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Introduction

Malignant gliomas are characterized by aggressive tumor growth with a mean patient survival of 15–18 months and frequently develop resistance to temozolomide (TMZ) which is the first-line treatment for patients with high-grade gliomas (1,2). Although TMZ has been well demonstrated to effectively prolong the survival of patients with brain tumors in clinical application, unfortunately, glioma cells showed resistance to TMZ in certain cases (3–6). Therefore, current therapy is far from satisfactory, indicating the need for new therapeutic agents and approaches which can prolong the survival of glioma patients.

Recently, extensive study has been conducted to evaluate the therapeutic efficacy and safety of natural compounds for treating cancer. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a natural product derived from the rhizome of rhubarb which has been used medicinally in China for more than 1,000 years (7). Rhein possesses diverse biological properties such as antioxidant (8–10), anti-inflammatory (11,12), antiviral (13), antibacterial (14), antifungal (15), anti-allergic (16) and anticancer (17–24). The reported mechanism of the antitumor activity of rhein in cancer cells is due to its ability to induce apoptosis and/or cell cycle arrest in corresponding cancer cells (20,21). Although rhein has many pharmacological effects, due to its inability to dissolve in water, the use of rhein in the clinic is limited.

Our previous studies showed that rhein lysinate (RHL), the salt of rhein and lysine that is easily dissolved in water, inhibited the proliferation of breast (25) and ovarian cancer (26), hepatocellular carcinoma (26), cervical cancer (27,28), and lung carcinoma cells (29), and human umbilical vein endothelial cells (HUVECs) (28). However, the IC50 was found to be higher in normal cells, meaning that the drug can be used to prevent the proliferation of cancer cells at a lower concentration, without a strong effect on normal cells. Yet, its effect on human glioma is still unknown.

In the present study, human glioma U87 cells and a xenograft model in BALB/c nude mice were used to examine the antitumor activity of RHL against human glioma, supporting the potential use of rhein as an anti-glioma medicinal agent.

Materials and methods

Chemicals and reagents

Rhein (purity, 98%) was purchased from Nanjing Qingze Medicine Ltd. (Nanjing, Jiangsu, China), while lysine was purchased from Beijing Solarbio Science and Technology Co. (Beijing, China). RHL was produced at the Department of Oncology of the Institute of Medicinal Biotechnology, the Chinese Academy of Medical Sciences (patent no. 200810089025.8). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (Shanghai, China). Dichlorodihydrofluorescein-diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR, USA). Antibodies targeting Bcl-2, BAX, Bim, cyclin D and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Secondary antibodies against rabbit or mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). The prestained protein marker p7708V was purchased from New England Biolabs Ltd. (Beijing, China). All other chemicals were of standard analytical grade.

Cell culture

The human glioma U87 cell line was cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The human glioma U87 cell line was obtained from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China).

In vivo therapeutic effects

Fourteen female BALB/c nude mice (20±2 g) obtained from Vital River Laboratories (Beijing, China), at the age of 4–6 weeks were used for human glioma U87 xenografts. Mice were maintained in a temperature-controlled room (22±2°C) with a 12-h light/12-h dark cycle and a relative humidity of 40–60%. The mice were given ad libitum access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Normal University. U87 tumors for implantation were initially grown by injection of U87 cells at a dose of 5×106 cells/mouse in two female BALB/c nude mice. A tumor piece of 2–3 mm in diameter was implanted subcutaneously into each experimental animal. After 9 days of tumor growth, the animals were randomly divided into groups (n=6) in a manner that minimized the difference in tumor size between the groups. Each animal received 200 μl of either phosphate-buffered saline (PBS) (vehicle control) or RHL by intraperitoneal injection every other day for a consecutive 14 days. At the end of the experiment, the body and tumor weight were measured.

Cell proliferation assay

Cell proliferation assays were performed using the MTT method, according to the manufacturer's instructions. The cells were seeded into 96-well plates (Costar, Cambridge, MA, USA) with 3,000 cells/well. Subsequent to overnight incubation, triplicate wells were treated with various concentrations of RHL (0, 20, 40, 80 and 160 μmol/l) for 48 h. Next, 20 μl of MTT solutions (5 mg/ml in PBS) were added to each well and incubated for 4 h at 37°C. The MTT formazan was dissolved in 150 μl DMSO and the absorbance was measured with a microplate reader (Multiskan MK3; Thermo Labsystems, Waltham, MA USA) at a wavelength of 570 nm.

Cell growth curve

Cells were plated into 96-well plates at a density of 500 cells/well. Six plates were seeded with cells. Triplicate wells/plate were treated with various concentrations of RHL (0, 20, 40, 80 and 160 μmol/l). One plate was assayed by MTT method as described above each day until the sixth day. The cell growth curve for various concentrations of RHL was constructed.

Detection of reactive oxygen species (ROS) by DHCF-DA

Intracellular hydrogen peroxide levels were monitored by fluorescence microscopy and flow cytometry after staining with DCFH-DA. Cells were plated at 250,000 cells/flask. Following a 24-h incubation period, the cells were treated with various concentrations of RHL (0, 20, 40, 80 and 160 μmol/l). Forty-eight hours later, DCFH-DA (10 μmol/l) was then added to each flask. After a 1-h incubation period, the cells were monitored using an Olympus inverted fluorescence microscope (Tokyo, Japan), and were subjected to flow cytometric analysis with FACSCalibur and CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA). The percentage of cells displaying increased dye uptake was used to reflect an increase in ROS levels.

FITC-Annexin V/PI apoptosis assay

Cells were plated at 250,000 cells/flask. Following a 24-h incubation period, the cells were treated with various concentrations of RHL (0, 20, 40, 80 and 160 μmol/l). Forty-eight hours later, the cells were collected and resuspended in 200 μl binding buffer. Then, 10 μl FITC-labeled enhanced Annexin V and 10 μl propidium iodide (PI) were added. Upon incubation in the dark (15 min at room temperature or 30 min at 4°C), the samples were diluted with 300 μl binding buffer. Cells were subjected to flow cytometric analysis with FACSCalibur and CellQuest software.

Western blot analysis

Cells were harvested and washed with PBS. The whole cellular extracts were prepared by incubating cells on ice in a lysis buffer and a cocktail of phosphatase inhibitors (Roche, Indianapolis, IN, USA). The cell lysates were cleared by centrifugation at 12,000 × g for 20 min. Protein concentrations were determined by Bradford assay. Equal amounts of lysate (40 μg) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked in TBST containing 5% non-fat skim milk at room temperature for 2 h and probed with primary antibodies overnight at 4°C. Then the membranes were blotted with an appropriate horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology). Proteins were visualized using enhanced chemiluminescence western blotting detection reagents (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Statistical analysis

Results are expressed as the means ± SD. Treatment effects were compared using one-way ANOVA and differences between the means were considered to indicate a statistically significant result when P<0.05.

Results

Inhibition of human glioma U87 xenograft growth in BALB/c nude mice

Treatment was started on day 9 after tumor transplantation. RHL was administered by intraperitoneal injection at doses of 50 mg/kg every other day for consecutive 14 days. Control mice were administered PBS vehicle only. The body weight of the animals showed no significant differences between the control and treated groups (Fig. 1A). The growth of tumors in the RHL-treated BALB/c nude mice was significantly suppressed compared with the controls (Fig. 1B and C). Treatment with RHL inhibited the growth of human glioma U87 xenografts by 31.9%. These findings suggest that RHL at a well-tolerated dose markedly inhibited tumor growth.

Figure 1 Effects of rhein lysinate (RHL) on the growth of human glioma U87 xenografts in BALB/c nude mice. Each animal was intraperitoneally administered with 200 μl of either PBS (vehicle control) or RHL every other day for consecutive 14 days after tumor transplantation. *P<0.05, compared with the control group. (A) Body weight, (B) tumor weight, (C) images of the tumors.

Growth inhibition of RHL in human glioma U87 cells

The growth inhibitory effect of RHL on human glioma U87 cells was examined with MTT assay. Cells were cultured for 48 h (Fig. 2A) in the presence of various concentrations of RHL. The U87 cells showed a decreased cell proliferation in a dose-dependent manner after treatment with RHL. In addition, RHL (160 μmol/l) inhibited the proliferation of the U87 cells by 40% at 48 h. Compared with the control group, RHL at 20 and 40 μmol/l inhibited U87 cell proliferation on day 6. Nevertheless, RHL at 80 and 160 μmol/l inhibited U87 cell proliferation from initial administration (Fig. 2B)

Figure 2 (A) Growth inhibition of human glioma U87 cells by RHL. Cells were exposed to rhein lysinate (RHL) at various concentrations for 48 h and proliferation was determined by MTT. (B) Growth curve of human glioma U87 cells treated with RHL. Cells were exposed to RHL at various concentrations for a consecutive 6 days and proliferation was determined by MTT each day. Data are from three independent experiments. *P<0.05, compared with the control group.

Induction of apoptosis by RHL in human glioma U87 cells

Induction of apoptosis by RHL was confirmed by FITC-Annexin V/PI staining. RHL at 80 μmol/l induced apoptosis in the glioma U87 cells. The ratio of apoptosis was significantly enhanced when cells were incubated with 160 μmol/l RHL for 48 h. This suggested that apoptosis was the predominant mode of RHL-induced cell death (Fig. 3).

Figure 3 Induction of apoptosis by rhein lysinate (RHL) in human glioma U87 cells. Cells were treated with RHL for 48 h at the indicated concentrations (1, 0 μmol/l; 2, 20 μmol/l; 3, 40 μmol/l; 4, 80 μmol/l; 5 160 μmol/l RHL). Cells were labeled with a combination of FITC-Annexin V and PI followed by flow cytometric analysis. The percentage of apoptotic cells (upper right quadrant and lower right quadrant) is indicated.

Induction of ROS production by RHL in human glioma U87 cells

Induction of ROS production by RHL was confirmed by DCFH-DA staining and detected by fluorescence microscopy and flow cytometry. Fluorescence intensity/cell was significantly enhanced when cells were treated with RHL for 48 h in dose-dependent manner. This suggested that RHL induced ROS production in a dose-dependent manner in glioma U87 cells (Fig. 4).

Figure 4 Induction of ROS production by rhein lysinate (RHL) in human glioma U87 cells. Cells were treated with RHL for 48 h at the indicated concentrations (1, 0 μmol/l; 2, 20 μmol/l; 3, 40 μmol/l; 4, 80 μmol/l; 5, 160 μmol/l RHL). Cells were labeled with DCFH-DA followed by inverted fluorescence microscopy (A) and flow cytometric analysis (B). The percentage of DCFH-DA-positive cells is indicated.

Downregulation of Bcl-2 and cyclin D and upregulation of BAX and Bim in the RHL-induced apoptosis in glioma U87 cells

In order to investigate the mechanism of RHL against human glioma U87 cells, we examined whether RHL treatment modulates the levels of apoptosis-associated and cell cycle-associated proteins. As shown in Fig. 5, expression of Bcl-2 was markedly downregulated by RHL treatment in the U87 cells, whereas the levels of BAX and Bim were increased. In addition, we also examined the expression of cyclin D. The level of cyclin D was decreased after RHL treatment.

Figure 5 Effect of rhein lysinate (RHL) on the expression of proteins associated with apoptosis and the cell cycle. Cells were treated with RHL at different concentrations for 48 h and then lysed and subjected to western blotting as described in Materials and methods. *P<0.05, compared with the control group.

Discussion

Malignant glioma is one of the most deadly human malignancies worldwide and its incidence has increased in recent years (30). In spite of the progress in the prognosis and treatment of glioma, the dismal outcome has not improved substantially over the last three decades. The survival of glioma patients is still quite short, particularly when invasion and metastasis occur, even when aggressive surgical resection, chemotherapy, and radiotherapy are carried out. Temozolomide (TMZ) is a well-tolerated orally bioactive alkylating agent used in glioma patients which has been adopted as the first-line treatment in patients with high-grade gliomas. Although TMZ has been well demonstrated to effectively prolong the survival of brain tumor patients in clinical application (1,2), unfortunately, glioma cells show resistance to TMZ in some cases (3–6). Therefore, new therapeutic agents and approaches are highly required for prolonging the survival of glioma patients.

Our previous studies showed that rhein lysinate (RHL), the salt of rhein and lysine that is easily dissolved in water, inhibited the proliferation of breast (25), ovarian cancer (26), hepatocellular carcinoma (26), cervical cancer (27,28), and lung carcinoma cells (29) and human umbilical vein endothelial cells (28). However, the IC50 value is higher in normal cells, meaning that the drug can be used to prevent the proliferation of cancer cells at a lower concentration, without a strong effect on normal cells. In addition, the in vivo and in vitro therapeutic efficacy of RHL on human glioma have not been evaluated. As known, human glioma U87 xenografts are useful models for testing the therapeutic effects of anti-tumor agents in vivo (31–33). Human glioma U87 cells and a xenograft model in BALB/c nude mice were used to examine the antitumor activity of RHL against human glioma in the present study. RHL exerted a significant inhibitory effect on the growth of human glioma U87 xenografts in BALB/c nude mice. There was no significant body weight loss in the treated groups compared with the control group. All animals survived the duration of the experiment. The results indicate that RHL shows a high efficacy against human glioma U87 cells.

RHL showed a potent cytotoxic effect on U87 cells. In the cell proliferation assays, RHL (160 μmol/l) inhibited U87 cell proliferation by 40% at 48 h (Fig. 2A). In the cell growth curve assays, RHL at concentrations of 80 and 160 μmol/l inhibited U87 cell proliferation from the initial administration (Fig. 2B). In the cell apoptosis assays, RHL displayed a highly potent apoptosis-inducing effect on the U87 cells. It is evident that the induction of apoptosis appeared to be the predominant mode of RHL-induced cell death particularly in the high dose-RHL group (160 μmol/l).

Excessive intracellular ROS production induced by a toxicant within the mitochondria or cytoplasm can damage many types of biological macromolecules such as membrane lipids, DNA and enzymes. Furthermore, ROS induce mitochondrial depolarization and permeability transition (34,35). In the present study, the DCF fluorescence assay showed that the intracellular ROS level in the RHL-treated cells was higher than that of the control cells. This observation suggests that RHL exposure caused oxidative stress, mitochondrial permeability transition, and apoptosis of U87 cells. Therefore, excessive ROS induction by RHL exposure may be a key early factor in the cellular damage and apoptosis of U87 cells.

As known, members of the Bcl-2 protein family act as key regulators of cellular apoptosis and are important determinants of cellular sensitivity or resistance to chemotherapy drugs (36–38). Overexpression of Bcl-2, an anti-apoptotic member of this family, is commonly observed in human cancers, and Bcl-2 overexpression correlates with chemoresistance in this disease. The anti-apoptotic protein, Bcl-2, has been associated with inhibition of apoptosis and cell survival mechanisms. The Bax and Bim proteins are pro-apoptotic members of this family, and their increased expression is often associated with increased apoptosis in target cells (39,40). Next, we studied the molecular mechanism of apoptosis induced by RHL in human glioma U87 cells. As shown in Fig. 5, when the U87 cells were treated with RHL for 48 h, levels of Bax and Bim proteins were significantly upregulated compared with the control group in a dose-dependent manner. In contrast, compared with the control group, the level of Bcl-2 protein in the cells treated with RHL was significantly downregulated in a dose-dependent manner. Furthermore, the level of cyclin D1 was decreased in the RHL group in a dose-dependent manner. Cyclin D1 is a protein required for progression through the G1 phase of the cell cycle. During the G1 phase, it is synthesized rapidly and accumulates in the nucleus, and is degraded as the cell enters the S phase. Cyclin D1 is a regulatory subunit of cyclin-dependent kinases CDK4 and CDK6. The protein dimerizes with CDK4/6 to regulate G1/S phase transition and entry into the S phase (41). It can be concluded that RHL also inhibited cell proliferation by blocking the G1/S phase of the cell cycle.

Our results showed that RHL is highly effective against the growth of human glioma U87 xenografts in BALB/c nude mice. In vitro, RHL induced apoptosis in human glioma U87 cells by decreasing Bcl-2 and increasing the expression of BAX and Bim. The downregulation of the level of cyclin D was also involved in the anticancer effects of RHL.

Acknowledgments

The present study was supported by grants from the National Natural Science Foundation of China (81001439), and the General Program of the Natural Science Foundation of Hebei Province of China (H2012401030).

References