The aim of the present study was to investigate the effect of ZnPcS2P2-meditated sonodynamic therapy (SDT) on U251 human glioma cells and to identify its underlying biological mechanism. The growth inhibition rate was determined by MTT assay. The apoptotic rate was examined by flow cytometry. Fine structures were observed with transmission electron microscopy (TEM). Generation of reactive oxygen species (ROS) was detected spectrophotometrically. Caspase-3, -8 and -9 expression was detected by Western blot analysis. The growth inhibition rate of U251 human glioma cells indicated that ZnPcS2P2-meditated SDT had a better growth inhibition rate of tumor cells at a concentration of 5.0 μg/ml ZnPcS2P2, at a 4-h incubation time with ZnPcS2P2, and at 6 h re-incubation following SDT. At 6 h after SDT, the growth inhibition rate of cells was significantly higher compared to other groups, apoptosis could be detected in SDT by flow cytometry. TEM examination revealed morphological features of apoptosis or necrosis. Furthermore, caspase-3, -8 and -9 expression following SDT was found to be increased by Western blot analysis. Finally, generation of ROS in cells was also elevated. In conclusion, ZnPcS2P2-SDT is capable of inducing U251 cell apoptosis or necrosis and has satisfying antitumor effects. The mechanism of ZnPcS2P2-meditated SDT involves ROS generation in U251 cells, which initiates subsequent apoptosis through the mitochondrial and death receptor pathways.
Gliomas are the most common types of tumors of the central nervous system and are resistant to numerous treatments, including radiation, chemotherapy or other adjuvant therapies. Although considerable progress has been made in the treatment of glioma, its prognosis remains poor (
The sonosensitizer is the key factor of SDT. Photosensitizers have been used in SDT as well as in photodynamic therapy (PDT). Certain drugs, including hematoporphyrin, photofrin II, ATX-70 and ATX-S10, have been demonstrated to induce cell killing when activated by ultrasound exposure. It has previously been indicated that these chemicals, originally generated in PDT, were therefore applicable as sonosensitizers for tumor treatment in combination with ultrasound. In the present preliminary study, we used a new domestic photosensitizer, di-sulfo-di-phthalimidomethyl phthalolcyanine zinc (ZnPcS2P2) (
As we know, apoptosis is often initiated by an extrinsic (activated caspase-8) or an intrinsic pathway (activated caspase-9) (
ZnPcS2P2 was a gift from the Department of Chemistry, Institute of Research on Functional Materials, Fuzhou University (China). Its chemical structure is shown in
U251 human glioma cells were obtained from the Shanghai institute of Cytobiology (Institute of Chinese Academic Medical Scinence, China) and were continuously cultured in DMEM (Gibco BRL; Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 mmol/l L-glutamine in a humidified incubator at 37°C and 5% CO2. Cells in the exponential growth phase were used for all experiments.
A multi-function physical therapy ultrasound device (Tianshi Technologies Ltd. Co., Beijing, China) was used to generate ultrasound at 1 MHz. Ultrasonic intensities (0.5 W/cm2) were measured by a stainless-steel ball radiometer (diameter 0.32 cm) (
The monoclonal anti-β-actin antibody and anti-caspase-3, -8 and -9 antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin-V-FITC Apoptosis Detection Kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from Calbiochem (La Jolla, CA, USA). All other chemicals and cell-culture reagents were purchased from Laohekou Jing hong Chemical Co. Ltd. (China). All solvents used in chemical reactions were anhydrous and obtained as such from commercial sources. All other reagents were used as supplied, unless otherwise stated.
U251 human cells were transferred to 6 wells in the centre of a 96-well plate at a density of 8×103 cells/well and in the same way, transferred to 36 sections of a 96-well plate. The following day, each of the 6 plates were changed by DMEM with various final concentrations of ZnPcS2P2 (0.625, 1.250, 2.500, 5.000, 10.000 and 20.000 μg/ml), cells were continuously cultured in a humidified incubator at 37°C and 5% CO2 for 4 h, then subjected to ultrasound treatment at 1.0 MHz and 0.5 W/cm2 for 2 min. Cells were re-incubated for up to 4 h and subjected to MTT assay.
Similarly, cells were transferred to 36 sections of a 96-well plate, were re-incubated with a final concentration of ZnPcS2P2 5.0 μg/ml for (1, 2, 3, 4, 5 and 6 h) and then underwent ultra-sound treatment. Cells were subsequently evaluated by MTT assay following re-incubation for 4 h.
Cells of 36 sections of a 96-well plate (with a final concentration of ZnPcS2P2 5.0 μg/ml) were incubated for up to 4 h, followed by ultrasound treatment, and subjected to MTT assay at various time-points post-SDT (3, 6, 12, 24, 48 and 96 h).
The growth inhibition rate was calculated as: inhibition rate (%) = (1-OD treatment group/OD control group) x 100%.
In the control group, cells were neither treated with ZnPcS2P2 nor with ultrasound. In the ZnPcS2P2 group, cells were treated with ZnPcS2P2 alone (final concentration 5.0 μg/ml, incubation time 4 h). In the ultrasound group, cells were treated with ultrasound alone. In the SDT group, cells were treated with ZnPcS2P2 (5.0 μg/ml, incubation time 4 h) and then subjected to ultrasound treatment at 1.0 MHz and 0.5 W/cm2 for 2 min. Cells were re-incubated for up to 6 h and subjected to MTT assay following treatment.
Following treatment, cells were re-incubated for up to 6 h, 4 groups of cells (control, ZnPcS2P2 alone, ultrasound alone and ZnPcS2P2 + ultrasound) were re-suspended in a concentration of 5×106 cells/ml and washed twice with pre-cooling of the phosphate-buffered saline (PBS). Following washing with Annexin-V binding buffer, cells were stained with FITC-conjugated Annexin-V and propidium iodide (PI) reagents for 15 min as per the manufacturer’s instructions and further examined by flow cytometry (FACScalibur; Becton-Dickinson). The percentages of dead cells and those undergoing apoptosis were analyzed using Cell Quest Software.
For the electron microscope examination, cells from each group were re-incubated for up to 6 h and then washed twice with PBS and fixed in a mixture of 2.5% glutaraldehyde and osmium tetroxide. Cells were then dehydrated in progressive 10-min steps for 3 times in ethanol/water (70, 90 and 96%, respectively), followed by isoamyl acetate. Finally, cells were stained with uranyl acetate (Guangzhou Chemical Reagent Factory, China) and lead citrate (Guangzhou Chemical Reagent Factory). Ultra-thin sections were examined under a transmission electron microscope (JEM-1220, Japan).
To examine protein expression, cells of various groups were separately washed, collected and homogenized in a lysis buffer (10 mM Tris-HCl, pH 8.0, 0.32 mM sucrose, 5 mM EDTA, 2 mM DTT, 1 mM phenylmethyl sulfonylfluoride and 1% Triton X-100), followed by centrifugation. Proteins in various groups were separately electrophoresed on sodium dodecyl sulfate (SDS) polyacrylamide gel (12%), the gel-separated proteins were transferred to nitropure nitrocellulose membranes (Santa Cruz Biotechnology, CA, USA), and the membranes were probed overnight at 4°C with primary antibodies. Each of the targeted proteins was immunostained by anti-β-actin and anti-caspases-3, -8 and -9 (each 1:1000) antibodies. Following probing, the membranes were washed 3 times and incubated for 1 h at room temperature with the respective alkaline phosphatase-conjugated secondary antibodies (Sigma) prior to visualization using a chemiluminescence detection kit (Sigma).
The production of intracellular ROS was assayed spectrophotometrically with dichlorofluorescin diacetate (DCFH-DA) as described previously (
Statistical evaluation was performed using the least significant difference t-test, Dunnett test or paired t-test using the SPSS 11.0 Software system. Data are presented as the means ± standard error (SE). P<0.05 was considered to indicate a statistically significant difference.
A MTT assay revealed that when the concentration of ZnPcS2P2 was >5.0 μg/ml, the growth inhibition rate was less concentration-dependent (
SDT will be most effective when the ZnPcS2P2 concentration in the glioma cells is at its maximum. As shown in
The percentage of apoptotic cells was calculated by flow cytometry using the Annexin-V/PI double-staining assay (
The TEM observation of U251 tumor cells immediately following SDT treatment is shown in
The SDT-induced apoptotic pathway was investigated by Western blot analysis of caspase expressions. Caspases-8, -9 and -3 are key factors in the extrinsic and intrinsic apoptosis pathway, thus, we measured the activities of these caspases. As shown in
The production of ROS was determined by a fluorescence spectrophotometer with DCFH-DA (
Sonodynamic therapy (SDT) is the foundation on which ultrasound is capable of activating the sonosensitizer in order to generate cytotoxic substances and further kill tumor cells. Owing to the fact that the sonosensitizer has high accumulation in tumor tissue, SDT can not only maximally kill tumor cells, but also protect normal brain tissue from impairment (
Until now, the majority of sonosensitizers in SDT are not chemosynthetic compounds. In the present experiment, ZnPcS2P2, a porphyrin compound created by artificial chemosynthesis that has stable physical and chemical properties, was used. In a previous study, ZnPcS2P2-mediated PDT improved the effects of killing leukemia HL60 and K562 cells (
In order to evaluate the effects of ZnPcS2P2-mediated SDT, we first aimed to pinpoint optimal concentrations and incubation times of ZnPcS2P2 in order to ensure the best result of ZnPcS2P2-mediated, SDT-induced U251 glioma cell death by MTT assay. Our experimental results indicated that the inhibition rate of tumor cells increased gradually with the elevation of drug concentration to some extent. When the concentration of the drug was ≥5.0 μg/ml, the inhibition rate of the tumor did not significantly increase (
Apoptosis is one of the major modes of death of neurospongioma. Li
To further clarify the apoptosis phenomena by morphological change, we observed the subcellular structure in U251 cells following SDT. In the SDT group, it was revealed that a section of the cell microvilli had vanished, the volume of the cellular nucleus became small and chromatin gathered densely, thus displaying the characteristics of apoptosis or necrosis (
Apoptosis is characterized by a number of well-defined features, including phosphatidyserine exposure, membrane blabbing, activation of caspase, chromatin condensation and DNA fragmentation (
To further study the mechanisms of the ultrasound activationof ZnPcS2P2 to kill U251 cells, we detected the level of cellular ROS. ROS, which include hydroxyl radicals, superoxide anions, singlet oxygens and hydrogen peroxides, are by-products of cellular metabolism. Excessive intracellular ROS is capable of damaging critical biomolecules and eventually results in several biological effect disorders, including alterations in signal transduction and gene expression for mitogenesis, mutagenesis and cell death (
In conclusion, ZnPcS2P2-SDT is capable of inducing cell apoptosis and necrosis in U251 cells. Ultrasound therapy, combined with ZnPcS2P2, has satisfying antitumor effects. ROS generation in U251 cells plays a crucial role in ZnPcS2P2-SDT-induced cell death and initiates subsequent apoptosis through mitochondrial and death receptor pathways following ZnPcS2P2-SDT.
This study was supported by the National Natural Science Foundation (30970834; 81072079), Technological Key Research Projects of Heilongjiang province (GC10C304-1).
The chemical structure of ZnPcS2P2.
Experimental setup.
Selection of drug conditions. (A) Analysis of the growth inhibition rate of various concentrations of ZnPcS2P2, (B) incubation times and (C) time-points, post-SDT by MTT assay. The results revealed that when the concentration of ZnPcS2P2 was >5.0 μg/ml and incubation time was >4 h, the growth inhibition rate did not markedly increase, and the inhibition rate was significantly higher at 6 h post-SDT. (D) Growth inhibition rates of the 4 groups of cells (control, ZnPcS2P2 alone, ultrasound alone and ZnPcS2P2 + ultrasound), determined by MTT assay. The inhibition rate in the SDT group was significantly higher than the other 3 groups. Data are represented as the means ± standard error (n=6). *P< 0.05 denotes statistically significant differences vs. control. SDT, sonodynamic therapy.
Flow cytometric analysis of apoptosis in U251 cells. (A) Control group, (B) ZnPcS2P2 alone group, (C) ultrasound alone group, (D) ZnPcS2P2 + ultrasound group. In each panel, the upper right quadrants represent the percentage of secondary necrosis cells (Annexin-V binding and for PI uptake). The lower right quadrant contains the apoptotic cells (positive for Annexin-V and negative for PI, demonstrating cytoplasmic membrane integrity) undergoing SDT-induced apoptosis and necrosis in U251 cells. Data are expressed as the means ± standard error (n=6). (E) #P<0.01 denotes statistically significant differences vs. control, *P<0.01 denotes statistically significant differences vs. control. SDT, sonodynamic therapy; PI, propidium iodide.
TEM images of U251 cells
Western blot analysis of cleaved caspases-9,-8 and -3 in various groups at 6 h post-sonication. β-actin was used as the control.
Bar graph showing the changes of the levels of ROS in various groups. Data are expressed as the means ± standard error (n=6). *P<0.05 denotes statistically significant difference vs. the other 3 groups, #P<0.05 denotes statistically significant difference vs. control group and ZnPcS2P2-alone group. ROS, reactive oxygen species.