed and amplified..
Part3.Inhibitory effects of Effects of Two Prostate-specific antigen Promoter mediated ODC and AdoMetDC antisense RNA-expressing adenovirus on the synthesis of polyamine and cell growth in prostate cancer cells in vitro
Aim:This study was designed to investigate the effects of two Prostate-specific antigen Promoter mediated antisense vector targeting ornithine decarboxylase(ODC) and S-adenosylmethionine decarboxylase(AdoMetDC) on the synthesis of polyamine and its potential for prostate cancer therapy..
Methods:The two recombinant Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas adenoviruses were constructed to infect various cancer cell lines including prostate cancer LNCaP,Du145,colon cancer HT-29,lung cancer H1299,liver cancer HepG2,and cervical cancer HeLa.At 24 h post-infection,the GFP expression level and cell proliferation were analyzed to determine the optimal transfection doses used in subsequent studies.The effects of the Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas on the expression of cellular ODC and AdoMetDC,and on cell cycle,apoptosis and p21 levels were analyzed by Western blotting and cytometry.A Matrigel invasion assay was used to analyze the effects of the two recombinant viruses on tumor cell invasion.The effects on the polyamine content were also determined,and the relationships between the inhibition of cellular ODC and AdoMetDC and decreases in cellular polyamine were also investigated using a polyamine recovery assay.
Results:the Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas at an MOI of 90 significantly inhibited the proliferation of prostate cancer cells,which could not be recovered by adding exogenous putrescin.It also inhibited the expression of ODC and AdoMetDC,and significantly decreased the content of three kinds of polyamines.The G1 phase of prostate cancer cells was delayed,but no increase in apoptosis was detected.The down-regulation of ODC and AdoMetDC increased p21 expression.
Conclusions:Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas specifically inhibited the expression of ODC and AdoMetDC,the synthesis of polyamine,and induced p21 expression,resulting in cell growth arrest in G1 phase in prostate cancer cells,but not other cells.
Part4.Inhibitory effects of Two Prostate-specific antigen Promoter mediated ODC and AdoMetDC biantisense virus on colorectal cancer cell growth in vivo.
Aim:To evaluate Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas inhibitory effect on prostate cancer in vivo.
Methods:Firstly,to detect the effect of the antisense ODC and AdoMetDCas on prostate tumorigenicity:the prostate cancer cells were infected with Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas at a MOI of 90 for 48 hours,harvested,washed three times with PBS,and resuspended in 1640 medium. The cell suspensions(5×10~6) in a total volume of 100μl were then injected subcutaneously into 6-week-old BALB/C nude male mice.Tumor volume was measured every week and calculated with the following formula: volume=M1×M2×M2×0.5236(M1,long axis;M2,short axis).Secondly,to examine the effect of the antisense ODC and AdoMetDCas on the formed prostate tumor:the mouse prostate tumors were established by injecting 5×10~6 cells(in medium) s.c.into nude male mice.When tumors had reached 5～7mm in diameter,the mice were treated with the recombinant adenovirus every third day.Tumor size was measured twice a week.
Results:Expression of Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas inhibited the growth of prostate cancer cells in vivo,as evidenced by the reduction in tumor incidence and tumor sizes,when compared with those of rAd-GFP treated or no virus-treated tumors.Mice implanted with unmanipulated human prostate carcinoma cells developed a single big cancer and mice received prostate cells transfected with Ad-GFP also formed single tumor.However,the total volume of tumor in the mice received human prostate carcinoma cells transfected with Ad-GFP was significantly smaller than that in the mice received unmanipulated prostate cells. Importantly,the mice received human prostate carcinoma cells transfected with the Ad-P61-AdoMetDC-ODCas and Ad-PPA8-AdoMetDC-ODCas almost failed to develop visible cancer throughout the experimental period.. Ad-P61-ODC-AdoMetDCas and Ad-PPA8-ODC-AdoMetDCas can also inhibit the growth of the formed prostate tumors.
Conclusion:Antisense ODC and AdoMetDCas plays a role in the prostate tumorigenicity,on the other hand it has inhibitory effect on the formed prostate tumors.
引文
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