Dihydroartemisinin was obtained from Bide Pharmaceutical Corporation (Guangzhou, Guangdong Province, China). Working solutions were prepared by dissolving the compound in dimethyl sulphoxide (DMSO) before experiments. The final concentration of DMSO was less than 1% in all experiments. Lipofectamine2000 and Mitotracker Red were purchased from Invitrogen (Carlsbad, CA, USA). Rhodamine 123 (Rho123), Hoechst 33258 and PI were obtained from Sigma (St.Louis, MO, USA). Cell Counting Kit (CCK-8) was purchased from Dojindo Laboratories, Kumamoto (Japan). Annexin V(FITC) apoptosis detection kit was obtained from Bender Medsystems (Austria) and Ac-DEVD-AFC was purchased from Alexis (Switzerland). Z-VAD.fmk, a broad spectrum caspase inhibitor, was purchased from BioVision (CA, USA). SCAT3 plasmid was kindly provided by Prof. Miura [24
Cell culture and transfection
The human lung adenocarcinoma cell line (ASTC-a-1) was obtained from the Department of Medicine, Jinan University (Guangzhou, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Grand Island, USA). For fluorescence studies, the cells were transferred in 35-mm dish 24–48 h after transfection. The cell line stably expressing SCAT3 was obtained from our previous studies [25
] and all the cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) with 5% CO2
at 37°C in a humidified incubator.
Cell viability assay
The cell viability after treatment with DHA was measured by Cell Counting Kit-8 (CCK-8) assay. ASTC-a-1 cells were suspended at a final concentration of 1 × 104 cells/well and cultured in 96-well flatbottomed microplate. After exposure to DHA for 48 h, CCK-8 (10 μl) was added to each well of a 96-well flatbottomed microplate containing 100 μl of culture medium and DHA (0, 1, 5, 10, 20, 30 μg/ml DHA, respectively) mixture, and the plate was incubated for 1 h at 37°C. Moreover, cells were incubated with 20 μg/ml of DHA for different periods of time (0, 6, 12, 24, 48 and 72 h). Viable cells were counted by absorbance measurements at 450 nm using auto microplate reader (infinite M200, Tecan, Austria). The OD450value was proportional inversely to the degree of cell apoptosis. All experiments were performed in triplicate on three separate occasions.
Morphological examination for apoptosis
Cells were grown on the coverslip of a 35 mm chamber. After being treated with 20 μg/ml DHA for 48 h, the cells were washed with PBS three times and incubated with 1 μM Hoechst 33258 and 1 μg/ml PI for 20 min at room temperature in the dark, respectively. Cells were then washed three times with PBS and visualized under a Zeiss fluorescent microscope (Axiovert 200 M). The images of Hoechst 33258 were recorded using a digital camera (Nikon, Tokyo, Japan) with 1280 × 1280 pixels resolution. PI was excited with 543 nm and recorded with a 600–650 nm long-pass filter. Moreover, after being incubated with 10, 20 μg/ml DHA for 48 h at 37°C, a laser scanning confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany) was used to examine ultrastructural changes in ASTC-a-1 cells.
Annexin V and PI staining method
The appearance of phosphatidyl-serine on the extracellular side of the cell membrane was quantified by annexin V/PI staining. After 20 μg/ml DHA treatment for 24 and 48 h, control cells, STS-treated cells and DHA-treated cells were stained with 5 μl of annexinV-FITC and 10 μl PI (10 μg/ml) for 10 min at room temperature as recommended by the manufacturer. Cells were subjected to fluorescence-activated cell sorting (FACS) analysis using a flow cytometer (FACS. Arla BD, USA) with apoptotic cells being annexin V-positive/PI-negative.
Determination of mitochondrial size
Cells were cultivated on a coverslip of a 35 mm chamber. At indicated times, after being treated with 20 μg/ml DHA, the cells were washed with PBS three times and incubated with 0.1 μM Mito-tracker Red for 30 min at room temperature in the dark. The cells were then washed three times with PBS and visualized under confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany). Mito-tracker Red was excited at 633 nm and the emitted light was recorded through a 650 nm long-pass filter. For every condition tested, the size of 100–200 mitochondria in at least 50 different cells is from three independent experiments. The determination of the mitochondrial size was carried out using Zeiss Rel3.2 image processing software (Zeiss, Jena, Germany).
Measurement of Mitochondrial Membrane Potential (ΔΨm)
Rho123 was used to evaluate changes in ΔΨm. After being incubated with DHA for 48 h, the fluorescence images of cells stained with potential-sensitive dye Rho123 were monitored in real-time using a confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany). Rho123 was excited with 488 nm and the emission fluorescence was recorded through a 454–600 nm filter. Cells were washed with PBS three times, and stained with the Rho123 at 5 μM for 20 min in the dark at room temperature. Subsequently, cells were examined using a confocal microscope after being washed with PBS three times.
The ΔΨm was also measured by flow cytometry (FCM). Cells were grown at a density of 1 × 105 cells in 12-well flatbottomed microtiter plates. After treatment without DHA or with DHA for 12, 24, and 48 h, cells preincubated with 1 ml DMEM were collected and washed with PBS three times. Cells in 1 ml PBS were stained with 1 μM Rho123 for 30 min in dark at room temperature. After that, cells were collected by centrifugation (1200 rpm, 3 min) and were washed with PBS three times and then resuspended in 1 ml PBS. Fluorescence emitted from the Rho123 was detected with a flow cytometer (FACS. Arla BD, USA). Results were expressed as the proportion of cells exhibiting high mitochondrial membrane potential which was estimated by reduced fluorescence intensity from Rho123.
Confocal and FRET acceptor photobleaching technique
Fluorescence imaging and FRET were performed on a confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany). All the quantitative analysis of the fluorescence images was performed by Zeiss Rel3.2 image processing software (Zeiss, Jena, Germany). For time-lapse imaging, culture dishes were mounted onto the microscope stage equipped with a temperature-controlled chamber (Zeiss, Jena, Germany).
According to the FRET technology, acceptor photobleaching in single living cell was used to monitor the effect of DHA on the caspase-3 activation. ASTC-a-1 cells stably expressing SCAT3 were cultured in coverslip of a 35 mm chamber after a treatment of DHA for 48 h. The acceptor photobleaching of SCAT3 was performed with the highest intensity of 514 nm laser on a confocal microscope. SCAT3 was excited at 458 nm from an Ar-Ion laser and CFP emission was collected through 470–500 nm band-pass filters, and Venus (FRET-acceptor) emission was recorded through a 530 nm long-pass filter.
Fluorescence spectral analysis inside living cells
ASTC-a-1 cells stably expressing SCAT3 were cultured in the 96-well flatbottomed microplate for 24 h. Then, cells were incubated with various concentrations of DHA (0, 5, 10, 20 μg/m1) for indicated times (0, 12, 24, 48 h) and 1 μM STS for 6 h in the presence or absence of 10 μM Z-VAD.fmk. The emission spectra of the SCAT3 were detected in living cells after the addition of DHA and STS by auto microplate reader (infinite M200, Tecan, Austria). In the meantime, we also needed to detect the emission spectra of the empty cells that were not transfected and considered them as the background emission spectra. The step length of the scanning spectra is 2 nm. The excitation wavelength of SCAT3 was 409–427 nm and the emission fluorescence channel was 454–600 nm band-pass.
Fluorometric assay for Caspase-3 activity
For the detection of caspase-3 activity, PBS-washed cell pellets (derive from either the medium or the adherent cells) were resuspended in extract buffer [25 mM HEPES (pH7.4), 0.1% TritonX-l00, 10% glycerol, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, and 10 mg/ml Leupeptin] and vortexed vigorously. 20 μl of extract (corresponding to 10% of the sample) were incubated with the caspase-3 fluorogenic substrates Ac-DEVD-AFC at 100 μM final concentration at room temperature, and caspase-3 activity was measured continuously by monitoring the release of fluorigenic AFC at 37°C. The excitation wavelength of AFC was 400 nm and the emission wavelength was 530 nm using auto microplate reader (infinite M200, Tecan, Austria).
Protein extraction and Western Blot analysis
The activation of caspase-3 in DHA-treated ASTC-a-1 cells was examined by Western blotting which visualized the processing of the inactive caspase proform to the catalytically active, smaller units. For Western blot analysis, the cells were treated with 1 μM STS for 12 h, 20 μg/m1 DHA for 48 h and co-treatment with 20 μg/m1 DHA and 10 μM Z-VAD.fmk for 48 h. Collected cells were washed with cold PBS. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton-100, 1 mM PMSF and protease inhibitor cocktail set I). Protein content was quantitated using Bradford assay. Equivalent amounts of total protein were resolved by 15% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Co., Billerica, MA, USA) following the conventional protocols. Before being immunoblotted, the membranes were blocked in 5% nonfat milk in TBST buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. The rabbit polyclonal anti-caspase-3 (Cell signaling, Cat. No. 9746) was used at a dilution of 1:1,000 for 2 h at room temperature and secondary anti-rabbit IgG-HRP (Rockland, Gilbertsville, PA, USA) was used at 1:1,000 for 1 h. Detection was performed using the LI-COR Odyssey Infrared Imaging System (LI-COR, Inc., USA).
The results are expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS10.0 software for multiple comparisons. A value of P < 0.05 was considered to be statistically significant.