Treatment Of Ovarian Cancer By Ultrasound-Microvesicle-Mediated Transfection Of The Fcy::Fur Suicide Gene Driven By Htert Promoter

Ovarian cancer is a common female genital system malignant tumor type with the highest mortality rate. Despite the fact that the clinical treatment for it has been developing dramatically in the past two decades, the average 5-year survival rate for a patient is less than 30%. Gene therapy has become a new treatment modality for ovarian cancer with the development of molecular biology and gene technology. Targeted expression of remedial genes and high transfection efficiency are the key points as well as the emphasis and nodus of gene therapy. As a new treatment modality for ovarian cancer, gene therapy has sparked a renewed interest with the emergence of novel targeted therapeutic strategies and the development of non-viral gene carriers such as nanoparticles and ultrasound-microvesicle.In the present study, we first analyzed the feasibility and transfection efficiency of ectogenous plasmid transfected into ovarian cancer SKOV3 cells by ultrasound-mediated microvesicle destruction. Then, we constructed a recombinant expression plasmids pGL3-hTERT-Fcy::Fur which contained hTERT promoter and Fcy∷Fur suicide gene and analyzed its expression in human ovarian cancer cells and human embryonic lung fibroblast cells. Eventually, we explored the therapeutic effect and the targeting of suicide gene using ultrasound-mediated microvesicle destruction as genetic carrier to transfect the recombinant plasmids pGL3-hTERT-Fcy::Fur into SKOV3 cells in vitro and transplanted tumor in vivo with the hope of providing an faithful experimental data for gene therapy for ovarian cancer. 4 parts are included in the present study.Part one Parameters screening and feasibility analyzing of the ectogenous plasmids transfected into skov3 cells using ultrasound-mediated microvesicle destruction as genetic carrierObjective To screen the optimal parameters of ultrasound intensity and microvesicle concentration and to investigate the feasibility of ectogenous plasmid transfected into SKOV3 cells (Human ovarian cancer cell line) using ultrasound-mediated microvesicle destruction as genetic carrier.Methods⑴6 groups SKOV3 cells were exposed continuously to different ultrasound intensity of 0.5,0.75,1.0,1.25,1.5 and 2.0 W/cm2 for 60 s respectively in order to screen out the optimal ultrasound intensity that do no conspicuous harm to SKOV3 cells by trypan blue staining.⑵A bottle of self-made lipid microvesicle was lysised into 5 ml PBS and its initial concentration was detected, then, the microvesicle,s initial concentration was diluted for 5 and 10 times by PBS. SKOV3 cells were under the impact of different combinations of the screened ultrasound intensity with microvesicle of different concentration so as to screen out the optimal parameters combination.⑶SKOV3 cells were divided into 5 groups: naked plasmid group (NP) , plasmid with microvesicle group (MV), plasmid with ultrasound group(US), plasmid with ultrasound and microvesicle group(MV & US), plasmid with liposome group(LP). pGL3-Promoter-EGFP plasmid were transfected into them via each mediator mentioned above, the transfection efficiency of each group was calculated.Results⑴The cells death rate is<10% when the ultrasound intensity is below 0.75 W/cm2.⑵When exposed to combination of the microvesicle concentration of 0.4×108/ml or 0.8×108/ml with the ultrasound intensity of 0.5 W/cm2, the cells death rate is <10%.⑶Few fluorescence cells were observed in either MB group or US group and no significant difference was found between them (p=0.64). However, large quantities of fluorescence cells were observed in both MB&US group and LP group, with an average number of 79.9±17.6 and 62.7±22.5 respectively, and significant difference was found between that of the two groups (p=0.041). The transfection efficiency of MB&US group is the highest than that of any of the others.Conclusions⑴The SKOV3 cells can tolerate the ultrasound at a 0.5 W/cm2 intensity and the microvesicle at a 0.8×108/ml or 0.4×108/ml concentration.⑵Ectogenous plasmid can be transfected into SKOV3 cells efficiently by ultrasound-mediated microvesicle destruction.⑶ultrasound-mediated microvesicle destruction is an targeting gene delivery system.Part two Construction, verification and expression of the recombinant Plasmid containing hTERT promoter and Fcy∷Fur suicide geneObjective To construct, verify the recombinant plasmid containing hTERT promoter and Fcy∷Fur suicide gene and detect its expression in SKOV3 [human ovarian cancer cell line, human telomerase reverse transcriptase (hTERT positive)] and MRC-5 (human embryonic lung fibroblast cell line, hTERT negative).Methods⑴The cDNA sequence (CDS) encoding the whole open reading frame of the Fcy::Fur gene was generated by a PCR strategy taking the pORF5-Fcy::Fur plasmid containing the complete CDS for Fcy::Fur as a template. A 5' primer containing the NCO I and a 3' primer containing the Xba I site were used to generate a 1139bp PCR product from BamH I to EcoR I. After being subcloned and completely sequenced to verify the correct reading frame and lack of other mutations, the Fcy::Fur cDNA was then introduced as an Nco I to Xba I fragment into the unique Nco I to Xba I sites of the P2XEB plasmid, from which Fcy::Fur can be expressed under the regulation of the hTERT promoter, thus acquiring the pGL3-hTERT- Fcy::Fur recombinant plasmid, which was further identified by restriction enzyme digestions. The control recombinant plasmid pGL3-SV40-Fcy::Fur (containing SV40 constitutive promoter, non tissue-specific) was constructed in much the same way through insertion of the Fcy::Fur PCR product into plasmid pGL3-promoter. All the recombinant plasmids were routinely transformed into Escherichia coli JM109, amplified, extracted, and finally identified by restriction enzyme analysis and gene sequencing by Shanghai Sangong technological Limited company.⑵After being identified and sequenced to verify the correct reading frame and lack of other mutations, the recombinant plasmids were transfected into SKOV3 and MRC-5 to detect its expression by reversed transcriptive-polymerase chain reaction and Westen-blot analysis.Results⑴pGL3-hTERT-Fcy::Fur and pGL3-SV40-Fcy::Fur were constructed successfully. Furthermore, DNA sequencing results showed that both the sequence of hTERT promoter and Fcy::Fur gene are identical with the array published in GeneBank and their insertion direction was correct.⑵Both the results of RT-PCR and Westen-blot indicated Fcy::Fur gene driven by hTERT promoter was expressed higher in SKOV3 than in MRC-5, significant difference was found between them.Conclusion The recombinant plasmid pGL3-hTERT-Fcy::Fur containing hTERT promoter and Fcy∷Fur suicide gene was constructed successfully, which has different expression levels in ovarian cancer cell and normal cell.Part three Ultrasound-microvesicle-mediated transfection of pGL3-hTERT-Fcy::Fur exhibits specific proliferation inhi- bition and apoptosis induction effects on SKOV3 ovarian cancer cells in vitroObjective To explore the specific lethal effects of Fcy::Fur gene driven by the hTERT promoter on SKOV3 ovarian cancer cells in vitro by ultrasound-microbubble-mediated transfection.Methods Both SKOV3 and MRC-5 Cells were transfected with pGL3-hTERT-Fcy::Fur, pGL3-SV40-Fcy::Fur or pGL3-Promoter plasmid (not containing the Fcy::Fur gene) respectivly using the ultrasound- microvesicle as gene vector. 48h after incubation with 5-FC at various concentrations (0, 32, 65, 130, 250 and 500 mg/L), cell counting kit-8 (CCK-8) assay was employed to analyze the proliferation inhibitory rate of cells in each group. Acridine orange/ethidium bromide (AO/EB) staining and transmission electron microscopy were used for the analysis of cell apoptosis. High performance liquid chromatogram(HPLC)was introduced to analyze the concentration change of 5-FC. hTERT expressions and its promoter activity in SKOV3 and MRC-5 cells were detected respectively by reversed transcriptive-polymerase chain reaction (RT-PCR) and luciferase analysis.Results⑴Transfection of pGL3-hTERT-Fcy::Fur Plasmid led to specific proliferation inhibition and induced specific apoptosis in SKOV3. The SKOV3 and MRC-5 cells that were transfected with pGL3-SV40-Fcy::Fur showed an proliferation inhibitory rate that was not significantly different (P=0.87), nevertheless, the proliferation inhibitory rate of pGL3-hTERT-Fcy::Fur/SKOV3 is higher than that of pGL3-hTERT-Fcy::Fur/MRC-5 (P=0.036). pGL3-Promoter showed no dramatic influence on proliferation of either SKOV3 or MRC-5 cells co-cultured at a 5-FC concentration that was below 250 mg/L (the proliferation inhibitory rates were below 10%). when co-cultured with 5-FC at a concentration of 250 mg/L, apoptosis rate was significantly higher in pGL3-hTERT-Fcy::Fur/SKOV3 cells than in pGL3-hTERT- Fcy::Fur/MRC-5 cells, P=0.028, yet, there was no significant difference between the apoptosis rate in pGL3-SV40-Fcy::Fur/SKOV3 cells and pGL3-SV40-Fcy::Fur/MRC-5 cells, P = 0.82. Moreover, swollen mitochondria and apoptotic body appeared in pGL3-hTERT-Fcy::Fur/ SKOV3 cells, while no significant changes were detected in pGL3-hTERT-Fcy::Fur/ MRC-5 cells.⑵The result of HPLC indicated that 5-FC concentration was lower in SKOV3 than that in MRC-5 cells 48 hour after transfection by pGL3-hTERT-Fcy::Fur.⑶Positive and negative expression of hTERT mRNA were deteched in SKOV3 and MRC-5. The SKOV3 cells showed a more potent promoter activity(26.2%)that was 40 times higher than that in MRC-5 cells(0.65%).Conclusions⑴Fcy::Fur suicide gene driven by the hTERT promoter exhibits specific proliferation inhibition and apoptosis induction effects on hTERT positive SKOV3 while has little lethal effects on hTERT negative MRC-5.⑵The specific proliferation inhibition and apoptosis induction effects of the pGL3-hTERT-Fcy::Fur plasmid in SKOV3 cells was due to its potent hTERT promoter activity which resulted in much higher transcription of the Fcy::Fur gene, on the contrary, the low hTERT promoter activity led to its lower transcription in MRC-5 cells. Part four Treatment of transplanted ovarian cancer by ultrasound-mediated pGL3-hTERT-Fcy∷Fur-loaded microv- esicle destructionObjective To explore the targeting anti-tumor effects on transplanted ovarian cancer using ultrasound-microvesicle as gene transfer to mediate pGL3-hTERT-Fcy∷Fur gene transfection.Method⑴Transduction targeting mediated by ultrasound-mediated microvesicle destruction. 25 nude mice modle of ovarian cancer were assigned randomly to 5 groups, each group received different treatment.①Ultrasound targeted destroy the gene-loaded microvesicle group (US&MV), 0.2 ml pGL3-hTERT-Fcy∷Fur-loaded microvesicle was injected into nude mice through tail vein, then, ultrasound irradiation was released to destroy the microvesicle on the region of tumor.②Ultrasound irradiated gene group (US), 0.2 ml culture fluid containing gene was injected into nude mice, then, ultrasound irradiation was released on the region of tumor.③Ultrasound irradiated void microvesicle group(US&VMV), 0.2 ml microvesicle not containing gene was injected into nude mice, then, ultrasound irradiation was released.④Gene-loaded microvesicle group (MV), gene-loaded microvesicle was injected into nude mice, but ultrasound irradiation was not released.⑤Control group (CT), 0.2 ml physiological saline was injected.⑵Transcription targeting mediated by hTERT promoter. 20 nude mice modle of ovarian cancer were assigned randomly to 4 groups.①pGL3-hTERT-Fcy∷Fur-loaded microvesicle group.②pGL3-Pro-Fcy::Fur-loaded microvesicle group.③pGL3- Promoter loaded microvesicle.④Control group. Both gene-loaded microvesicle and physiological saline were injected into nude mice through tail vein, ultrasound irradiation was released on the region of tumor and liver. Each group,s treatment was performed everyday and last for 7 days. 0.3 ml 5-FC dilution administering(500 mg/kg)through intraperitoneal injection followed the treatment from the 2nd day on, 5 days after the last treatment, all the mice were sacrificed and the tumors were harvested to measure volumes, masses and calculate the tumor inhibition rate. Apoptotic cells were detected by TUNEL analysis.Results⑴The results of transduction targeting.①There is no significant difference about the mean tumor volume, mass and cell apoptosis rate between MV and CT group, P>0.05.②There is no significant difference about the mean tumor volume, mass and cell apoptosis rate between US and US&VMV group, P>0.05. Nevertheless, significant difference about the index mentioned above were found in the two groups when compared with CT group.③Compared with any group of the others, the mean tumor volume and mass was the lowest, while its tumor inhibition rate and cell apoptosis rate was the highest in US&MV group, P<0.05.⑵The results of transcription targeting.①Compared with pGL3-Promoter loaded microvesicle group, there is significant difference about the mean tumor volume, mass and cell apoptosis rate in both the pGL3-hTERT-Fcy∷Fur-loaded microvesicle group and pGL3-Pro- Fcy::Fur-loaded microvesicle group, P<0.05.②There is no significant difference about the tumor cell apoptosis rate between pGL3-hTERT- Fcy∷Fur-loaded microvesicle group and pGL3-Pro-Fcy::Fur-loaded microvesicle group, P>0.05. Nevertheless, there is significant difference about the liver cell apoptosis rate between them, the apoptos rate is higher in the latter.Conclusions①Ultrasound-mediated gene-loading microvesicle destruction is a targeting gene vector. The technique is possible to be adopted as a novel tool gene transfer for gene therapy.②Fcy∷Fur gene driven by hTERT promoter has targeting lethal effects on transplanted ovarian cancer while do little harm to normal cells.③Ultrasound- mediated Fcy∷Fur-loading microvesicle destruction exhibits efficient anti-cancer effect on transplanted ovarian cancer, the therapeutic mode is possible to be adopted as a novel tool for ovarian cancer.