Malignant glioma is the most common primary brain tumor and patients with this tumor have poor prognoses.1 Despite the availability of numerous treatment strategies, including surgery, radiotherapy and chemotherapy, the median survival time is 12–15 months for glioblastoma multiforme (WHO grade 4 astrocytoma) and 2–3 years for anaplastic astrocytoma (WHO grade 3 astrocytoma).1 Thus, there is an urgent need for better therapies for treatment of these malignant brain tumors.
Tetrandrine (C38H42O6N2, MW 622.730) is a bisbenzylisoquinoline alkaloid that is extracted from the root of Stephania tetrandra S. Moore, a herbaceous perrenial vine of the family Menispermaceae that has been used in traditional Chinese medicine.2 Tetrandrine has anti-inflammatory, immunosuppressive and cytoprotective effects.2–4 It suppresses T and B cells and inhibits the production of cytokines such as histamine, prostaglandins and tumor necrosis factor-α.5, 6 Tetrandrine has been used for treatment of silicosis and can suppress inflammatory reactions associated with rheumatoid arthritis and uveitis or hepatitis in animals that is provoked by bovine serum albumin, interleukin-1 (IL-1) or endotoxin.2, 7 It also prolongs survival time of mice following allogenic cardiac transplant.8 Tetrandrine also blocks calcium channels, decreases portal venous pressure and blood pressure4, 6, 9 and has antioxidant effects, which inhibits lipid peroxidation and platelet aggregation and reduces ischemia/reperfusion injury.10, 11
Recent research has shown that tetrandrine inhibits the proliferation and induces the apoptosis of several cancers, including breast cancer, lung cancer, neuroblastoma, Burkitt's lymphoma, hepatoma and leukemia.3, 12–16 The mechanisms of these antitumor effects are not fully understood. Tetrandrine increases the expression of p53, p21 and bax, causes p53 nuclear translocation, induces pRB hypo-phosphorylation, increases cell apoptotic signals such as Apo-1 (also called CD95 or FasR), induces the Cdk inhibitor p1, decreases the expression of cyclin D1, increases the release of mitochondrial cytochrome c, activates caspases-3, -8 and -9, increases cleavage of PARP (poly(ADP-ribose) polymerase) and reduces Bcl-XL.13, 14, 16 Furthermore, tetrandrine decreases the level of glutathione and causes cells to arrest in the G1 phase of the cell cycle.6, 13, 14 All of these effects suggest that tetrandrine induces cellular apoptosis, although the exact mechanism is still uncertain.
There are several reports on the effects of tetrandrine on gliomas, and most of these have focused on glioma cell ion channels.17–22 Tetrandrine suppresses the expression of large-conductance, calcium-activated potassium channels in human glioma cells19, 20 and inhibits calcium mobilization in rat glioma cells.18 We hypothesized that tetrandrine might inhibit the proliferation of glioma cells because ion channels in unexcitable cells are involved in proliferation and volume regulation20 and tetrandrine induces cellular apoptosis via inhibition of BK channels, which reduces K+ efflux and depolarization.6, 13, 15 There are no previous reports on the effects of tetrandrine on glioma cell proliferation. One publication reported that tetrandrine eliminates radiation-induced cell cycle perturbation and enhances the radiosensitivity of human glioblastoma cells.23 Another recent study reported that tetrandrine inhibits adjuvant-induced angiogenesis in vitro and suppress angiogenesis in streptozotocin-induced diabetic rodents.24 Heretofore, no studies have investigated the effects of tetrandrine on the neovascularization of tumors.
In this study, we investigated the effects of tetrandrine on the proliferation and apoptosis of glioma cells, the in vivo antitumor effects of tetrandrine on gliomas and the effect of tetrandrine on glioma angiogenesis.
Material and methods
Cell lines and cell culture
The cell lines were the rat RT-2 glioma cell line and the ECV304 human umbilical vein endothelial cell line. RT-2 is derived from an avian sarcoma virus-induced brain tumor in the Fischer 344 rat.25 ECV304 is an immortalized human umbilical vein endothelial cell (HUVEC) line.26, 27 Both RT-2 and ECV304 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Seromed, Berlin, Germany) that was supplemented with 10% fetal calf serum (FCS; Biological Industries, Israel), 2 mM L-glutamate, 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator.
Preparation of tetrandrine
Tetrandrine was purchased from Sigma-Aldrich Chemical (St. Louis, MO) and made into a fine suspension by dissolution in 0.1 N HCl at a concentration of 25 mg/mL. Then it was passed through a 0.22 μm filter for bacteriologic sterilization. Ultrasonification of the thawed preparation and adjustment of the pH to 6.8 was performed immediately before use.
Cytotoxic effects of tetrandrine on RT-2 glioma cells
The sensitivity of RT-2 cells to tetrandrine was determined in vitro by an MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assay. For this assay, 5 × 103 cells were seeded in triplicate wells of a flat-bottomed 24-well microtiter plate and cultured overnight before treatment. Cells were exposed to various concentrations (0–75 μM) of tetrandrine for 24 or 72 hr. After removal of tetrandrine, cells were incubated for a total of 5 days. The extent of cell proliferation and viability was determined by the MTT assay. The tetrandrine concentration at which 50% of cells were killed was designated as the LC50.
Analysis of tetrandrine-induced apoptosis by flow-activated cell sorter (FACS) flow cytometry
Following 24- or 72-hr treatment with various concentrations of tetrandrine (0–75 μM), 1 × 106 cells were trypsinized and washed twice with phosphate-buffered saline (PBS). Cells were stored in 1 mL 80% ethanol/PBS at −20°C for subsequent experiments. For FACScan analysis, cells were centrifuged at 14,000 rpm for 5 min and washed twice with PBS. Then, cells were incubated with 0.5 mL 0.5% Triton X-100/PBS and 5 μg RNase A for 30 min, then stained with 0.5 mL 50 μg/mL propidium iodide/PBS in the dark. Analysis was performed with FACScan flow cytometry (FACSCalibur, Becton Dickinson Immunocytometry System, San Jose, CA).
One million cells cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) were treated with 25 μM tetrandrine for 24 hr or were untreated (controls). Then, adherent cells were stained using the MEBSTAIN Apoptosis kit Direct (Immunotech, Marseille, France). All procedures were conducted according to manufacturer's instructions. Slides were observed on a Zeiss Axioskopz epifluorescence microscope (Carl Zeiss Jena GmbH, Zeiss Gruppe, Germany). Apoptotic fractions were counted and compared.
Reverse transcription-polymerase chain reaction (RT-PCR) for analyses of expression of vascular endothelial growth factor (VEGF) in RT-2 glioma cells treated with tetrandrine
After the treatment with 0–25 μM of tetrandrine for 24 hr, the expression vascular endothelial growth factor (VEGF) of the RT-2 glioma cells was determined by RT-PCR. First, the RNA of the cells was extracted using REzol™ C&T (Promega, Madison, WI). About 1 μg of total RNA was reverse transcribed using SuperscriptII reverse transcriptase (Gibco BRL, Grand Island, NY) according to the manufacturer's instructions. The expression levels of VEGF mRNA were determined relative to the expression of GAPDH mRNA. Amplified products were obtained in the exponential phase for both sets of primers at 35 cycles. Differences in the expression of the RT-PCR products were analyzed by ABC-Tiger Gel V2.0 (software from Taigen Bioscience Corp., Taipei, Taiwan). The sense and antisense primers of VEGF were 5′-CTGTGTGCCCCTAATG-3′ and 5′-CTCCGCTCTGAACAAG-3′. The sense and antisense primers of GAPDH (control) were 5′-CACCACCAACTGCTTAG-3′ and 5′-CTTCACCACCTTCTTGATG-3′.
Whole cell extract preparation and western blot analyses
After cells were treated with various concentrations (0, 1, 5, 10, 25 μM) of tetrandrine for 24 hr, they were lysed in buffer containing 20 mM HEPES at pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 0.5 μg/mL leupeptin, 1μg/mL aprotinin and 100 μg/mL 4-(2-aminoethyl) benzenesulfonyl fluoride. The cell lysate was rotated at 4°C for 30 min, and then centrifuged at 10,000 rpm for 10 min after which precipitates were discarded. Protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as reference standard. Cellular protein (20–50 μg) was loaded onto 10% SDS-polyacrylamide gels. Protein bands were then transferred electrophoretically to PVDF membranes (Micron Separations, Westborough, MA). Membranes were probed with anti-VEGF or anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) antibody, followed by a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Detection of antibody reactions was performed with Western blotting reagent ECL (Santa Cruz Biotechnology). Resultant chemiluminescence was demonstrated by exposure of the filter to Kodak Medical X-ray film (Eastman Kodak Company, Rochester, NY). Differences in expression of proteins were analyzed by ABC-Tiger Gel V2.0 (software from Taigen Bioscience Corp., Taipei, Taiwan).
Cytotoxic effects of tetrandrine on the ECV304 human umbilical vein endothelial cells
An in vitro proliferation assay was used to analyze the antiangiogenic effect of tetrandrine. In triplicate wells of a flat-bottomed 12-well microtiter plate, 1 × 104 ECV304 HUVECs (prepared as described above) were seeded and cultured overnight before tetrandrine treatment. The ECV304 cells were exposed to 0–250 μM tetrandrine for 24 or 72 hr. Then the MTT-based colorimetric assay was used to measure cell viability.
Animal experiments were approved by the Department of Clinical Research at the National Taiwan University Hospital and were conducted according to the guidelines of the Laboratory Animal Center of the National Taiwan University College of Medicine. Fischer 344 rats, weighing 200–350 g, were used for the experiments. The C57/BL6 mice weighing 18–35 gm were used for the matrigel plug assay. The rats and mice were housed with free access to food and water and were exposed to a 12:12 hr day-night cycle (lights on between 0600 and 1800 hr) at 20°C. The rats were anesthetized with 10 mg/kg xylazine and 80 mg/kg ketamine hydrochloride and the mice were anesthetized with 3 mg/kg xylazine and 80 mg/kg ketamine hydrochloride before experimentation.
Matrigel plug assay
Mice were injected subcutaneously with 500-μL BD Matrigel™ Matrix (BD Biosciences, Bedford, MA) containing PBS, basic fibroblast growth factor (bFGF, 1 nM) (Peprotech, Rocky Hill, NJ) or tetrandrine (100 μM) plus bFGF (1 nM). PBS and bFGF served as negative and positive control.28 Mice were euthanized after day 7, plugs dissected and angiogenesis was analyzed using hemoglobin measurement. Hemoglobin was measured by the tetramethylbenzidine (TMB) method,29 when compared with a standard curve, and the values normalized to plug weight. Some of the plugs were paraffin-embedded for histological examination which was used to correlate with the TMB method. Five-micrometer sections were deparaffinized and stained using Masson's trichrome reagent.29
Tetrandrine treatment of subcutaneous RT-2 tumors in rats
To study the antitumor effects of tetrandrine on RT-2 gliomas in rats, subcutaneous tumors were induced by injection of 1 × 105 RT-2 cells (in 10 μL of PBS) into the right flanks of Fischer rats. These rats were then given various treatments starting immediately (small subcutaneous glioma model) or at day 5 (large subcutaneous glioma model) post tumor cell inoculation.7, 8, 30, 31 The experiment for the study of the effect of tetrandrine on the small subcutaneous gliomas consisted of 4 groups. Group A received no treatment, Group B received oral gavage of the vehicle, Group C received oral gavage of 50 mg/kg/day of tetrandrine and Group D received oral gavage of 150 mg/kg/day tetrandrine. The experiment for the study of the effect of tetrandrine on the large subcutaneous gliomas consisted of 4 groups. Group A-1 received no treatment, Group B-1 received oral gavage of the vehicle, Group C-1 received oral gavage of 50 mg/kg/day of tetrandrine and Group D-1 received oral gavage of 150 mg/kg/day tetrandrine. Treatment was given once daily for 4 weeks. Animal survival over time was followed for all groups.
Tumor growth rates
Tumor size was determined twice weekly until each rat died. A blinded observer measured tumor length and width. Tumor volume was calculated from the formula V = 1/2 (d1 × d2 × d3), where d1, d2 and d3 are tumor diameters as measured with calipers in mutually perpendicular directions. Average daily tumor volumes from each group were compared. Group averages were not calculated after one or more animals in a group died.
Tetrandrine treatment of intracerebral gliomas in rats
Intracerebral tumors were induced by implanting tumor cells into the brains of Fischer 344 rats (10 rats per group) by stereotactic surgery. Each rat was fixed in a stereotactic frame, a burr hole was drilled and tumor cells were injected into the right caudate-putamen (CPu) (coordinates: 2.5 mm lateral, 1 mm anterior to the bregma, 4 mm below the dura) with a Hamilton syringe. About 5 × 103 tumor cells (suspended in 5 μL of PBS) were injected. The syringe remained in place for 3 min and then was slowly withdrawn for an additional 3 min. These rats received various treatments starting immediately (small intracerebral glioma model) or at day 3 (large intracerebral glioma model) post tumor cell inoculation. The experiment for the study of the effect of tetrandrine on the small intracerebral gliomas consisted of 3 groups. Group E received no treatment, Group F received oral gavage of the vehicle and Group G received oral gavage of 150 mg/kg/day of tetrandrine. The experiment for the study of the effect of tetrandrine on the large intracerebral gliomas consisted of 3 groups. Group E-1 received no treatment, Group F-1 received oral gavage of the vehicle and Group G-1 received oral gavage of 150 mg/kg/day tetrandrine. The treatment was continued until a total of 4-week treatment was completed or the animal died, whichever came first. Survival rates and survival time was recorded.
Effects of tetrandrine on the serum biochemistry and internal organs of the rats
To study the toxicity of tetrandrine, the rats received either no treatment or oral gavage of 150 mg/kg/day tetrandrine for 4 weeks (6 rats in each group). Four weeks after initiation of treatment, the blood was sampled and the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN) and creatinine were measured to monitor the hepatic and renal functions using an automatic analyzer (model 717, Hitachi, Tokyo, Japan). The results were presented as international unit per liter (IU/L) or mg/dl. In addition, the liver and small intestine were also harvested and subjected to histological study (H & E stain) to examine the tissue damage.
Immunohistochemical studies of large subcutaneous gliomas treated with tetrandrine
Animals with subcutaneous tumors received various treatments 5 days after tumor cell inoculation and tumors were harvested 4 weeks after initiation of treatments. For immunohistochemical analysis, these tumors were embedded in AMES Ornithine carbamyl transferase embedding compound (Miles, Elkhart, IN) and frozen at −70°C. For immunohistochemical staining, 8 μm cryostat sections of the tumors were air-dried for 1 hr at room temperature. Sections were fixed in acetone at 4°C for 5 min, washed with PBS and then incubated with 3% H2O2 in methanol for 30 min. The sections were then dried and incubated with blocking solution for 30 min. Next, the specific antibody was diluted in 1% bovine serum albumin (BSA) in PBS. Mouse anti-rat CD31 antibody (PharMingen, San Diego, CA) was used to monitor changes of CD31 expression in endothelial cells and another nonspecific monoclonal antibody was used as a negative control. The antibodies were layered onto the section and incubated at 4°C for more than 12 h. After reacting with a secondary antibody, the sections were processed with DAKO LSAB®2 System HRP (DAKO Corp., Carpinteria, CA) according to the manufacturer's instructions. The slides were then counterstained with hematoxylin, mounted with a coverslip and viewed under a light microscope. The number of CD31-stained sites in the gliomas was counted, with counts representing microvessel density.32 Low power light microscopy (magnification 40× and 100×) was used to scan the heterogeneous tumor sections for areas with high neovascularization. Any single positive-stained cell or cluster of endothelial cells that was clearly separate from adjacent microvessels, tumor cells and other connective tissue elements was considered to be a vessel. The presence of red blood cells or a vessel lumen was not required for a structure to be classified as a microvessel. Microvessels were counted in the 3 areas of highest vascular density on a 200× field (20× objective lens and 10× ocular lens). Microvessel density (MVD) was expressed as the mean number of vessels in these areas.
One-way analysis of variance (ANOVA) by Scheffe's multiple comparison was used for statistical analyses of the extent of glioma cell cytotoxicity and apoptosis, the proliferation of the ECV304 HUVECs induced by various concentrations and exposure times of tetrandrine, and the difference in tumor size among the groups. Fisher's exact test was used to analyze animal survival rates. The Kaplan-Meier method was used to assess animal survival time, and the log-rank statistic was used to test differences between groups. The Student t-test was used for analysis of the difference in serum biochemistry and hemoglobin levels in matrigel plugs. The difference in microvessel density among various groups was analyzed by the Mann-Whitney U test. Statistical significance was p < 0.05.
Cytotoxic effects of tetrandrine on RT-2 glioma cells
In our initial experiments, we performed an MTT-based colorimetric assay to determine the cytotoxic effect of tetrandrine on RT-2 glioma cells. Figure 1 shows the survival curve of glioma cells treated with various concentrations of tetrandrine for 24 or 72 hr. The viability of cells treated with tetrandrine was concentration- and time-dependent (p < 0.05). The LC50 was 22.6 μM for the 24 hr treatment and 11.3 μM for the 72 hr treatment.
Tetrandrine-induced apoptosis of glioma cells
Next, we used FACS flow cytometry to determine the effect of tetrandrine on glioma cell apoptosis. Our results show that apoptosis was directly related to tetrandrine concentration and drug exposure time (Fig. 2a) (p < 0.05). Control cells (no tetrandrine) had average apoptotic fractions of 3.8–4.3%. Treatment with 25 μM tetrandrine for 24 or 72 hr induced apoptosis in an average of 19.9 and 24.8% of cells, respectively. Treatment with 75 μM tetrandrine for 24 or 72 hr induced apoptosis in an average of 38.3 and 52.7% of cells, respectively. We also examined the extent of apoptosis in cells treated with 25 μM tetrandrine for 24 hr by use of TUNEL staining (Fig. 2b). In this experiment, the apoptotic fraction was 20.5 ± 2.1%, significantly higher than that of untreated control cells (1.1 ± 0.9%) (p = 0.001). These results indicate that tetrandrine induces apoptosis of glioma cells in a concentration- and time-dependent manner.
Effect of tetrandrine on expression of VEGF in RT-2 glioma cells
VEGF is important in vasculogenesis and angiogenesis, so we examined its expression in RT-2 glioma cells treated with various concentrations of tetrandrine for 24 hr by use of RT-PCR (Fig. 3a). Low-dose (0–1 μM) tetrandrine had no effect on VEGF expression, but treatment with 5, 10 and 25 μM tetrandrine for 24 hr resulted in 19, 25 and 70% suppression (respectively) relative to the control. Further, western blot analysis also showed tetrandrine suppressed the expression of VEGF (Fig. 3b). Treatment with 10 and 25 μM tetrandrine for 24 hr resulted in 65 and 91% suppression (respectively) of VEGF expression relative to the control. These data indicate that tetrandrine suppresses the expression of VEGF in RT-2 glioma cells in a concentration-dependent manner.
Tetrandrine induced cytotoxicity in ECV304 human umbilical vein endothelial cells
Next, we analyzed the effect of tetrandrine on endothelial cells by using an in vitro proliferation assay. Tetrandrine suppressed the proliferation of ECV304 HUVECs in a concentration-dependent manner (Fig. 3c) (p < 0.05). The LC50 was 34.2 μM for the 24 hr treatment and 25.1 μM for the 72 hr treatment. These results clearly indicate that tetrandrine inhibits the proliferation of ECV304 human umbilical vein endothelial cells in vitro.
Tetrandrine suppressed bFGF-induced angiogenesis in vivo
To study the effect of tetrandrine on the angiogenesis in vivo, the matrigel plug assay was done (Fig. 3d and 3e). The hemoglobin level in the matrigel plug containing bFGF was significantly higher than that containing PBS (p = 0.001). In contrast, the hemoglobin level in the matrigel plug containing both tetrandrine and bFGF was significantly lower than that in the matrigel plug containing bFGF only (p = 0.001), although it was still higher than that containing PBS (p = 0.005). The data indicated that tetrandrine could suppress the bFGF-induced angiogenesis in vivo.
Effect of tetrandrine on subcutaneous gliomas
We subcutaneously inoculated rats with 105 RT-2 cells over the right flank, and then applied different treatments immediately or 5 days later. During the 4-week treatment period, animals given tetrandrine-exhibited good activity, normal food and water intake and had no body weight loss, indicating good tolerance of this treatment. Animals experienced significant loss of body weight and decreased appetite as the tumors became large.
For the small subcutaneous gliomas (Fig. 4a), all rats in Groups A (no treatment) and B (treated with vehicle) died, with survival times of 46.4 ± 8.1 days and 48.1 ± 8.6 days, respectively. The survival time of the rats in Groups A and B was not significantly different (p = 0.67). In contrast, Group C (50 mg/kg/day of tetrandrine) had significantly longer survival time than Groups A and B (Group C vs. Group A or B, p = 0.001), with 20% of the rats having long-term survival. Animals in Group D (150 mg/kg/day of tetrandrine) had significantly longer survival times than in the other 3 groups (Group D vs. Group A or B, p = 0.0001; Group D vs. Group C, p = 0.0075). In Group D, 70% of the rats had long-term survival and the survival time of rats that ultimately died was 68 days longer. The survival rate of Group D rats was significantly higher than that of Groups A, B and C (Group D vs. Group A or B, p = 0.002; Group D vs. Group C, p = 0.035). These results indicate that treatment with 150 mg/kg/day of tetrandrine has antitumor effects on the small subcutaneous gliomas, increases survival rate, and prolongs survival time, whereas low dose tetrandrine had mild antitumor effects.
For the large subcutaneous gliomas (Fig. 5a), all rats in Groups A-1 (no treatment), B-1 (treated with vehicle) and C-1 (50 mg/kg/day of tetrandrine) died, with survival times of 48.4 ± 8.4 days, 47.0 ± 8.3 days and 56.6 ± 11.7 days, respectively. The survival time of the rats in Groups A-1 and B-1 was not significantly different (p = 0.89). In contrast, animals in Group C-1 (50 mg/kg/day of tetrandrine) had significantly longer survival times than those in Groups A-1 and B-1 (Group C-1 vs. Group A-1, p = 0.042; Group C-1 vs. Group B-1, p = 0.025). Animals in Group D-1 (150 mg/kg/day of tetrandrine) had significantly longer survival times than in the other 3 groups (Group D-1 vs. Group A-1, B-1, or C-1, p = 0.0001). In Group D-1, 50% of the rats had long-term survival and the survival time of rats that ultimately died was 62 days longer. The survival rate of Group D-1 rats was significantly higher than that of Groups A-1, B-1 and C-1 (p = 0.02). These results indicate that treatment with 150 mg/kg/day of tetrandrine has antitumor effects on the large subcutaneous gliomas, increases survival rate and prolongs survival time. The animal survival of the small and large subcutaneous gliomas treated with 150 mg/kg/day tetrandrine was further compared, and we found that tetrandrine was more effective for the small subcutaneous gliomas than the large ones, as shown by the longer animal survival time in the former (p = 0.03), but there was no difference of the animal survival rate between the small and large glioma groups (p = 0.14).
Figure 4b shows the growth rate of the small subcutaneous RT-2 tumors in rats following various treatments. The tumor size was not significantly different in Groups A, B and C from day 10 to 35 (p > 0.07). In contrast, the tumor size of rats in Group D was significantly smaller than that of animals in Groups A and B from day 21 to 35 (p < 0.05) and smaller than that of animals in Group C at day 17 (p = 0.04). Figure 5b shows the growth rate of the large subcutaneous RT-2 tumors in rats following various treatments. The tumor size from day 10 to 21 was not significantly different in the 4 groups (p > 0.08). There was also no difference in tumor size of rats in Groups A-1, B-1 and C-1 from day 25–31 (p > 0.2). In contrast, the tumor size of rats in Group D-1 was significantly smaller than that of animals in Groups A-1 and B-1 from day 25 to 31 (p < 0.05) and smaller than that of animals in Group C-1 from day 31 to 35 (p < 0.05). These results indicate that treatment with 150 mg/kg/day of tetrandrine suppresses the growth of subcutaneous RT-2 tumors.
Effect of tetrandrine on intracerebral gliomas
The rats were intracerebrally inoculated with 5 × 103 RT-2 cells, followed by various treatments starting immediately (small intracerebral glioma model) or at day 3 (large intracerebral glioma model) post tumor cell inoculation. The treatment was continued until a total of 4-week treatment was completed or the animal died, whichever came first.
For the small intracerebral gliomas (Fig. 6a), all rats in Groups E (no treatment) and F (vehicle) died and had similar survival times of 20.4 ± 2.8 and 20.0 ± 3.2 days, respectively (p = 0.67). In contrast, the survival time of Group G (150 mg/kg/day of tetrandrine) was significantly longer than that of the other 2 groups (Group G vs. Group E or F, p = 0.0001), with 2 of the 10 rats having long-term survival. The results indicated that treatment with 150 mg/kg/day of tetrandrine exerted antitumor effects on the small intracerebral gliomas and prolonged the animal survival.
For the large intracerebral gliomas (Fig. 6b), all the rats in Groups E-1 (no treatment), F-1 (vehicle only) and G-1 (150 mg/kg/day tetrandrine) died and had survival times of 19.4 ± 3.5, 20.2 ± 3.7 and 35.6 ± 4.1 days, respectively. The survival time was not significantly different for rats in Groups E-1 and F-1 (p = 0.57), but rats in Group G-1 had significantly longer survival times (Group G-1 vs. Group E-1 or F-1, p = 0.0001). These results indicate that 150 mg/kg/day of tetrandrine has antitumor effects on the intracerebral gliomas and prolongs the animal survival. The animal survival of the small and large intracerebral gliomas treated with 150 mg/kg/day tetrandrine was further compared, and we found that tetrandrine was more effective for the small subcutaneous gliomas than the large ones, as shown by the longer animal survival time in the former (p = 0.04), but there was no difference of the animal survival rate between the small and large glioma groups (p = 0.24).
Effects of tetrandrine on the serum AST, ALT, BUN and creatinine levels in rats
To study the toxicity of tetrandrine, the serum AST, ALT, BUN and creatinine were measured 4 weeks after initiation of tetrandrine treatment. The serum ALT level was 96.8 ± 14.8 IU/L in the rats treated with 150 mg/kg/day tetrandrine for 4 weeks, which was higher than that in the control group (54.8 ± 7.7 IU/L) (p = 0.005). The serum AST, BUN and creatinine levels were 127.8 ± 28.1 IU/L, 13.5 ± 3.3 mg/dl and 0.5 ± 0.1 mg/dl, respectively in the rats treated with tetrandrine, which were not different from those in the control group (123.2 ± 18.7 IU/L, 10.0 ± 3.2 mg/dl and 0.6 ± 0.2 mg/dl, respectively) (p = 0.4, 0.05, 0.1, respectively)
Histological examination of the livers and small intestines in the rats treated with tetrandrine
To study the toxicity of tetrandrine, the livers and small intestines were harvested 4 weeks after initiation of tetrandrine treatment and subjected to H & E staining (Fig. 7). The livers andsmall intestines showed no significant damage after 4-week tetrandrine treatment, when compared with those receiving no treatment.
Effect of tetrandrine on microvessel density in large subcutaneous gliomas
Finally, we used immunohistochemical methods to determine the effect of tetrandrine on the microvessel density (MVD) of gliomas (Fig. 8). Animals with subcutaneous gliomas received various treatments 5 days after tumor cell inoculation and tumors were harvested 4 weeks after initiation of treatments. The MVD was not significantly different for Group A-1 (104.6 ± 8.3) and Group B-1 (98.2 ± 9.0) (p = 0.1, Mann-Whitney U test). In contrast, the MVD of Group C-1 (37.0 ± 6.4) and Group D-1 (14.2 ± 3.1) was significantly less than that of Group A-1 or B-1 (p = 0.01, Mann-Whitney U test). The MVD of Group D-1 was also significantly less than that of Group C-1 (p = 0.01, Mann-Whitney U test). These results indicate that high dose (150 mg/kg/day) or low dose (50 mg/kg/day) tetrandrine suppresses glioma angiogenesis and that a high dose has a stronger effect.
The effects of tetrandrine on the proliferation and apoptosis of glioma cells have not been studied previously. We found that tetrandrine has cytotoxic effects and elicits a concentration- and time-dependent inhibition of glioma cell proliferation, with an LC50 in the micromolar range. Our LC50 value is comparable with previously reported LC50 values for tetrandrine against breast cancer, lung cancer, neuroblastoma, Burkitt's lymphoma, hepatoma and leukemia.3, 12–16, 33 Defective control of apoptosis plays a central role in tumor pathogenesis,34 so we also studied the effect of tetrandrine on induction of glioma cell apoptosis. Our results show that tetrandrine induces apoptosis in RT-2 glioma cells as has been observed in other cancer cells.13, 12–16, 33 Tetrandrine-induced apoptosis of the RT-2 glioma cells also occurs in a concentration- and time-dependent manner, with higher concentrations and prolonged exposure times eliciting greater cellular apoptosis. Our tetrandrine-induced cytotoxicity and apoptosis data suggest that prolonged treatment with tetrandrine, with a serum level in the micromolar range, has potential therapeutic value for treatment of glioma. Our results also demonstrate that treatment with certain levels of tetrandrine have greater effects on cytotoxicity than apoptosis. This suggests that mechanisms other than apoptosis may contribute the cytotoxic effect of tetrandrine against glioma cells.
We further investigated the in vivo effects of tetrandrine on gliomas in rats. Our results show that tetrandrine slows the growth of subcutaneous tumors, prolongs survival time, and increases survival rate and that a high dose (150 mg/kg/day) is more effective than a low dose (50 mg/kg/day). Further, we found tetrandrine was more effective for the small subcutaneous gliomas than the large ones (tetrandrine treatment was started at day 5 post tumor cell inoculation). The less antitumor effect of on large tumors was a common problem seen in many other treatment strategies for the malignant tumors in clinical situation. Only 2 previous studies have reported in vivo antitumor effects of tetrandrine. One of these studies found that intraperitoneal injection of tetrandrine (30 mg/kg, every other day) had no antitumor effects on human breast cancer xenografts in athymic nude mice.12 Another report demonstrated that intraperitoneal injection of 10 mg/kg/day tetrandrine for 5 days (starting from the day of intravenous injection of tumor cells) reduced the number of pulmonary metastatic foci of colon cancers by 40%.35 However, tumor model used in the colon cancer study represents small tumor model because tumors were treated immediately after injection of tumor cells. In our study, we increased the dose of tetrandrine to 150 mg/kg/day and started treatment either immediately or 5 days after tumor cell inoculation. The delayed treatment is more closely resembles the clinical situation. There may be several explanations for the differences between our results and the results of previous antitumor studies with tetrandrine including the use of different animal models, inoculation with different amounts of tumor cells, use of different types of tumor cells and differences in the plasma levels of tetrandrine. We did not measure the plasma level of tetrandrine in rats treated with 150 mg/kg/day. In the literature, a report had studied the plasma level of tetrandrine after intraperitoneal injection of 30 mg/kg of tetrandrine in mice.36 The plasma peak concentration of tetrandrine achieved 1.98 μM and more than 1.0 μM plasma concentration lasted until 18 hr after administration of tetrandrine.36 Although the dosage and the route of administration of tetrandrine were different from what we used, the data might give us a hint about the plasma tetrandrine level in the rats treated with tetrandrine in our experiment. Because the effect of tetrandrine on subcutaneous gliomas does not represent its effect on the intracerebral gliomas, we studied the latter. In these gliomas, 150 mg/kg/day of tetrandrine had therapeutic effects on the intracerebral gliomas but this antitumor effect was not as significant as those we observed on the subcutaneous gliomas. In addition, under the treatment of 150 mg/kg/day of tetrandrine, the rats with large intracerebral gliomas had shorter survival time than those with large ones, although statistically the difference of the animal survival rate between these 2 groups was not different. Intracerebral gliomas may require a higher dose of tetrandrine because treatment of these tumors requires entry of tetrandrine into the central nervous system, which may be restricted by the blood-brain barrier, the molecular size of tetrandrine, the lipid solubility of tetrandrine and other factors. In the present study, we did not measure the concentration of tetrandrine in the brain, intracerebral glioma or the cerebrospinal fluid.
Drug safety is important for using any kind of drugs in animals or humans. Treatment of 150 mg/kg/day tetrandrine for 4 weeks only mildly increased the serum ALT level in the rats, without causing significant change of the serum AST, BUN or creatinine. The histology of the livers and small intestines in the tetrandrine-treated rats showed no obvious damage. These data suggested such a dosage of tetrandrine caused mild liver toxicity, without significant damage to the kidney or small intestine. However, our treatment only lasted for 4 weeks, so we cannot make any general comments about drug safety. Previous researchers have used similar tetrandrine dosages and similar or longer treatment durations.7, 30, 31 Rats treated with 150 mg/kg/wk tetrandrine for 9 months30 or 60 mg/kg tetrandrine on alternate days for 40 days also showed no evidence of toxicity.31 Mice treated with 150 mg/kg/day tetrandrine for 2 weeks also had no signs of toxicity.8 Thus, we consider that the dose of tetrandrine used in our study has low toxicity and can be used safely in animals. Clearly, additional preclinical studies and clinical trials are necessary to assess the safety of tetrandrine.
The mechanisms of the antitumor activity of tetrandrine are not yet fully understood. Induction of apoptosis appears to be 1 of the mechanisms, because tetrandrine causes apoptosis of glioma cells and other types of cancer cells.3, 12–16, 33 However, the mechanisms of the tetrandrine-induced apoptosis are also unclear. In recent years, tetrandrine has been found to inhibit angiogenesis.24 Tetrandrine inhibits choroidal angiogenesis and air-pouch granuloma angiogenesis in diabetic rodents and fetal bovine serum-, IL-1α-, VEGF- or platelet-derived growth factor (PDGF)-BB-stimulated tube formation in vascular endothelial cells.24 There are no previous reports on the effect of tetrandrine on angiogenesis in malignant tumors. Angiogenesis can promote tumor growth and increase the number of channels for tumor cell metastases. The number of vessels in cancers correlates with the prognosis of cancer patients.32, 37 Similarly, angiogenesis plays a significant role in the growth of gliomas and is very prominent in malignant gliomas.38 The number of vessels in gliomas correlates with the degree of malignancy, and angiogenesis is 1 of the pathological characteristics of glioblastoma multiforme.38–40 In our study, tetrandrine reduced the number of CD31 positively-stained cells in gliomas. CD31, also called platelet endothelial cell adhesion moldecule-1 (PECAM-1), is an adhesion molecule that is involved in angiogenesis and is abundantly expressed on endothelium (concentrated in intercellular junction) and platelets.41 CD31 is involved in tube formation and inhibition of PECAM-1 suppresses tumor angiogenesis.42, 43 Thus, tetrandrine-induced reduction of CD31 expression in gliomas indicates that this drug may have antiangiogenesis effects in gliomas.
Various cytokines and growth factors stimulate angiogenesis, and the expression of these cytokines and growth factors correlates with the onset of pathological angiogenesis.44 These angiogenic factors are related to vascular cell proliferation, invasion of the vascular cells and differentiation of neovasculature.44 These angiogenic factors act on specific receptors, induce proliferation of endothelial cells, stimulate endothelial cells to produce proteolytic enzymes that destroy the matrix and cause endothelial cell migration and invasion of adjacent tissues.45–47 Among the numerous angiogenic factors, VEGF is well known as an important factor that is essential for tumorigenesis of several types of human cancers.48–50 In gliomas, angiogenesis is related to the amount of VEGF that is secreted.38, 51 In our study, we found that tetrandrine suppresses VEGF expression in glioma cells and induces cytotoxic effects on of the HUVECs in a concentration- and time-dependent manner. Further, the in vivo matrigel plug assay revealed tetrandrine could suppress the bFGF-induced angiogenesis. We also demonstrated that tetrandrine suppresses angiogenesis in gliomas in vivo, as shown by the decreased MVD in gliomas treated with high dose tetrandrine (150 mg/kg/day), relative to the MVDs of untreated gliomas and of gliomas treated with low dose tetrandrine (50 mg/kg/day). Thus tetrandrine seems to inhibit glioma-induced angiogenesis, and this may contribute to its antitumor effects on gliomas.
In summary, our study demonstrates that tetrandrine has concentration- and time-dependent cytotoxicity to gliomas and induces apoptosis of glioma cells. In addition, tetrandrine exerts an antitumor effect on subcutaneous and intracerebral gliomas, as determined by measurements of tumor growth rate and animal survival time. The mechanisms of these antitumor effects might be related (at least partly) to the inhibition of angiogenesis in gliomas. This is the first report that documents the effects of tetrandrine on the growth and angiogenesis of gliomas. However, clinical studies are needed before making any recommendation about the use of tetrandrine in the treatment of gliomas.