| Summary: | 碩士 === 國立成功大學 === 臨床藥學與藥物科技研究所 === 100 === Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer. The incidence of this type of skin cancer has increased greatly for the past decade. The treatment for treatment of squamous cell carcinoma include surgery, radiotherapy, chemotherapy, immunotherapy and gene therapy.
Human WWOX gene, encoding the WWOX/FOR/WOX1 family proteins, is mapped to a fragile site on chromosome 16q23.2. WOX1 protein is considered as a candidate tumor suppressor protein. TP53 gene is located on the short arm of chromosome 17p13.1. It is well-known that p53 protein is important for cellular response to a wide variety of stressful stimulation. During the development of skin cancer, loon-term ultraviolet radiation (UVR) exposure can alter gene activity in keratinocytes, resulting notably p53 mutantion.
The technology of gene delivery plays an important role in the evelopment of gene therapy. Several methods have been developed to transfer DNA into cells for gene therapy. One of the non-invasive gene delivery methods is the use of gene vectors or carriers. Polyethylenimine (PEI) is a cationic polymer-based gene carrier,. Many researches demonstrated that PEI can transfer genes into different kind of cells under different transfection conditions.
The aim of this study was to examine the feasibility of using PEI as gene carrier to deliver WWOX gene TP53 gene to squamous cell carcinoma. The PEI/DNA complexes of different N/P ratios were characterized by gel retardation assay. Transfection efficiency, degree of cell apoptosis and protein expression were analyzed in SCC cells and HaCaT cells, in order to determine optimal transfection conditions for PEI/DNA complexes to be used in in vivo experiments.
Results of gel retardation assay showed that WWOX gene formed complexes with PEI when N/P ratios were greater than 4:1. It was demonstrated that transfection efficiency were related to the amount of DNA, but not to DNA types in both cell lines, and transfecton efficiency of PEI was lower than that of Lipofectamine. In SCC-15 cells, degree of apoptosis induced by WWOX gene were not related with DNA types. However, degree of apoptosis induced by p53 gene were dose-dependent. On the other hand, degree of apoptosis induced by both WWOX gene and p53 gene in HaCaT cell was associated with DNA content but not with DNA types. Degree of apoptosis induced by PEI/DNA complex was more prominent than lipo/DNA complex in HaCaT cells, while they were comparable in SCC-15 cells.
Based on the above data, vector/DNA 2μg/mL (N/P ratio 8:1) was selected for the following evaluation. The results displayed that WWOX protein expression was significantly increased in comparison with control groups in both cell lines. For HaCaT cell, protein expression level was comparable between PEI and Lipo as vectors, while in SCC-15 Cell, protein expression level by PEI-mediated gene delivery was greater than Lipo. Direct injection of vector/DNA complexes into tumor-bearing mice demonstrated that PEI/WWOX pEGFPC1, PEI/WWOX-pEGFPC1+ PEI/p53-pDsRedN1, Lipo/WWOX-pEGFPC1 and PEI/p53-pDsRedN1 complexes significantly inhibited tumor growth in comparison to the control. Among these groups, the effect from PEI/WWOX-pEGFPC1 complex was the most prominent.
In summary, the current study demonstrated that both WWOX gene and p53 gene can be effectively delivered to squamous cell carcinoma by PEI and Lipofectamine and effectively reduced tumor size in vivo. The effect of PEI was comparable to Lipofectamine in terms of transfection efficiency, apoptotic effect and protein expression, with the advantages of easy accessibility and cost-effectiveness. It is concluded PEI with WWOX gene and p53 gene complexes is a potential therapeutics for squamous cell carcinoma.
Keywords：Squamous cell carcinoma (SCC), WWOX gene, p53 gene, gene delivery, polyethylenimine(PEI)
|
|---|
