Introduction
Brain glioma is the most common form of neural malignancy in the nervous system. High grade glioma is the leading cause of brain cancer-related mortality due to its high invasive ability and malignant proliferation (1,2). Glioma incidence accounts for approximately 40–50% of all intra-cranial tumors (3). Although current treatment has modestly improved patient survival, patients with gliomas still have a very poor prognosis and low cure rates. It was reported that patients with anaplastic astrocytoma have a median survival of 2–3 years, but for those with more aggressive glioblastomas, the median survival is only 12–15 months (4,5). Currently, surgery is considered as an important initial clinical approach for glioma, yet it is difficult to completely remove diffuse tumor cells. Radiotherapy and chemotherapy after initial surgical resection are regarded as an effective therapeutic plan to prevent disease recurrence (6). However, most attempts to increase the efficacy of radiotherapy and chemotherapy are hampered by unacceptable late toxicities (7). In addition, the drug-resistance of tumor cells finally results in the abrogation of the effects of current chemotherapeutic agents (8). Accordingly, to improve the prognosis and quality of life of patients with gliomas, the identification of new anticancer agents against glioma cells requires further investigation.
Anticancer agents derived from botanical herbs such as camptothecin and paclitaxel are the drugs of first choice in many tumor therapies, accounting for more than 30% of all anticancer drugs (9,10). Many scholars in China have come to believe that it is important to screen antitumor active ingredients from botanical herbs, particularly from traditional Chinese medicines.
Dracorhodin perchlorate (Dp) is a synthetic analogue of the antimicrobial anthocyanin red pigment, dracorhodin, which is isolated from the exudates of the fruit of Daemonorops draco. It is a traditional Chinese medicine (11). Increasing evidence has demonstrated that dracorhodin has various pharmacologic activities including antimicrobial and anti-viral roles, but it is not stable in solution (12). Dp, one of its important derivatives, was found to inhibit cancer cell proliferation and induce apoptosis in prostate cancer PC-3 cells (13), gastric tumor SGC-7901 cells (14), breast cancer MCF-7 cells (15), and melanoma A375-S2 cells (16). In addition, previous studies have indicated that Dp-mediated apoptosis in human leukemia HL-60 cells was triggered through upregulation of the ratio of mitochondrial proteins, Bax/Bcl-XL, and activation of caspases (17). However, according to the best of our knowledge, no literature has reported on the exact effects of Dp on glioma cells. In light of previous studies, we speculate that Dp may be an effective therapeutic candidate for glioma treatment. Therefore, in this study, we investigated the inhibitory effects of Dp on human glioma U87MG and T98G cells and the underlying molecular mechanisms. Our findings demonstrated that Dp effectively suppressed cell proliferation, arrested cell cycle progression, and induced cellular apoptosis in glioma cells via the signaling pathway of caspase-3/-9.
Materials and methods
Reagents
Dp was obtained from the National Institute for Food and Drug Control (Beijing, China), and its stock solution was prepared in dimethyl sulfoxide (DMSO) and stored at −20°C for less than one month. As vehicle, 0.1% DMSO was added to the control cells, and this concentration did not show any effect on the morphology and proliferation of glioma cells. Annexin V-FITC/propidium iodide kit for apoptosis detection and Caspase-Glo-3/9 assay kit were purchased from Promega Corporation (Madison, WI, USA). ApoAlert cell fractionation kit and the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenz-imidazolylcarbocyanine iodide) mitochondrial membrane potential detection kit were purchased from Cell Technology Co. (Mountain View, CA, USA). The primary antibodies against cytochrome c, p53, p21, Cdc2, P-Cdc2, Cdc 25A, Bim, Bax, Bcl-2, procaspase-3/-9, cleaved caspase-3/-9 and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA).
Cell lines
Human glioma cell lines U87MG and T98G were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, and they were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA) at 37°C in a humidified incubator with 5% CO2.
Cell viability assay
The effect of Dp treatment on the proliferation of U87MG and T98G cells was determined by MTT assay. Briefly, the cells were seeded in 96-well plates at a density of 1×104 cells/well in a final volume of 150 µl of culture medium with 10% FBS. After 24 h of incubation, Dp at various concentrations (0, 10, 20, 40, 80, and 160 µM) was added to each well for 24, 48 and 72 h. Subsequently, 20 µl of MTT (5 mg/ml solution in 1X phosphate-buffered saline) was added to each well for a 4-h incubation at 37°C. The supernatant was then removed, and 150 µl DMSO was added to each well. Then, cell viability was measured by an auto-mated spectrophotometric plate reader (PerkinElmer, USA) at a wavelength of 570 nm. The percentage of cell survival was calculated according to a previously published formula (18): Survival (%) = (mean experimental absorbance)/(mean control absorbance) × 100. The experiments were independently performed at least three times.
Determination of cell cycle progression
The effect of Dp on cell cycle progression in the U87MG and T98G cells was carried out as described previously (19), with slight modifications. Briefly, the U87MG or T98G cells (2×105) were plated in 6-well plates, and treated without or with 40 and 80 µM Dp for 48 h. Then, the cells were trypsinized with 0.25% trypsin-EDTA solution and collected by centrifugation at 1×800 g. After washing with phosphate-buffered saline (PBS), the cells were stained with cell cycle staining solution containing propidium iodide, 1% Triton X-100, and 0.1 mg/ml ribonuclease A for 30 min at room temperature. Subsequently, cell cycle distribution was analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).
Determination of apoptosis
Cellular apoptosis was evaluated by using Annexin V-FITC and propidium iodide (PI) double-staining assay as described previously (20), with slight modifications. Briefly, the U87MG and T98G cells were incubated with the indicated concentrations of Dp for 48 h, and collected for washing with PBS twice. Then, the cells were re-suspended in 500 µl binding buffer (0.14 M NaCl, 10 nM HEPES, and 2.5 mM CaCl2, pH 7.5) containing 5 µl of Annexin V-FITC and 10 µl of PI for 30 min at room temperature in the dark, followed by determination with flow cytometry (Becton Dickinson). The percentage of viable (Annexin V-FITC-negative and PI double negative), early apoptotic (Annexin V-FITC-positive and PI-negative), and late apoptotic and necrotic cells (Annexin V-FITC-positive and PI-positive) were analyzed with CellQuest software.
Measurement of mitochondrial membrane potential (Δψm)
JC-1, a cationic dye, was used to evaluate the extent of mitochondrial membrane potential damage as previously described (21). Briefly, U87MG and T98G cells were incubated in the absence or presence of 40 and 80 µM of Dp for 24 h, and washed with PBS (pH 7.4), followed by incubation with JC-1 staining solution at 37°C for 30 min. After washing with serum-free medium, the cells were analyzed with a laser confocal scanning microscope (Olympus FY, Japan) to measure JC-1 aggregates (red) in intact mitochondria and JC-1 monomer (green) in apoptotic cells with depolarization of Δψm. The ratio of red/green fluorescence intensity was calculated to obtain the Δψm.
Determination of cytochrome c release
To investigate the effect of Dp on cytochrome c, ApoAlert cell fractionation kit was used to separate cellular cytoplasmic and mitochondrial fractions after U87MG and T98G cells were treated with 0, 40, and 80 µM of Dp for 48 h. Then, 10 µg of cytosolic protein was separated on 10% SDS-PAGE gel and analyzed by western blot analysis using monoclonal anti-cytochrome c and anti-β-actin antibodies as previously described (22). The densities of the cytochrome c bands were normalized to the corresponding β-actin bands.
Western blotting
Human glioma U87MG and T98G cells were treated with 0, 40, and 80 µM of Dp for 48 h, and cell lysates were prepared as described previously (23). Subsequently, protein concentration was measured by the BCA method, and 10 µg of total protein from each sample was separated by SDS-PAGE. Then, proteins were transferred to polyvinylidene difluoride membranes, and blocked in 5% skimmed milk powder overnight at 4°C. The membranes were washed with TBST, and separately incubated overnight at 4°C with the desired primary antibodies against p53, p21, Cdc2, P-Cdc2, Cdc25A, Bim, Bax, Bcl-2, procaspase-3/-9, cleaved caspase-3/-9, and β-actin used a loading control, followed by an additional washing with TBST. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Protein bands were detected using an enhanced chemiluminescence reagent and system (Amersham Biosciences Corporation, Piscataway, NJ, USA). Band intensities were quantified by performing optical density analysis with Quantity One software (Bio-Rad, Hercules, CA, USA).
Determination of caspase-9 and caspase-3 activity
Caspase-9/-3 activity was detected as described previously (24), with slight modifications. In brief, U87MG and T98G cells were seeded in 96-well plates, and then treated with 0, 40, and 80 µM of Dp for 48 h, followed by addition of 100 µl of Caspase-Glo-9 or Caspase-Glo-3 reagent to each well for 2 h of incubation at room temperature. Subsequently, a microplate luminometer (Promega) was used to determine the luciferase activity in the cells. Each sample was independently performed at least three times.
Statistical analysis
All data are expressed as mean ± standard deviation (SD), and statistical analyses were performed with SPSS 13.0 software. The mean values of two groups were compared using the Student's t-test, and a value of P<0.05 was considered to indicate a statistically significant result.
Results
The anti-proliferative effect of Dp on human glioma cell lines
The potential of Dp to suppress cell proliferation in the human glioma cell lines U87MG and T98G was first evaluated by MTT assay. The results indicated that Dp treatment for 24 h produced a significant dose-response inhibitory effect on U87MG and T98G cells with its corresponding concentration ranging from 10 to 160 µM (Fig. 1A). Additionally, a time-dependent inhibitory effect was also found when these cell lines were incubated with 40 µM Dp for 24, 48, and 72 h (Fig. 1B).
Dp induces cell cycle arrest in glioma cells
To investigate the mechanism involved in the suppressive effect on cell proliferation by Dp, we tested whether Dp had any effect on cell cycle progression by using flow cytometry. The results demonstrated that Dp treatment induced cell cycle arrest at the G0/G1 phase with a parallel significant reduction in the S and G2/M phase when U87MG and T98G cells were treated with 40 and 80 µM Dp for 48 h (Fig. 2A). These results suggest that cell cycle progression of U87MG and T98G cells was markedly blocked by Dp treatment.
In addition, previous studies have confirmed that tumor suppressor gene p53 plays a critical role in the regulation of its downstream genes and cell cycle progression (25). Accordingly, we further investigated p53 protein expression and cell cycle-related biomarkers including p21, Cdc25A, Cdc2, and P-Cdc2. The results of western blot analysis indicated that the expression levels of p53 and p21 protein were significantly upregulated in the U87MG and T98G cells 48 h after Dp treatment (Fig. 2B–D). However, cell cycle-associated proteins Cdc25A, Cdc2, and P-Cdc2 were concurrently found to be downregulated in both cell lines. The findings suggest that Dp mediated cell cycle arrest possibly through modulation of the expression of p53, p21, and cell cycle-related proteins.
Dp induces apoptosis in glioma cells
To further investigate whether the decreased cell viability caused by Dp was related with apoptosis, Annexin V-FITC and propidium iodide staining was performed. As shown in Fig. 3A, Dp induced dose-dependent apoptosis when U87MG and T98G cells were exposed to various concentrations of Dp for 48 h. The apoptotic percentage in the U87MG and T98G cells treated with 40 µM Dp for 48 h was 20.93±3.42 and 17.99±2.87%, respectively. In addition, a higher percentage of apoptosis was observed when these cells were treated with 80 µM Dp for 48 h. However, in the vehicle controls, the percentage of apoptosis was only 4.85±1.28 and 5.13±1.59% in the U87MG and T98G cells (Fig. 3B).
Since mitochondrial membrane potential (Δψm) damage occurs before apoptosis, we further investigated the change of Δψm by JC-1 staining that is widely used to detect mitochondrial depolarization by a decrease in the ratio of red/green fluorescence intensity (26). The results indicated that in the Dp-treated U87MG and T98G cells, the number of cells with red staining was decreased while a concomitant increase in cells with green staining was noted, while the control cells showed strong red fluorescence and weak green fluorescence (Fig. 3C). These results demonstrated that there was a significant decrease in the ratio of red/green fluorescence intensity in the treated U87MG and T98G cells compared to the control cells (Fig. 3D), which indicated that Dp treatment resulted in the damage of Δψm in the U87MG and T98G cells.
Dp induces the release of cytochrome c and activates the apoptotic pathway
To further investigate the effect of Dp on mitochondrial integrity, we measured the cytochrome c release from mitochondria in the U87MG and T98G cells exposed to 0, 40, and 80 µM Dp for 48 h. The results indicated that the expression level of cytosolic cytochrome c was enhanced with an increasing concentration of Dp in both cell lines (Fig. 4), which suggested that Dp treatment induced cytochrome c release from the cellular mitochondria.
Additionally, we further detected the effects of Dp on apoptotic proteins Bim, Bax, and Bcl-2. The results of western blotting indicated that Dp treatment significantly upregulated the expression levels of pro-apoptotic proteins Bim and Bax compared to the control when U87MG and T98G cells were exposed to 40 and 80 µM Dp for 48 h. However, anti-apoptotic protein Bcl-2 expression was found to be highly downregulated in these two cell lines, which suggested that Dp treatment suppressed Bcl-2 expression and abrogated its anti-apoptotic effect.
Dp activates caspase-9 and caspase-3 in the glioma cancer cells
Increasing studies indicate that the caspase family plays a pivotal role in induction of cellular apoptosis, in particular for the initiation and execution of apoptosis (27). Therefore, we detected the enzymatic activation of procaspase-3/-9 during Dp-mediated apoptosis in the U87MG and T98G cells. The results of the western blot analysis demonstrated that a gradual reduction of intact procaspase-3/-9 was observed 48 h after the cells were treated with 0, 40, and 80 µM Dp, while their proteolytic cleaved forms, caspase-3/-9, were synchronously found to be upregulated (Fig. 5A). Further quantification analysis indicated that the expression of procas-pase-3/-9 protein was significantly decreased, while cleaved caspase-3/-9 was significantly increased when compared to the controls (Fig. 5B and C). These results suggest that Dp treatment induced the activation of caspase-3/-9, which was further confirmed by their activity analysis. As shown in Fig. 5D and E, the results of the Caspase-Glo-3/9 assays demonstrated that their activity was significantly enhanced in the Dp-treated U87MG and T98G cells in a concentration-dependent manner compared to the control. These findings suggest that Dp induced cellular apoptosis in U87MG and T98G cells probably via the caspase-dependent pathway.
Discussion
Malignant gliomas are the most common and fatal primary tumors of the central nervous system, and account for approximately 70% of the 22,500 new cases diagnosed in the United States each year (28,29). Even when a combination of surgery, radiotherapy and chemotherapy is applied, the outlook for patients with malignant gliomas remains very poor. Resistance to apoptosis is a characteristic of many types of cancer including malignant glioma, and finally abrogates the effects of radiotherapy and chemotherapy (18). Therefore, development of novel therapeutic strategies and discovery of new drugs are essential to improve the prognosis of patients with malignant gliomas (29). In the present study, we investigated Dp for its capability to suppress cell proliferation in human glioma cells. Our results indicated that Dp had strong inhibitory effects on glioma U87MG and T98G cells through cell cycle arrest at the G1 phase and induction of apoptosis.
Dracorhodin is a major constituent found in the resin of Daemonorops draco and is a traditional Chinese medicine for wound healing, pain alleviation, and bleeding control (30). Recently, dracorhodin and its derivant, Dp, were found to have similar pharmacologic activities, and many researchers have focused on their potential antitumor activity (12). Previous studies found that Dp inhibits cell growth and induces apoptosis in various cancer cells (14–17). In this study, we firstly found that Dp effectively suppressed the proliferation of human glioma U87MG and T98G cells. Furthermore, we also found that the growth inhibitory effect of Dp on glioma cells was associated with arrest of the cell cycle and induction of apoptosis.
Previous studies have demonstrated that Dp inhibited cell proliferation in various cancer cell lines, yet the effects of Dp treatment on cell cycle progression have not been evaluated. In this study, we confirmed that Dp treatment resulted in cell cycle arrest at the G1 phase in U87MG and T98G cells, concurrently reducing the percentage of cells at the S and G2/M phase (Fig. 2). To further investigate the mechanism of Dp-mediated cell cycle arrest in glioma cells, we detected the expression levels of cell cycle-associated proteins such as p53, p21, Cdc25A, Cdc2 and its phosphorylated form. The results of western blot analysis indicated that the expression levels of p53 and p21 proteins were significantly upregulated by Dp treatment. It is well-known that p53 functions as a focal point for determining whether cells respond to various types and levels of stress and treatment with cell cycle arrest, DNA repair, autophagy, cell metabolism, senescence and apoptosis (31). Previous studies have indicated that p21 is one of the most important downstream genes of p53, and contributes to the regulation of cell cycle progression at the G1/S phase (32), and recruitment of co-activators including Tip 60/hMOF and CBP/p300 is essential for the activation of p21 in a p53-dependent manner (33,34). Therefore, in the present study, we speculated that cell cycle arrest at G1 phase in glioma cells after Dp treatment was mediated by the regulation of p53 and p21 protein expression. However, whether Dp treatment results in the translocation of p21 in the subcellular location was not investigated, and needs to be clarified in future research. Previous studies indicate that Cdc25A and Cdc2 play an important role in cell cycle progression at the G1/S or G2/M checkpoint (35,36). Similar to previous studies, the downregulation of Cdc25A, Cdc2 and P-Cdc2 expression at least contributed to the inhibition of cell cycle progression in the U87MG and T98G cells.
The induction of apoptosis is regarded as an effective approach for cancer control and therapy. Accordingly, we investigated whether Dp-mediated anti-proliferation effects in glioma cells was due to the occurrence of apoptosis apart from cell cycle arrest. Our results indicated that a significant increase in the percentage of apoptosis in U87MG and T98G cells was observed after Dp treatment. Further investigation demonstrated that the damage of mitochondrial membrane potential and the following release of cytochrome c from the mitochondria confirmed the occurrence of apoptosis in these cells, similar to a previous study (15). Research has confirmed that p53 is an important regulator of apoptosis, and its interaction with Bcl-2 or Bcl-XL results in the assembly of pro-apoptotic proteins Bax and Bak in the mitochondrial membrane to form pores, which finally causes the release of cytochrome c and other apoptotic activators from the mito-chondria (37). It was reported that the mitochondrial pathway (also called the intrinsic pathway) of apoptosis is inhibited or triggered by anti-apoptotic proteins such as Bcl-2 and Bcl-xL or pro-apoptotic proteins such as Bax, Bak, Bim and Bid (38). In the present study, the downregulation of Bcl-2 expression, and the synchronously upregulated expression of Bim and Bax in the U87MG and T87G cells probably contributed to the release of cytochrome c and the following apoptotic events, which is similar to previous studies in human gastric tumor cells and breast cancer cells (14,15).
It is well-known that the caspase family plays a critical role in apoptotic initiation and execution (39). In the present study, our findings showed that Dp treatment resulted in the downregulation of procaspase-9 and procaspase-3 proteins in the U87MG and T98G cells, and concurrently upregulated the expression levels of cleaved caspase-9 and caspase-3, which indicated that procaspase-9 and procaspase-3 were degraded by proteolytic cleavage, and their corresponding cleaved fragments (cleaved caspase-9/-3) could be detected by western blotting. These findings indicated that caspase-9 and caspase-3 were activated by Dp treatment during apoptosis in the U87MG and T98G cells. Caspase-Glo-3/9 assays also confirmed that their activities were both significantly increased in these cells, which were indispensable for their roles in apoptosis. Previous studies also demonstrated that Dp treatment led to apoptosis in gastric cancer SGC-7901 cells (14), breast cancer MCF-7 cells (15), and leukemia HL-60 cells (17) via degradation of caspase-1, -3, -8, and -9, and increased their activities. Additionally, caspase-9 activation by a complex containing Apaf-1 and cytochrome c can trigger the activation of other caspase family members including caspase-3, -6, and -7, finally resulting in the occurrence of apoptosis (40,41).
Taken together, our findings indicated that Dp treatment effectively inhibited cell proliferation and arrested the cell cycle at G1 phase, and concurrently decreased the percentage of cells at the S and G2 phase in glioma U87MG and T98G cells. The arrest of the cell cycle was regulated through the mediation of the cell cycle regulatory proteins including the upregulation of p53 and p21 protein production, and synchronous reduction in the protein expression levels of Cdc25A, Cdc2 and its phosphorylated form. In addition, we found that Dp treatment induced cellular apoptosis in the U87MG and T98G cells in a concentration-dependent manner, and resulted in mitochondrial membrane potential loss and the release of cytochrome c. Moreover, further investigation indicated that the expression levels of pro-apoptotic Bim and Bax proteins were enhanced, while the level of anti-apoptotic Bcl-2 protein was simultaneously decreased by Dp treatment, resulting in the activation of caspase-9 and caspase-3, and finally inducing cellular apoptosis in the U87MG and T98G cells probably via the mitochondrial pathway. Therefore, our data suggest that Dp is possibly a potential candidate for glioma treatment.