Study On The Therapy For Colorectal Cancer With Double Suicide Gene Driven By VEGF Promoter And Survivin Antisense Oligonucleotide

ObjectivesThe recombinant adenovirus containing cytosine deaminase and typeâ… herpessimplex virus thymidine kinase gene driven by vascular endothelial growth factorpromoter (Ad-VEGFP-CDglyTK) and survivin antisense oligonucleotide(ASODN)was used as vector to infection colorectal cancer Lovo cell line in vitro and in vivorespectively. Then we studied the lethal effect in vitro and the effect of tumorrestrained in vivo. Survivin ASOND can inhibit the expression of survivin andinduce apoptosis of colorectal cancer and eliminate the cytoprotective action ofVEGF in the middle of vasoformation, which induce apoptosis of endothelial celland degenerescence of micrangium. Double suicide gene driven by VEGF promoterchoose to kill and wound the colorectal cancer cell which secrete VEGF, anddecrease the secretion of VEGF. So double suicide gene driven by VEGF promoterand survivin ASODN can inhibit the growth of tumor through synergistic action. Methods1. The study of the lethal effect in vitro of survivin ASODN with colorectal cancerLovo cellWe take the different concentration survivin ASODN transfection to Lovo celland survey the survival rate of tumor cell and detect the expression of survivinmRNA by the method of RT-PCR.Then we detect the expression of survivin proteinof colorectal cancer Lovo cell by the method of Western blot. We detect the cellcycle by flow cytometer in the end.2. Study on the lethal effect in vitro to colorectal cancer Lovo cell with doublesuicide gene driven by VEGF promoter and survivin ASODNWe do the recovery and amplification of recombinant adenovirusAd-VEGFP-CDglyTK, which contain Green fluorescent protein (GFP) reportgene.Then We purified the recombinant adenovirus by density gradientcentrifugation of cesium chloride and carry out the determination of rite. Lovo cellwas infected by recombinant adenovirus. Then the Lovo cell was cultured with theprodrug 5-Fluorocytosine and Ganciclovir.We detect the transfection efficiency ofLovo cell and survival rate of Lovo cell and bystander effect of double suicide gene.The cellular morphology was also observed. We observed the hyperplasy andapoptosis of Lovo cell treated by survivin ASODN and double suicide gene drivenby VEGF promoter.3. Study on the antitumor effect in vivo to colorectal cancer Lovo cell in nude micewith double suicide gene driven by VEGF promoter and survivin ASODNThe 4-week-old female Balb/c nude mice was inoculated as hosts for Lovo cellxenografts, a total of 0.5×10⁷ Lovo cells in 0.2ml PBS was injected subcutaneouslyusing an 18-guage needle, and tumors were allowed to grow for 2 weeks beforerandomizing mice by size. All tumors were at least 0.5cm and mice were grouped according to treatment regimen.The nude mice was injected survivin ASODN andthe recombinant adenovirus according to differert groups. Thereafter,5-Fluorocytosine and Ganciclovir were injected to peritoneal cavity of the animalmodel. Then we measured the weight of the solid tumor and calculated thesuppression rate of tumor. Moreover, hematoxylin and eosin stain of tumor tissueswere performed for histological examination and immunohistochemistry method wasused to detect to microvessel density (MVD) of tumor tissues.Results1. The study of the lethal effect in vitro of survivin ASODN with colorectal cancerLovo cell1.1 The survival rate ofcolorectal cancer Lovo cell treated by survivin ASODNThe results of survival rate of different concentration of survivin ASODN groupand survivin SODN group and control group was measured. It had conspicuousdifference between survivin ASODN group with control group and between survivinASODN group with survivin SODN group by analysis of variance(P＜0.05). It hadno conspicuous difference between survivin SODN group with control group(P＞0.05). The results hinted that colorectal cancer Lovo cell was inhibited bysurvivin ASODN conspicuously.The depressant effect was most obviously while theconcentration of survivin ASODN is 400ng/ml.1.2 The expression of survivin mRNA of colorectal cancer Lovo cell treated bysurvivin ASODNThe production of RT-PCR displayed that the strap of survivin was showed inall groups. The expression of survivin mRNA was lowest in all groups, which wasstatistical significance(P＜0.05). It had no conspicuous difference among the controlgroup and the lip group and survivin SODN group(P＞0.05). The difference of expression of suvivin mRNA was most obviously while the concentration of survivinASODN was 400ng/ml and 800ng/ml.1.3 The expression of survivin protein of colorectal cancer Lovo cell treated bysurvivin ASODNThe production of Western blot displayed that the expression of survivin proteinhad no conspicuous difference among the control group and the lip group andsurvivin SODN group(P＞0.05). The expression of survivin protein was lower thanabove-mentioned groups while the concentration of survivin ASODN was 200ng/ml.The difference of expression of suvivin protein was most obviously while theconcentration of survivin ASODN was 400ng/ml and 800ng/ml, which had statisticalsignificance(P＜0.05). The expression of survivin protein in 400ng/ml survivinASODN group is 16.33±1.48.1.4 The change of cell cycle of colorectal cancer Lovo cell treated by survivinASODNThe production of flow cytometry displayed the apoptosis peak of all groups:control group(0.91±0.29)%, survivin SODN group(0.87±0.22)%, 200ng/mlsurvivin ASODN group (18.56±2.80)%, 400ng/ml survivin ASODN group (30.47±1.73)%. It had conspicuous difference between survivin ASODN group withcontrol group and between survivin ASODN group with survivin SODN group byanalysis of variance(P＜0.05), which hinted that survivin ASODN could induceapoptosis of colorectal cancer cell powerfully. The ratios of G₁ phase cells werecontrol group(75.22±2.13)%, survivin SODN group(72.17±2.63)%, 200ng/mlsurvivin ASODN group (43.24±2.75)% and 400ng/ml survivin ASODN group (9.37±2.67)%. The ratios of S phase cells were not conspicuous difference in allgroups(P＞0.05). The ratios of G₂/M phase cells were control group(4.46±1.44)%,survivin SODN group(5.43±1.43)%, 200ng/ml survivin ASODN group (13.40± 2.04)% and 400ng/ml survivin ASODN group (39.85±2.66)%. The ratios of G₂/Mphase cells had conspicuous difference between survivin ASODN group with controlgroup and between survivin ASODN group with survivin SODN group by analysisof variance(P＜0.05). The phenomenon was called G₂/M phase blockage. SurvivinASODN could induce G₂/M phase blockage of colorectal cancer Lovo cell by meansof dose-dependent.2. Study on the lethal effect in vitro to colorectal cancer Lovo cell with doublesuicide gene driven by VEGF promoter and survivin ASODN2.1 the transfection efficiency of recombinant adenovirus in vitro to colorectalcancer Lovo cellWe observed that the Lovo cell infected by recombinant adenovirus showedgreen flourescene in fluorescence microscope and the Lovo cell no infected byrecombinant adenovirus do not showed green flourescene. We take differentconcentration MOI of Ad-VEGFP-CDglyTK to Lovo cell and observed that thetransfection efficiency of recombinant adenovirus gradually increased with theincreasing of the MOI of Ad-VEGFP-CDglyTK. GFP was expressed more than 90%as the MOI of the recombinant adenoviruses was 100. It showed that therecombinant adenoviruses constructed by our laboratory had higher transfectionefficiency.2.2 The survival rate of colorectal cancer Lovo cell infected by recombinantadenovirusesThe survival rate of Lovo cell measured by MTT showed that the survival rateof Lovo cell infected by the recombinant adenoviruses gradually decreased with theincreasing of the drug concentration after 5-FC or GCV or 5-FC+GCV was taken. Itwas called concentration dependent to prodrug therapy. The survival rate of Lovocell was (56.47±2.39)% as 1μg/ml concentration of GCV. The survival rate of Lovo cell was (53.35±2.20)% as 40μg/ml concentration of 5-FC. The survival rateof Lovo cell was (27.23±1.46)% as GCV and 5-FC was used association. It showedthat the survival rate of Lovo cell was conspicuous difference between singleprodrug with double prodrug as fixed concentration of 5-FC and GCV.2.3 Bystander effect of CDglyTK geneThe results of bystander effect showed as follow. Lovo cell was infected bydifferent proportion recombinant adenoviruses. When the proportion of recombinantadenoviruses, the survival rates of Lovo cell were showed: GCV group (65.25±1.58)%, 5-FC group (63.63±1.71)% and GCV+5-FC group (45.60±2.07)%. Thesurvival rates of above-mentioned groups were lower than control group, whichhinted its statistical significance (P＜0.05). It had no conspicuous difference (P＞0.05)between GCV group with 5-FC group. We observed that the survival rate of Lovocell gradually decreased with the increasing proportion of Lovo cell infected byrecombinant adenoviruses and it had conspicuous difference between control groupwith other groups (P＜0.05). It had conspicuous difference between GCV+5-FCgroup with GCV groups and between GCV+5-FC group with GCV groups 5-FC(P＜0.05). It hinted that the bystander effect of double suicide gene driven by VEGFpromoter was stronger than single suicide gene. CD gene and TK gene showedsynergistic effect.2.4 Changing of coloreactal cancer Lovo cell in TEM after treatmentCell shrinkage, chromatin gathered along the nuclear membrane, cell budding.Chromatin condensation, fragmentation with intensive electron density and apoptoticbody were observed by TEM. And the others exhibited necrosis.2.5 The cell proliferation of colorectal cancer Lovo cell treated by double suicidegene driven by VEGF promoter and survivin ASODNThe results showed the survival rates after treatment of all groups: control group (98.35±1.22)%, survivin ASODN group (62.49±2.478)%,Ad-VEGFP-CDglyTK group (69.43±1.49)%, survivinASODN+Ad-VEGFP-CDglyTK group (31.48±2.38)%. It had conspicuousdifference beween control group with other groups (P＜0.05). The survival rate ofgene therapeutic alliance group was lower than survivin group andAd-VEGFP-CDglyTK group. It had statistical significance (P＜0.05). It showed thesynergistic effect between double suicide gene driven by VEGF promoter andsurvivin ASODN in inhibiting the proliferation of colorectal cancer Lovo cell.2.6 The change of apoptosis of colorectal cancer Lovo cell treated by double suicidegene driven by VEGF promoter and survivin ASODNThe production of flow cytometry displayed the apoptosis rates of all groups:survivin ASODN group (19.55±2.38)%, Ad-VEGFP-CDglyTK group (21.83±2.23)%, survivin ASODN+Ad-VEGFP-CDglyTK group (38.53±2.30)%. Theapoptosis rates of above-mentioned groups promoted apoptosis effect and hadconspicuous difference than control group (P＜0.05). It had no conspicuousdifference between survivin ASODN group and Ad-VEGFP-CDglyTK group(P＞0.05). The apoptosis rates of gene therapeutic alliance group had conspicuousdifference than survivin group and Ad-VEGFP-CDglyTK group (P＜0.05). It showedthe synergistic effect between double suicide gene driven by VEGF promoter andsurvivin ASODN in apoptosis of colorectal cancer Lovo cell.3. Study on the antitumor effect in vivo to colorectal cancer Lovo cell in nude micewith double suicide gene driven by VEGF promoter and survivin ASODN3.1 The growth of bearing cancer of nude mice and antitumor effect with genetherapeutic allianceLovo cell was inoculated in nude mice and then we may touch small foxtailmillet nodus after 4～5days. The nodus showed in every nude mice after 12 days. Achievement ratio of inoculating was 100ï¼…. We began the experiment when meandiameter achieved 50mm. The results showed the beginning volume of tumor had noconspicuous difference in all groups (P=0.389). The final weight of tumor and thesuppression rates showed after treatment: control group (516.58±10.58)mg;Ad-VEGFP-CDglyTK group (238.74±15.77)mg, (53.78±3.12)ï¼…; survivinASODN group (225.61±13.47)mg, (56.23±2.60)ï¼…;Ad-VEGFP-CDglyTK+survivin ASODN group (63.70±3.41)mg, (87.66±0.70)ï¼….It had conspicuous difference between control group with other groups (P＜0.05). Ithinted that double suicide gene driven by VEGF promoter and survivin ASODN hadantitumor effect and gene therapeutic alliance had stronger antitumor effect. Doublesuicide gene driven by VEGF promoter and survivin ASODN had synergistic effect.3.2 Pathological change of transplantation tumorThe pathological change showed the growth of tumor was inhibited especiallygene therapeutic alliance group. The tissue section of tumor showed lamellar zone ofnecrosis and massive inflammatory cell infiltrate.3.3 Detecting of microvessel density (MVD) of tumorThe results of MVD showed: control group 19.25±3.19, Ad-VEGFP-CDglyTKgroup 11.33±2.46, survivin ASODN group 10.42±2.35,Ad-VEGFP-CDglyTK+survivin ASODN group 3.33±1.56. The MVD of controlgroup was higher than other groups, which had statistical significance (P＜0.05). TheMVD of Ad-VEGFP-CDglyTK group and survivin ASODN group were higher genetherapeutic alliance group and It had conspicuous difference (P＜0.05).Conclusions1. Survivin ASODN transfection colorectecal cancer Lovo cell hinder the the processof transcription and translation of survivin mRNA and decreases the expression of survivin protein.2. Survivin ASODN may inhibit the proliferation of colorectal cancer Lovo cell. Thedepressant effect of survivin ASODN gradually increases with increasing of itsconcentration.3. Survivin ASODN may seal the expression of survivin gene and induce theapoptosis ofcolorectal cancer Lovo cell.4. Double suicide gene driven by VEGF promoter has conspicuous lethal effect invitro and the lethal effect is stronger than single suicide gene.5. The mechanism of action of double suicide gene driven by VEGF promoter isdirect lethal effect to target cell and bystander effect, and showed inhibiting theproliferation of target cell and induce the apoptosis of target cell after treatment.6. We Study the effect to colorectal cancer Lovo cell treated by double suicide genedriven by VEGF promoter and survivin ASODN in vitro. Double suicide gene drivenby VEGF promoter and survivin ASODN have synergistic effect in the process ofinhibiting the proliferation of colorectal cancer Lovo cell and inducing apoptosis ofcolorectal cancer Lovo cell.7. Double suicide gene driven by VEGF promoter and survivin ASODN haveantitumor effect in vivo and gene therapeutic alliance had stronger antitumor effect.Double suicide gene driven by VEGF promoter and survivin ASODN had synergisticeffect in vivo.