Study On The Targeting Therapy Of CD/TK Double Suicide Genes Mediated By Ultrasound Targeted Microvesicle (SonoVue) Destruction On Breast Cancer In Vitro

BackgroundBreast cancer is one of the major diseases that threaten the health of women, with the incidence increasing worldwide. However, conventional cancer therapies are accompanied by their intrinsic limitations, newly satisfied treatment methods are urgently needed. With the rapid development of molecular biology and genetic engineering, gene therapy represents a novel effective treatment model in cancer therapy, and suicide gene therapy has drawn greater attention in breast cancer treatment by its unique mechanism of action. There are several advantages of using E.coli cytosine deaminase (CD)/5-fluorocytosine (5-FC) and Herpes simplex virus thymidine kinase (HSV-TK) /ganciclovir (GCV) double suicide gene therapy systems, such as achieving synergetic effect, improving anti-tumor effect and avoiding drug resistance, etc.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 breast 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 ultrasound microvesicle.It has been revealed that KDR promoter over-expresses in the majority of the solid cancer cells and neogenetic vascular endothelial cells of neoplasma but not in normal cells, and many research results have demonstrated that KDR promoter could make genes of interest over-express exclusively both in tumor cells and its neogenetic vascular endothelial cells while reducing normal tissue toxicity.Viral vector and common non-viral vector liposome have shortages of high immunogenicity and toxicity, low transfection efficiency in vivo respectively, and lack of targeting. Ultrasound, which has been playing a very important role in targeted delivering of gene and active agents, merits further exploration. The aim is to use ultrasound and ultrasound echo contrast agents to offer a targeting delivering procedure, called the delivery system of ultrasound-mediated destruction and cavitation of targeted microbubbles. The core of the approach, which depend on the cavitation effect of microbubble under ultrasound field, is locally delivered pharmacological drugs or therapeutic genes which is coupled with microbubble or sacked within itself to target disease region while minimizing systemic toxic effects and achieving high concentrations. With the advantages of simplicity, invasiveness and easiness at bed, this delivery system is overwhelmingly welcomed to gene therapy, which currently was hampered due to bad targeting delivery and low transfection.Objectives1.To screen out the optimal parameter of ultrasound energy and to investigate the feasibility of ectogenous plasmid transfected into MCF-7 cells (human breast cancer cell line) using ultrasound-mediated microvesicle destruction as genetic carrier.2.To explore the specific lethal effects of CD/TK gene driven by the KDR promoter on MCF-7 cells in vitro by optimized ultrasound-microbubble-mediated transfection.Methods1. Sixteen groups of MCF-7 cells were exposed to different ultrasound intensity of 0.5,0.75,1.0,1.25W/cm2 for 10,30,45,60s with certain microbubble concentration respectively, in order to screen out the optimal ultrasound parameter that do no conspicuous harm to MCF-7 cells.2.MCF-7 cells were divided into 5 groups:naked plasmid group (NP), plasmid with SonoVue microvesicle group (MV), plasmid with ultrasound group (US), plasmid with ultrasound and SonoVue microvesicle group (US&MV), plasmid with liposome group (LP). PEGFP-C2 plasmid was transfected into them via each mediator mentioned above, the transfection efficiency of each group was calculated.3.MCF-7 cells producing KDR and LS174T cells without KDR were infected with the recombinant plasmid pEGFP-KDRP-CD/TK by optimal ultrasound parameter respectively. First, gene transfection ratio was compared according to green fluorescence expression. Then, CD/TK gene expression was detected by RT-PCR. Moreover, synergistic effect and "bystander effect" of CD/TK gene by using prodrugs (5-FC/GCV) were measured using an MTT cell inhibition assay.Results1.Cell viability decreased with the increase of ultrasonic intensity and the extension of irradiation time. The MCF-7 cell viability was still above 90% when the ultrasonic intensity was 0.75W/cm2 with irradiation time 45s or the ultrasonic intensity was 1 W/cm2 with irradiation time 30s. They were the best ultrasound parameters for MCF-7 cell transfection.2.Few green fluorescence cells were observed in either MV group or US group. However, large quantities of green fluorescence cells were observed in both US&MV group and LP group, with transfection ratio of (23.87±2.74)% and (22.03±2.29)% respectively, and no significant difference was found between them (P>0.05). The transfection efficiency of US&MV group is much higher than that of any of the MV or US group (P<0.001).3.Combinant plasmid pEGFP-KDRP-CD/TK was transfected into the MCF-7 and LS174T cells, and their transfection ratio was no difference by fluorescence microscopy, being (21.92±3.64)%and (20.97±2.74)% respectively. The difference was no significant (P>0.05).4.By RT-PCR, CD/TK fusion gene was proved to express in the KDR-positive cells (MCF-7), while silent in the KDR-negative cells (LS174T).5.In cytotoxicity experiment, the MCF-7 cells transfected with targeted vector pEGFP-KDRP-CD/TK were sensitive to prodrugs 5-FC and GCV. In single prodrug group 5-FC or GCV and combination group 5-FC/GCV, the cell inhibition ratio was (38.16±1.56)%, (37.29±1.95)% and (61.22±0.70)%, respectively. In combination group, cell inhibition ratio was significantly higher than single prodrug groups (P<0.001). Moreover, the inhibition ratio of each group was significantly higher than that of gene transfection (21.92±3.64)%(P<0.001). However, the transfected LS174T cells were not sensitive to prodrugs regardless of single 5-FC, GCV or combination of them (P>0.05). Conclusions1.The MCF-7 cells can tolerate the ultrasound at irradiation intensity 0.75W/cm2 and irradiation time 45s or irradiation intensity 1W/cm2 and irradiation time 30s.2.Ultrasound targeted microvesicle destruction (UTMD) is a safe, effective and targeting gene delivery system.3.Ultrasound-mediated microvesicle destruction shown effective-performance transfection ratio in MCF-7 cells and LS174T cells, and the transfection ratio of both cells wasn't different.4.KDR promoter can drive the expression of CD/TK double suicide gene targeted in MCF-7 cells because of high expression of KDR.5.Recombinant plasmid containing CD/TK fusion suicide genes under the control of KDRP exerted specific killing effect on MCF-7 tumor cells in vitro. Moreover, a double suicide gene/prodrugs approach (CD/5-FC and HSV-TK/GCV) is superior to a single approach.6.The antitumor mechanism of this therapeutic system included directly toxic effect on suicide gene modified cells and "bystander effect" on neighboring nonmodified cells.