Abstract
The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by magnetofection.
Highlights
A contemporary approach in cancer gene therapy is to target specific molecules or signaling pathways that are predominantly expressed in tumor cells, and is expected to have a good therapeutic index.[1]
Selection of the most effective small interfering RNA (siRNA) molecule against melanoma cell adhesion molecule (MCAM) and the construction of the plasmid DNA encoding shRNA against MCAM Three selected siRNA molecules were transfected into B16F1, B16F10, and 2H-11 cells with lipofection in order to determine their effect on MCAM mRNA and protein levels, as well as their influence on the biological properties of cells in vitro
In addition to the preparation of plasmid DNA encoding shRNA against MCAM (pMCAM), a control plasmid DNA encoding shRNA with scrambled nucleotide sequence of siRNA 801 was constructed (Figure 1a)
Summary
A contemporary approach in cancer gene therapy is to target specific molecules or signaling pathways that are predominantly expressed in tumor cells, and is expected to have a good therapeutic index.[1] The introduction of genetic material into target cells, with an effective and safe delivery system, still remains a challenge in the development of new gene therapy strategies. The use of viral vectors provides specificity, efficiency, long term expression, stability and integrity, due to safety issues, the development of new nonviral delivery systems is gaining in value.[2]. We and others have already demonstrated that magnetofection is an efficient nonviral transfection method in vitro and in vivo for reporter and therapeutic genes.[11,12,13] Besides plasmid DNA, small interfering RNA (siRNA), short hairpin
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