RETRACTED ARTICLE: Hypericin-mediated photodynamic therapy induces apoptosis of myoloma SP2/0 cells depended on caspase activity in vitro

Hypericin powder (Beijing Standard Herbs Medical Science & Technology Development Co. LTD., Beijing, China) with purity > 98% was determined by high-performance liquid chromatography (HPLC). A stock solution of hypericin (10 mM) was prepared with dimethyl sulfoxide (DMSO; Sigma) and stored at −20°C in the dark. Immediately before each experiment, the working solution of hypericin was diluted in RPMI1640 medium without serum to obtain a final concentration.

The mouse myeloma cell line SP2/0 (ATCC) was from American Type Culture Collection. The cells were cultured in RPMI1640 medium supplemented with 10% calf serum, 2.2 g/l NaHCO₃, 100 U/ml penicillin, and 100 g/ml streptomycin. Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C until the logarithmic growth phase based on the cell growth curve. The adherent cells were displaced by rinsing with 0.25% trypsin. Then, the adherent and suspension cells were harvested for the following experiments.

SP2/0 cells (5 × 10⁴ cells/ml) were seeded on 96-well plates (100 μl/well) and incubated with hypericin at different concentration (0.001, 0.01, 0.1, 1 and 10 μM) for 16 hours in the dark. Cells were given external 7 cm illumination treatment on a plastic diffuser sheet above a set of 6 yellow L18W/30 lamps (CH Lighting, Zhejiang, China) with the maximum spectrum range of 570–620 nm. Irradiation intensity was detected to be under 13000 lux by TES-1332A (TES, Taiwan, China). The total light doses were determined to be 2.82, 5.64, 8.46, 11.28 and 14.10 J/cm² by a photometer. During the illumination, the temperature of the samples ranged from 32 to 37°C. After PDT treatment, cells were maintained for 24 hours at 37°C in a humidified atmosphere of 95%, 5% CO₂ until further analysis.

In order to determine the optimal hypericin concentration or irradiation dose for PDT treatment, we performed MTT assay to analyze the proliferation of SP2/0 cells [29]. Briefly, SP2/0 cells at a final density of 5 × 10⁴ cells/ml (100 μl/well), were incubated with hypericin at a concentration gradient from 0.001 to 10 μM for 16 hours in the dark. The cell plates were illuminated with different light dose of 0, 2.82, 5.64, 8.46, 11.28 and 14.10 J/cm², and then incubated for 24 hours in the dark. To obtain the IC₅₀, SP2/0 cells were treated with indicated concentrations of hypericin (0, 0.0125, 0.025, 0.05 and 0.1 μM) for 16 hours in the dark. Subsequently, the cells were illuminated at a light dose of 11.28 J/cm² for 24 hours in the dark.

SP2/0 cells without any treatment were set as controls. The MTT assay was performed for SP2/0 cells at each condition according to the manufacturer’s instructions. In brief, 10 μl of MTT (5 mg/ml; Sigma) was added in each well for 4 hours at 37°C. After removal of the medium, the MTT crystals were dissolved with 100 μl DMSO for 15 min. The absorbance at 550 nm was measured by a Multiskan Spectrum 1500 (Thermo Electron Corporation, USA). The inhibition rate of cell proliferation was calculated as follows: (A550control − A550treated)/A550control × 100% and the IC₅₀ value was obtained by Logit method [30].

SP2/0 cells (1 × 10⁵cells/ml) were seeded into the 6-well plates (2 mL/well) and incubated with hypericin at the final concentrations of 0.025, 0.05 or 0.1 μM for 16 hours in the dark. Subsequently, the plates were illuminated at a light dose of 11.28 J/cm² for 24 h. The adherent and suspension cells were collected for the following morphological analysis according to the method described above.

A part of cells were harvested and observed by a phase-contrast microscopy. Some cells were collected for Hoechst 33342/propidium iodide (PI) co-staining [31]. SP2/0 cells were stained with Hoechst 33342 (5 μg/ml) and PI (5 μg/ml) at room temperature for 10 min, then examined under a fluorescence microscopy (E600, Nikon, Japan) with excitation at 360 nm [32]. Intact blue nuclei of viable cells, condensed/fragmented bright blue nuclei of apoptotic (early or late) cells and pink nuclei of necrotic cells can be observed. The number of apoptotic or necrotic cells was counted in a total of 200 cells from 5 random fields.

SP2/0 cells (1 × 10⁵cells/ml, 2 mL/well) treated with hypericin (0.025, 0.05 or 0.1 μM) and illumination (11.28 J/cm²) were collected for TEM examination. Samples were prepared as described previously [33]. The cells were fixed with 2.5% glutaraldehyde, followed by 1% OsO₄ and then dehydrated with acetone. They were embedded in Epon812-resin mixture. Serial sections (about 50 nm) were obtained by ultra-microtome. Sections were stained with sodium acetate and lead citrate and then viewed by a Hitachi H-7000/STEM electron microscope (Hitachi Inc., Tokyo, Japan) at 75 kV.

SP2/0 cells with hypericin (0.025, 0.05 and 0.1 μM) and illumination (11.28 J/cm²) were used for this experiment. The suspension cells and adhesive cells on the 6-well plates were all collected for DNA extraction following the manufacturer’s instructions of DNA Ladder Extraction Kit with Spin Column (Beyotime Institute of Biotechnology, China). The DNA extractions (40 μg) were separated on 1.0% agarose gel containing ethidium bromide (EB) and were visualized under UV light. Standard molecular weight markers (Fermentas, MBI) were run on the left-hand lane of the gel.

In order to explore the changes in the membrane potential, we carried out JC-1 staining for SP2/0 cells intervened by hypericin (0, 0.025 and 0.05 μM) and illumination (11.28 J/cm²). The collapse of the mitochondrial membrane was measured using a mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, China) [34]. JC-1 formed aggregates in the mitochondria at relatively high membrane potential exhibiting red-orange fluorescent at 590 nm. The JC-1 monomers distributed in the cytoplasm of cells with low potential and emitted a green fluorescent at 529 nm. After JC-1 staining, cells were analyzed by flow cytometry with green emission in channel 1 (FL1) and red emission in channel 2 (FL2). The relative ratio of JC-1 aggregate (FL2, red fluorescence intensity) to monomer (FL1, green fluorescence intensity) was calculated. A decrease in this ratio was defined as the depolarization of the mitochondrial membrane potential.

To evaluate the effects of caspase inhibitors (pan-caspase inhibitor Z-VAD-FMK or caspase-3 inhibitor Ac-DEVD-CHO) on apoptosis mediated by hypericin with PDT, the PI staining was carried out. The caspase inhibitors were dissolved in DMSO at a storage 20 mM concentration. SP2/0 cells were pre-incubated with the pan-caspase inhibitor (100 μM) or caspase-3 inhibitor (100 μM) for 1 h, and then exposed to 0.05 μM hypericin for 16 h in the dark. Preliminary experiment indicated that 0.125% DMSO had little effect on the cell death and proliferation. For PI staining, the caspase inhibitors were dissolved in 0.05% DMSO that was safe for SP2/0 cells. After illuminated at 11.28 J/cm² for 24 h, SP2/0 cells were harvested, fixed in 70% ethanol and stored at 4°C overnight. Then, cells were stained with PBS containing 40 μg/mL RNase and 10 μg/mL PI at room temperature for 30 min in the dark. Cells were analyzed using an FACS-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The cells during apoptosis were obtained from the distinct sub-G1 region of the DNA distribution histograms. At least 20,000 events were counted for each sample.

SP2/0 cells at a final density of 1 × 10⁵ cells/ml were grown in 6-well plates (2 mL/well) and incubated with the hypericin (0.025 and 0.05 μM) for 16 hours. The untreated cells were defined as control. After 24 h illumination at 11.28 J/cm², caspase-3 activity was determined using a caspase-3 activity kit (Beyotime, Nanjing, China) which was based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide into the yellow formazan product, p-nitroaniline. The absorbance at 405 nm was measured for cells treated with hypericin with PDT and controls. The formula was listed as follows:

Western blot analysis was performed as previously described [35-37]. Protein samples (40 μg) were separated by 8-12% (8% for PARP-1; 10% for AIF, β-actin; 12% for Bcl-2, Bax, Cytochrome c, Histone 2A, Caspase-3) sodium dodecyl sulfate-polyacrylamide gels electrophoresis and transferred to polyvinylidene fluoride membrane (Immobilon PVDF, Millipore). The membranes were blocked in 10% non-fat dried milk in TBS-T (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween) for 2 hours at room temperature. Then the membranes were incubated with the primary antibodies of anti-Bcl-2 rabbit monoclonal antibody (1:1000), anti-Bax rabbit monoclonal antibody (1:1000), anti-caspase-3 rabbit polyclonal antibody (1:1000), anti-PARP rabbit polyclonal antibody (1:1000), anti-β-actin mouse polyclonal antibody (1:1000), anti-cytochrome c rabbit monoclonal antibody (1:1000), anti-AIF rabbit monoclonal antibody (1:1000), and anti-H2AX rabbit monoclonal antibody (1:500), respectively, overnight at 4°C. All the antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Subsequently, membranes were washed three times with Tris-bufffered saline, 0.1% Tween-20 and then incubated with the peroxidase-conjugated secondary antibodies for 1 h. The membranes were washed three times again and developed using enhanced chemiluminescence (ECL, Amersham Biosciences). Bands were visualized using the ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-rad, USA). Protein expressions were quantified by densitometry analyzed using Quantity One 4.5.2 software (Bio-Rad, Hercules, USA).

All the data were displayed as mean ± standard deviation (SD). All the statistical analysis was performed by SPSS17.0 statistical software. Differences among groups were analyzed with S-N-K followed by One-Way ANOVA. P value < 0.01 was considered to be statistically significant.