| Part ?:Oncolytic adenovirus carrying sh RNA targeting SATB1 inhibits prostate cancer growth and metastasisObjective: To investigate effects of oncolytic adenovirus(OAd)ZD55-SATB1 on the inhibition of prostate cancer growth and metastasis In vivo and in vitro.Methods: The construction and identification of ZD55-SATB1.The viral plaques were purified and propagated on low passage HEK293 cells.The functional PFU titers were determined by plaque assay on low passage HEK293 cells.In vitro: the inhibitory effect of ZD55-SATB1 on SATB1 expression was evaluated by RT-PCR and Western blot analysis.The cytotoxicity of ZD55-SATB1 was detected by CCK8 assay and crystal violet staining assay.Cell invasion was detected by Matrigel invasion assay.The expression of E1 A protein in DU145 and LNCa P cell lines infected with viruses respectively was detected by Western blot.In vivo: DU145 cells were cultured and then develop tumor model by subcutaneous injection.When tumors reached 100-150 mm3,mice were divided randomly into four groups(8 mice for each group),which were treated by intratumoral injection of 100 ?L of ZD55-SATB1,ZD55-EGFP,Ad-SATB1 or PBS,respectively.Virus(5x108pfu/day)was administered by intratumoral injection every other day.The tumor was monitored every week by measuring tumor size for 49 days.At the end of the experiment,tumors were harvested for HE staining,immunohistochemistry and TUNEL assay.Results: 1.Crystal violet staining assay showed that ZD55-SATB1 exhibited obvious cytotoxicity in DU145 and LNCa P cells.After 96 h,the survival rate of the ZD55-SATB1group(31.6±3.31,36.6±3.63)% was significantly lower than those of the ZD55-EGFP group(61.7±2.42,65.4±2.324)%,Ad-SATB1 group(82.4±3.54,86.4±4.18)%,and ZD55-EGFP(61.65±4.53)%(P<0.05)for DU145 and LNCa P respectively.The cytopathic effect(CPE)of ZD55-SATB1 was much stronger than that of ZD55-EGFP and Ad-SATB1.ZD55-SATB1 showed obvious CPE in PZ-HPV-7 cells only when the dosage was increased to 100 MOI.2.CCK-8 assay showed that cell viability of ZD55-SATB1 group(31.6±3.31%,36.6±3.63%)was significantly lower than that of ZD55-EGFP group(61.7±2.42%,65.4±2.324%)and Ad-SATB1 group(82.4±3.54%,86.4±4.18%)for DU145 and LNCa P cells,respectively after 96 h.It was notable that significant CPE was not observed in PZ-HPV-7 cells treated with three different viruses.3.RT-PCR and Western blot assay:there was a significant decrease in SATB1 m RNA levels in DU145 and LNCa P cells 48 h after infection with ZD55-SATB1 compared to the Ad-SATB1,ZD55-EGFP,PBS group(P<0.05),SATB1 m RNA level reached(36.5±3.1,43.1±4.2)% and(51.9±3.2,61.9±3.4)% of the control for DU145 and LNCa P cell lines with ZD55-SATB1,Ad-SATB1 infection at a MOI of 10,respectively.In contrast,the ZD55-EGFP showed only a mild effect in reducing SATB1 m RNA expression.4.Western blot assay:SATB1 protein level reached(40.1±3.7,61.9±3.4)% and(45.9±3.2,67.1±3.7)% of the control for DU145 and LNCa P cell lines with ZD55-SATB1,Ad-SATB1 infection at a MOI of 10,respectively,whereas infection of ZD55-EGFP had little effect on SATB1 protein expression.These results demonstrate that ZD55-SATB1 could effectively knock down SATB1 gene expression in Prostate cancer cells.ZD55-SATB1 and ZD55-EGFP expressed E1 A protein,and Ad-SATB1 failed to express E1 A protein.The relative protein levels of E1 A in the prostate cancer cell lines DU145 and LNCa P treated with ZD55-SATB1 were significantly higher than those observed in human normal prostate epithelial cell line PZ-HPV-7(P<0.01),which indicated that ZD55-SATB1 has cancer specificity.5.Transwell cell invasive experiment shows that the number of cells that invaded the membrane in ZD55-SATB1 group was 61.3±17.25 and 25.4±6.2,significantly less than that of the Ad-SATB1 group(170.1±25.44 and 42.7±10.7,P <0.05),the ZD55-EGFP group(295.4±23.36 and 77.6±15.2,P <0.05)and the PBS group(490.5±33.36 and 112.6±20.1,P <0.05),these results suggest that ZD55-SATB1 significantly inhibited invasion of DU145 and LNCa P cells in vitro compared to other groups.6.ZD55-SATB1 inhibited the growth of prostate cancer xenograft tumors better than ZD55-EGFP and Ad-SATB1.The mean tumor size of ZD55-SATB1 group was 295.3±51.9 mm3,much smaller than that of ZD55-EGFP group(525.6±89.3mm3),Ad-SATB1(582.3±97.0 mm3)and PBS group(777.1±139.8 mm3),the difference was statistically significant(P<0.05).7.Lung tissues HE staining showed tumor metastasis was found in nude mice lung with 8 of PBS group,while two of ZD55-EGFP group,none of ZD55-SATB1 and Ad-SATB1 group,which indicated ZD55-SATB1 can effectively inhibit the distant metastasis of prostate cancer by RNA interference targeting SATB1 gene.8.TUNEL staining showed that the percentage of TUNEL-positive cells in ZD55-SATB1 treated group(87.3±4.8)% was significant higher than that in ZD55-EGFP treated group(51.2±3.4)%,Ad-SATB1 treated group(41.3±3.2)%and PBS treated group(20.1±1.9)%,which show that ZD55-SATB1 can promotes tumor cell apoptosis in vivoConclusion: We successfully engineered an oncolytic adenovirus ZD55-SATB1,which carry sh RNA targeting SATB1.The recombinant adenovirus ZD55-SATB1 showed excellent anti-tumor efficacy not only in vitro but also in xenograft prostate tumor in nude mice.These results suggest that ZD55-SATB1 may be a promising agent for the treatment of human prostate cancer.Part ?:Oncolytic virus carrying sh RNA targeting SATB1 combined with chemotherapy,endocrinotherapy treat hormone-sensitive prostate cancerObjective: To investigate the treatment effect of oncolytic adenovirus carrying sh RNA targeting SATB1(ZD55-SATB1)combined with chemotherapy,endocrinotherapy on a hormone-sensitive prostate cancer.Methods: 1.The viability and cytotoxicity of prostate cancer LNCa P cells was assessed by CCK-8 assay.2.Cell apoptosis was determined by Hoechst 33258 and TUNEL assay.3.Migration assay was performed to evaluate the migration of LNCa P cells.4.Invasion assay was performed to evaluate the invasion of LNCa P cells.5.The expression of SATB1 and apoptosis associated protein caspase-3?caspase-8?Bcl-2 in LNCa P cells were detected by Western Blot.6.Construct human prostate cancer LNCa P cells xenograft models,divided into eight groups: Control group(PBS),Endocrine therapy group(ET group: Castration operation),Drug treatment group(DTX group),adenovirus treatment group(ZD55-SATB1 group),Endocrine therapy combined with drug treatment group(ET+DTX group),Adenovirus combined with endocrine therapy group(ZD55-SATB1+ET group),Adenovirus combined with drug treatment group(ZD55-SATB1+DTX group),Adenovirus combined with endocrine therapy and drug treatment group(ET+DTX+ZD55-SATB1 group).7.Detect the volume of xenografts of nude mice in each group and draw tumor time-growth curve.8.H&E assay was detected to observe the cell growth of xenografts.9.Immunohistochemistry analysis was performed to evaluate the expression of apoptosis-related proteins including Bcl-2,caspase-3 and caspase-8.Results: 1.CCK-8 shown that the LNCa P cells were treated following with 5.0MOI ZD55-SATB1 plus 1.0ng/ml DTX,10 MOI ZD55-SATB1,10 MOI ZD55-EGFP,2ng/ml DTX,100ng/ml Sitelong and PBS after 48 h,and the survival rate were respectively(37.61±2.17)%,(65.32±5.06)%,(72.25±3.40)%,(55.94±3.97)%,(86.67±4.62)%,(81.17±2.19)%.Compared with ZD55-SATB1 group,DTX group,ZD55-EGFP group,Sitelong group,ZD55-SATB1 combined with DTX could markedly inhibit the proliferation of LNCa P cells,the differences had statistical significance(P<0.05).Compared with Sitelong group,ZD55-SATB1 combined with DTX group could markedly inhibit the proliferation of LNCa P cells,the differences had statistical significance(P<0.05).These results illustrated that ZD55-SATB1 combined with DTX without testosterone could markedly inhibit the proliferation of LNCa P cells compared with ZD55-SATB1 group,DTX group,ZD55-EGFP group,DTX group alone.2.Hoechst33258 shown that after treated with ZD55-SATB1(5.0MOI)plus DTX(1.0ng/ml),DTX(2.0ng/ml),ZD55-EGFP(10MOI),ZD55-SATB1(10MOI),Sitelong(100ng/ml),PBS group on LNCa P cells,at 48 h,the cell apoptosis rate(%)were respectively 64.00±6.52,52.67±8.14,33.33±3.59,36.52±7.84,5.71±2.26 and 6.01±4.64.Compared with monotherapy group,ZD55-SATB1 combined with DTX group could observably accelerate cell apoptosis,the differences had statistical significance(P<0.05).3.The average cell numbers crossed the membrane after 24 h in ZD55-SATB1(5.0MOI)plus DTX(1ng/ml),ZD55-SATB1(10MOI),ZD55-EGFP(10MOI),DTX(2ng/ml),Sitelong(100ng/ml)were 27.23±7.14?35.10±2.38?40.31±2.59?68.46±3.58?118.01±5.64 and 106.24±10.35,respectively.These results suggest that the combined treatment group significantly inhibited migration of LNCa P cells in vitro compared to other groups(P<0.05).4.Transwell cell invasive experiment shows that the number of cells that invaded the membrane in ZD55-SATB1(5.0MOI)plus DTX(1.0ng/ml)group was 12.31±0.41,significantly less than that of the ZD55-SATB1(10MOI)group(21.93±2.11),the ZD55-EGFP group(26.12±2.13),Sitelong group(76.82±4.31)and the PBS group(68.62±4.22),there were significant difference between the combined treatment group and the single treatment groups(P<0.01),these results suggest that combined treatment group significantly inhibited invasion of LNCa P cells in vitro compared to other groups.5.TUNEL assay shown that after treated with PBS,100ng/ml Sitelong,2 ng/ml DTX,10 MOI ZD55-EGFP,10 MOI ZD55-SATB1,5.0MOI ZD55-SATB1+1 ng/ml DTX on LNCa P cells,at 48 h,the cell apoptosis rate(%)were respectively(9.48±3.19)%,(4.36±2.23)%,(13.802±4.74)%,(12.29±5.26)%,(14.70±3.84)%,(31.13±6.88)%.Compared with the control group,DTX group,ZD55-EGFP group,ZD55-SATB1 group,ZD55-SATB1+DTX group had more strong red fluorescent markers which shown more apoptotic cells,the differences had statistical significance(P<0.05).6.Western blot shown that the expression of SATB1 in ZD55-SATB1(5.0MOI)plus DTX(1ng/ml),ZD55-SATB1(10MOI),ZD55-EGFP(10MOI),DTX(2ng/ml),Sitelong(100ng/ml)and PBS group were respectively 5.35±1.49,12.73±7.80,29.02±3.54,45.37±3.76,80.03±4.02,91.72±4.45,the expression of SATB1 was obvious decline in the combination group,there was significant difference between the combined treatment group and the single treatment groups,the differences had statistical significance(P<0.05).Moreover,ZD55-SATB1 combined with DTX could inhibit the expressioin of SATB1 while DTX had no effect on silenting SATB1 gene.On the other side,compared with the PBS group,the expression levels of caspase-3 and caspase-8 in each experimental group were up-regulated,and the expression of Bcl-2 protein was down-regulated,there was significant difference between the combined treatment group and the single treatment groups(P<0.05).7.The average tumor size of the ZD55-SATB1+DTX+ ET group was 264.92±28.88 mm3,which was much smaller than that of the ET+ZD55-SATB1 group(787.52±221.15mm3),the ET+DTX group(1589.47±300.91mm3),the ZD55-SATB1+DTX group(555.64±254.89 mm3),the ZD55-SATB1 group(1127.25±271.93 mm3),the ET group(2287.41±254.89 mm3),the DTX group(1679.58±258.82 mm3),and PBS group(2602.13±325.74 mm3).The xenograft tumor volume of adenovirus combined with endocrine therapy and drug treatment group were smaller than control group and monotherapy group,the differences had statistical significance(P<0.05).8.HE staining: there were a mass of tumor cells scattered in nests or clusters with the atypical and enlarged cell nuclei containing prominent nucleoli in ET group,DTX group,ZD55-SATB1 group,ET+DTX group,ZD55-SATB1+ET group,ZD55-SATB1+DTX+ET group.The necrosis tumor cells were more obvious in ZD55-SATB1+DTX+ ET group.9.Immunohistochemical staining shown that the expression levels of caspase-3 and caspase-8 were up-regulated,while the expression of Bcl-2 protein was down-regulated in combination group,compared with the DTX group,endocrinotherapy or oncolytic adenovirus treatment group alone,the differences had statistical significance(P<0.05).Moreover,the expression of CD31 in combination group was down-regulated,which indicate that the combination group could accelerate the expression of pro-apoptotic proteins,decrease the expression of inhibitor of apoptotic proteins and inhibit the formation of tumor tissue vessels.Conclusion: 1.Both oncolytic adenovirus and docetaxel had cell-killing effects on prostate cancer LNCa P cells while the testosterone could promote LNCa P cell proliferation.Meanwhile,the combined use of oncolytic adenovirus with docetaxel and androgen deprivation therapy had the synergistic anti-tumor effects.2.Oncolytic adenovirus,docetaxel and surgical castration could remarkably inhibit the growth of xenografts of LNCa P cells and the anti-tumor effect of combination group was more effectively than monotherapy group.The mechanisms probably include inhibiting the growth of tumor cells,inducing cell apoptosis and inhibiting angiogenesis. |
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