Several preclinical studies have already demonstrated that down-regulation of survivin expression or function could inhibit tumor growth, increase spontaneous and
induced apoptosis and sensitize tumor cells to anticancer agents. Phosphorylation of survivin at Thr 34 by the cyclin-dependent Daporinad molecular weight kinase cdc2 is believed to promote physical interaction between survivin and caspase-9, resulting in caspase-9 inhibition to reduce apoptosis. It was reported that the survivin mutant Thr34→Ala (survivin T34A) could abolish a phosphorylation site for cdc2-cyclin B1 and prevent survivin binding to activated caspase-9. This reduced tumor cell proliferative potential and led to caspase-dependent apoptosis in melanoma cell lines. It also increased the apoptosis of tumor cells, inhibited tumor angiogenesis and induced a tumor-protective immune response . It was found that greater efficiency was attained in
suppression of murine breast cancer by using a plasmid encoding the phosphorylation-defective mouse survivin T34A mutant complexed to DOTAP-chol liposomes (Lip-mS) . As a result, the present study was designed to determine whether Lip-mS could enhance the antitumor activity of CDDP chemotherapy and to explore the buy KU-60019 possible mechanisms of interaction between survivin targeting-agents and chemotherapy. Methods Cell lines and culture conditions The Lewis Lung Carcinoma (LLC) cell line of C57BL/6 mouse origin was purchased from the American Type Culture Collection (ATCC, Rockville, MD), cultured in DMEM (Gibco BRL, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum see more (FBS), and maintained in a humidified incubator at 37°C
in a 5% CO2 atmosphere. Plasmid DNA preparation The recombinant plasmid encoding the phosphorylation-defective mouse survivin threonine 34→alanine mutant (pORF9-msurvivinT34A, mS) and pORF9-mcs (null plasmid) were each purchased from InvivoGen Corporation (San Diego, CA, USA) and confirmed by restriction endonuclease analysis, PCR and DNA sequence analysis. The plasmid was prepared using the Endofree Plasmid Giga kit (Qiagen, Chatsworth, CA). Endotoxin levels of the prepared plasmid DNA were determined by Tachypleus Amebocyte Lysate (TAL). No genomic DNA, small DNA fragments, or RNA were detected in the plasmid DNA and the OD260/280 ratios of the DNA were between 1.8 and 2.0. The DNA was dissolved in sterile endotoxin-free water and stored at -20°C until use. Preparation of DOTAP-chol liposome/plasmid DNA DOTAP was purchased from Avanti Polar Lipids (Alabaster, AL) and highly purified cholesterol (Chol) was purchased from Sigma (St. Louis, MO). DOTAP-chol liposomes were prepared using the procedure described previously.