Therapeutic Effects of Hybrid Liposomes on Gastric Carcinoma Involve Apoptosis

Materials and Methods

Preparation of HLs. HLs were prepared by sonication of a mixture containing 90 mol% DMPC (NOF Co. Ltd., Tokyo, Japan) and 10 mol% C₁₂(EO)₂₃ (Sigma-Aldrich Co. Ltd., Missouri, USA) in 5% glucose solution using a bath type sonicater at 45°C with 300 W for 5 min (1 min/ml), and were then filtered with a 0.20 μm cellulose acetate filter (Advantec, Tokyo, Japan).

Dynamic light scattering measurements. The diameter of HLs was measured with a light scattering spectrometer (Otsuka Electronic, Osaka, Japan) using a He–Ne laser (633 nm) at a 90° scattering angle. The diameter (d_hy) was calculated using the Stokes-Einstein formula (Equation 1), where κ is the Boltzmann constant, T is the absolute temperature, η is the viscosity and D is the diffusion coefficient: dhy=κT∕3πηD (Equation 1)

Electron microscopy. An electron micrograph was obtained by means of negative-staining techniques. A sample solution of HLs in buffer was mixed with ammonium molybdate. The sample was then applied to a carbon grid and dried overnight at room temperature. Electron micrographs were taken on an electron microscope (JEM100FX, JEOL Co. Ltd., Tokyo, Japan).

Cell culture. Gastric carcinoma (CoRa 622 G6) cell line (13) was obtained from Hokkaido Institute of Public Health (Hokkaido, Japan). CoRa 622 G6 cells were grown in RPMI-1640 medium (Gibco BRL, MD, USA). The medium was supplemented with 10% fetal bovine serum (FBS; Hyclone, Nebraska, USA) and antibiotics (100 units/ml penicillin and 50 μg/ml streptomycin). These cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

Assessment of cell growth in vitro. Inhibitory effects in vitro were examined on the basis of the WST-1 assay (Cell Counting Kit; Dojindo Laboratories, Kumamoto, Japan) (14). CoRa 622 G6 cells were seeded at a density of 1.0×10⁴ cells/ml in a 96-well plate and cultured overnight in a humidified atmosphere of 5% CO₂ at 37°C. The cells were incubated for 48 h in the medium after adding HLs or single-component liposomes of DMPC. WST-1 solution was added and cells cultured for a further 2 h, and then the absorbance at 450 nm was measured by spectrophotometer (Molecular Devices, California, USA). The inhibitory effects of HLs on the growth of tumor cells was evaluated by A_mean/A_control, where A_mean and A_control denote the absorbance of water-soluble formazan, which was useful as an indicator of cell viability, in the presence and absence of sample solutions, respectively.

Caspase fluorometric protease assay. Activation of caspases was measured on the basis of caspase fluorometric protease assay (Medical and Biological Laboratories, Nagoya, Japan) (15). CoRa 622 G6 cells were seeded at a density of 2.5×10⁵ cells/ml in flask and cultured for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. The cells were treated with HLs ([DMPC]=3.42 mM, [C₁₂(EO)_n]=0.38 mM) for 10, 20, 30, 60 and 120 min. The cells were centrifuged at 3,000 rpm for 5 min, and resuspended in 50 μl of chilled cell lysis buffer. The cell lysates were incubated with N-acetyl-Asp-Glu-Val-Asp-(7-amino-4-trifluoromethylcoumarin) (Ac-DEVD-AFC; 50 μM; caspase-3 substrate), N-acetyl- Ile-Glu-Thr-Asp-(7-amino-4-trifluoromethylcoumarin) (Ac-IETD-AFC; 50 μM; caspase-8 substrate) or N-acetyl-Leu-Glu-His-Asp-(7-amino-4-trifluoromethylcoumarin) (LEHD-AFC; 50 μM; caspase-9 substrate) at 37°C for 2 h. The specific activities were determined fluorometrically at 390 nm excitation and 500 nm emission for AFC using Fluoroskan Ascent CF Fluorometer (Thermo Labsystems, Helsinki, Finland). Caspase activity was calculated by the following equation, Caspase activity=I_treatment–I_control, where I_treatment and I_control were the fluorescence intensity of AFC with and without HL, respectively.

Mitochondrial membrane potential. CoRa 622 G6 cells were seeded at a density of 2.5×10⁵ cells/ml in flask and cultured for 24 h in a humidified atmosphere of 5% CO₂ at 37°C. The cells were treated with HLs ([DMPC]=3.42 mM, [C₁₂(EO)_n]=0.38 mM) for 10-20 min. After centrifugation at 3,000 rpm for 5 min, cell pellets were incubated with JC-1 (16) staining solution (Cayman Chemical Company, Michigan, USA) for 20 min at 37°C, and then washed with wash buffer after discarding the medium. Pellets were suspended to culture medium, were seeded to a glass-bottom uncoated dish and were observed with a confocal microscope (TCS-SP Leica, Heidelberg, Germany). At low membrane potential, JC-1 continues to exist as monomer and produces a green fluorescence (emission at 527 nm); at high membrane potential, JC-1 forms J aggregates (emission at 590 nm) and produces red fluorescence.

Assessment of antitumor activity in vivo. The animals were handled in accordance with the guidelines for animal experimentation under Japanese law during the study. Female nude mice (BALB/cA Jcl-nu/nu, CLEA Japan Inc., Tokyo, Japan) were randomly grouped (n=5) on the basis of body weight on the day of tumor cell inoculation using the stratified randomization method. CoRa 622 G6 cells (5.0×10⁶ cells) were inoculated intraperitoneally. HLs (doses for DMPC of HLs were 136 mg/kg and 203 mg/kg) were intraperitoneally administered once each day for 14 days after 1 day of the peritoneal inoculation of CoRa 622 G6 cells. The mice were sacrificed 43 days after inoculation of tumor cells. The tumor nodules were counted macroscopically, and the volume of ascites was weighted.

Survival rate in vivo. Female nude mice (BALB/cA Jcl-nu/nu) were randomly grouped (n=5) on the basis of body weight on the day of tumor cells inoculation using the stratified randomization method and were treated with HLs as described above. The median lifespan was calculated using the following equation, median lifespan=(median survival days after treatment)/(median survival days of control group)×100.

TUNEL method. Detection of apoptotic cells was performed on the basis of the TUNEL method using an in situ apoptosis detection kit (ApopTag Plus Peroxidase; Intergen, New York, U.S.A). Tumors were removed from HL-treated sacrificed mice 43 days after the inoculation of tumor cells and were fixed in 10% formalin solution. Paraffin-embedded sections were cut, dewaxed in xylene and rehydrated through a series of ethanol to water. The sections were incubated with proteinase K for 15 min at room temperature and endogenous peroxidase was blocked with a solution of digoxigenin-conjugated nucleotides and terminal deoxynucleotidyl transferase (TdT) at 30°C for 60 min. Subsequently, anti-digoxigenin antibody was applied and sections were incubated for 30 min at room temperature. Detection of the antigen-antibody link was made through immunoperoxidase followed by 3,3′-diaminobenzidine chromogen. The sections were counterstained with 5% methyl green, rinsed in distilled water, mounted and were observed on the basis of TUNEL method using optical microscopy (Nikon TS-100; Tokyo, Japan).

Statistical analysis. Results are presented as the mean±S.D. Data were statistically analyzed using Student's t-test. A p-value of less than 0.05 was considered to represent a statistically significant difference.