Atelocollagen is a highly purified type I collagen of calf dermis with pepsin treatment (Koken Co., Ltd, Tokyo, Japan).
Synthetic 21-nt RNAs were purchased from Dharmacon (Lafayette, CO) in deprotected, desalted and annealed form. The sequence of our prepared human fibroblast growth factor (FGF)-4 (HST-1/FGF-4) siRNA was 5′-CGAUGAGUGCACGUUCAAGdTdT-3′; 3′-dTdTGCUACUCACGUGCAAGUUC-5′. Non-specific control siRNA duplex (VIII), luciferase GL3 siRNA duplex and luciferase GL2 siRNA were also purchased from Dharmacon, and were used as controls.
Formation of siRNA/Atelocollagen complex
The siRNAs and Atelocollagen complexes were prepared as follows. An equal volume of Atelocollagen (in PBS at pH 7.4) and siRNAs solution was combined and mixed by rotation at 4°C for 20 min. The complex was then kept at 4°C for 16 h before use. The final concentration of Atelocollagen in vitro and in vivo was 0.008 and 0.5%, respectively.
Stability of siRNA/Atelocollagen complex
An aliquot of 0.9 μg of siRNAs (luciferase GL3 duplex) and 0.5% Atelocollagen or cationic liposome (jetSI; Polyplus-transfection SAS, Illkirch Cedex, France) complexes were incubated in the presence of 0.1 μg/μl RNase A (NipponGene, Tokyo, Japan) for 0, 5, 15, 30, 45 and 60 min at 37°C. The solutions were extracted with phenol and phenol/chloroform/isoamyl alcohol (25:24:1). The siRNAs were precipitated with ethanol and agarose gel electrophoresed (3.5%) and visualized by ethidium bromide staining.
NEC8 cells (American Type Culture Collection, Rockville, MD) derived from human testicular tumor were maintained in DMEM with 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO2
. Increased expression of the HST-1/FGF-4
gene in this cell line has been reported previously (17
). B16-F10 melanoma cells continuously express luciferase (B16-F10-luc-G5; Xenogen Corp., Alameda, CA) and were maintained in DMEM with 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO2
Atelocollagen or liposome-mediated siRNA transfer
The siRNA/Atelocollagen (0.008%) complexes were prefixed to a 24-well plate (0.1–1.4 μg siRNA/50 μl/well) according to the method described previously (14
). The cultured cells were plated into the complex-prefixed 24-well plate at 3.5 × 104
cells/well and the effects of siRNA transfer were then observed. The cationic liposome-mediated transfer of siRNA was performed as described by the manufacturer.
Inhibition of cell growth
For monitoring the inhibition of cell growth, the TetraColor One cell proliferation assay reagent (Seikagaku Co., Tokyo, Japan) was used according to the recommended method. The color reaction was assessed by measuring the absorbance at 450 nm with an UVmicroplate reader.
Protein levels of human HST-1/FGF-4 in the culture supernatant and tumors were determined by using enzyme-linked immunosorbent assay (ELISA) using anti-human FGF-4 monoclonal antibody (R&D Systems, Minneapolis, MN). Absorbance was measured at a wavelength of 492 nm with a kinetic microplate reader (model 3550; Biorad, Richmond, CA).
For luciferase-based reporter gene assays, 24 μg pGL3 control vector (Promega, Madison, WI) was introduced into HEK 293 cells at 90% confluency in 10 cm dishes using LipofectAMINE™ 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. After transfection for 4 h, the cells were collected by trypsinization and plated in the 24-well dishes for siRNA transfection. Atelocollagen-mediated or conventional transfection of siRNAs into 293 cells was performed as detailed above. Cells were lysed (n = 4) on day 2 and analyzed for luciferase activity (Bright™-Glo Luciferase Assay System; Promega). Inhibition of luciferase production was normalized to the level of vehicle-treated cells. GL2 siRNA was used as control.
Analysis of siRNA delivery using in vivo imaging
B16-F10-luc-G5 cells were subcutaneously injected (1 × 105
cells per site) into athymic nude mice. Two days later, luciferase GL3 siRNA alone, siRNA mixed with liposome, siRNA complexed with Atelocollagen and Atelocollagen alone were injected into the tumors. For preparing the siRNA/Atelocollagen complex, an equal volume of Atelocollagen (1.0% in PBS at pH 7.4) and siRNA solution was combined and mixed by rotating for 20 min at 4°C. The siRNAs and their complexes were directly injected into the tumor (2.5 μg siRNA/50 μl/50 mm3
tumor). The final concentration of Atelocollagen was 0.5%. The siRNA concentration used in the liposome experiments was 2.5 μg/tumor equivalent to that used in the Atelocollagen experiments. Each group contains four animals. In vivo
bioimaging was conducted on a cryogenically cooled IVIS system (Xenogen Corp.) using LivingImage acquisition and analysis software (18
). Tumor growth was not affected by these treatments. As a control for GL3 siRNA, GL2 siRNA was used.
Reporter gene labeling of tumor cells
NEC8 cells were transfected with a complex of 2 μg pEGFPLuc plasmid DNA (Clontech, Palo Alto, CA) and 30 μl lipofection reagent (LipofectAMINE™ 2000; Invitrogen). Stable transfectants were selected in geneticin (400 μg/ml; Invitrogen) and bioluminescence was used to screen transfected clones for luciferase gene expression using the IVIS system. Clones expressing the luciferase gene were named NEC8-luc.
In vivo imaging study for orthotopic xenografts model
A total of 1.0 × 106 NEC8-luc cells were injected into mice intratesticularly. Cells were suspended in 50 μl of a serum-free medium and injected using a 26-gauge needle into both testes of 8-week-old athymic nude mice obtained from CLEA Japan (Shizuoka, Japan). Ten days after the injection of cells, tumor cell-bearing nude mice were randomly divided into four treatment groups (FGF-4 siRNA alone, FGF-4 siRNA complexed with Atelocollagen, control siRNA complexed with Atelocollagen and Atelocollagen alone). Each group consisted of four animals. The siRNAs and their complexes were injected directly into the testes (2.5 μg siRNA/50 μl/testis). The final concentration of Atelocollagen was 0.5%. Tumor growth was monitored by measuring light emission from individual mice 21 days after siRNA administration. Three days after siRNA administration, tumors were harvested and subjected to ELISA analysis for the detection of FGF-4 protein. Animal experiments in the present study were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute.
The results are given as means ± SE. Statistical analysis was conducted using the analysis of variance with the Bonferroni correction for multiple comparisons. A P-value of 0.05 or less was considered to indicate a significant difference.