Study On The Effects Of A Prostate-specific Antigen Promoter Mediated ODC And AdoMetDC Antisense RNA Expressing Adenovirus In The Treatment Of Prostate Cancer

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 adenovirusAim: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 adenovirusAim:To construct a prostatic androgen-dependent promoter mediated ODC and AdoMetDC antisense RNA-expressing adenovirusMethods: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 vitroAim: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.