Mechanism and efficacy of nanoparticle-mediated wt-p53 gene therapy in breast cancer model.

摘要

p53, a tumor suppressor gene, encodes a nuclear phosphoprotein involved in the control of cell growth and apoptosis. Breast cancer is associated with a high degree of mutations in the p53 gene that leads to loss of apoptotic control over cell proliferation. Introduction of wt-p53 gene into tumor cells has been shown to restore their normal p53 protein functions, and hence is suggested as a potential therapeutic approach for breast cancer therapy. In this thesis, biodegradable nanoparticles were investigated as a gene expression vector and their efficacy was evaluated using wt -p53 gene in a breast cancer mouse model. The hypothesis of this research was that the nanoparticle-mediated sustained wt-p53 gene delivery would result in sustained gene effect in the tumor tissue and hence would inhibit tumor growth. Initially, the effect of various formulation parameters were evaluated and then the optimized formulation of nanoparticles loaded with wt-p53 gene was evaluated for antiproliferative activity in human breast cancer cell line (MDAMB-435S) and for tumor inhibition in mouse model of breast cancer. It was demonstrated that particle size, DNA-loading and its release, and surface characterics of nanoparticles affect gene expression. Nanoparticles loaded with wt-p53 DNA demonstrated greater and sustained antiproliperative activity in vitro as compared to that with naked DNA and DNA-liposome complex. The greater efficacy of wt-p53 DNA-loaded nanoparticles was attributed to sustained intracellular DNA delivery and gene expression. A single-dose intratumoral administration of wt-p53 DNA-loaded nanoparticles demonstrated significant inhibition of tumor growth in MDAMB-435-induced subcutaneous breast cancer mouse model that also resulted in prolonged animal survival than controls. The mechanism of inhibition of tumor growth with wt-p53-DNA-loaded nanoparticles was attributed to higher apoptosis of tumor cells than that in controls, and also the induction of antiangiogenic protein, thrombospondin-I that inhibited tumor angiogenesis. The studies thus demonstrate the efficacy of nanoparticles as a non-viral gene expression vector and their potential application in breast cancer therapy.