J Neurooncol DOI 10.1007/s11060-014-1477-3
LABORATORY INVESTIGATION
Synergy of enediyne antibiotic lidamycin and temozolomide in suppressing glioma growth with potentiated apoptosis induction Xing-Qi Li • Zhi-Gang Ouyang • Sheng-Hua Zhang Hong Liu • Yue Shang • Yi Li • Yong-Su Zhen
•
Received: 20 July 2013 / Accepted: 30 April 2014 Ó Springer Science+Business Media New York 2014
Abstract The present work evaluated the synergistic efficacy of an enediyne antibiotic lidamycin (LDM) plus temozolomide (TMZ) against glioma in vitro and in vivo. LDM plus TMZ inhibited the proliferations of rat glioma C6 cells and human glioma U87 cells more efficiently than the single usage of LDM or TMZ. In addition, LDM also potentiated the apoptosis inductions by TMZ in rat C6 cells and human U87 cells. Meanwhile, the results of TdT-mediated dUTP Nick End Labeling assay for subcutaneous U87 tumor sections indicated an enhanced apoptosis induction in vivo by LDM plus TMZ, which confirmed the high potency of the combination for glioma therapy. As determined by Western blot, apoptosis signal pathways in C6 cells and U87 cells were markedly affected by the synergistic alteration of P53, bax, procaspase 3, and bcd-2 expression. In both subcutaneous U87 xenograft and C6 intracerebral orthotopic implant model, TMZ-induced glioma growth suppression was dramatically potentiated by LDM. As shown, the combination therapy efficiently reduced the tumor volumes and tumor weights of the human Xing-Qi Li and Zhi-Gang Ouyang have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11060-014-1477-3) contains supplementary material, which is available to authorized users. X.-Q. Li College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China X.-Q. Li Z.-G. Ouyang S.-H. Zhang H. Liu Y. Shang Y. Li Y.-S. Zhen (&) Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China e-mail:
[email protected]
glioma U87 xenograft. Kaplan–Meier assay revealed that LDM plus TMZ dramatically prolonged the life span of C6 intracerebral tumor-bearing rats with decreased tumor size. This study indicates that the combination of LDM with TMZ might be a promising strategy for glioma therapy. Keywords Lidamycin Temozolomide Synergism Glioma suppression Apoptosis induction
Introduction Glioblastoma is the most common primary brain cancer. The malignant glioma often lead to all kinds of handicaps of neuro-cognitive function, which heavily damaged life quality and eventually threaten the life safety of glioma patients [1, 2]. Although many advances have been achieved in cancer therapy, the prognosis of patients with nervous system tumors remains poor, and the median survivals of malignant glioma patients are usually 12–15 months [3, 4]. Moreover, the location and the heterogeneous biological complex conferred brain tumor dramatic chemotherapyresistance and radiotherapy-resistance [4, 5]. As known, temozolomide (TMZ) is an efficient drug for malignant glioma treatment with limited adverse effects. TMZ can significant increase the median survival, and it has evolved into the first-line therapies of glioma [6, 7]. However, the therapeutic effects of TMZ are far less ideal. The intrinsic and acquired resistances of glioma cells weakened and even exterminated the benefits of TMZ to glioma therapies [8, 9]. To improve the efficacy of glioma therapies, combinational administrations of TMZ with conventional chemotherapeutics, molecular targeted drugs or radiotherapy have been introduced to experimental glioma therapies and clinic strategies [10–12].
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The enediyne antibiotics are a family of some natural products with highly potent antitumor activity. The unique molecular structure of enediyne antibiotics attracted much attention to explore their intricate activities. The diyne functional groups of the enediyne antibiotics produced more potent antitumor activities relative to the widely used chemotherapy drugs [13]. Lidamycin (LDM, also named C-1027), a member of the enediyne antitumor antibiotic family, is produced by a streptomyces strain isolated in China [14]. LDM showed extremely potent cytotoxicity and tumor growth inhibitory effect in mice [15–17]. The tumor cell DNA damage potential induced by enediyne LDM resides in its unique abilities to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. LDM binding to DNA minor groove can abstract hydrogen atoms from the deoxyribose of the backbone by the highly reactive benzenoid diradical species of LDM chromophore, which eventually results in DSBs [18–21]. With the unique and complex mechanism of action, LDM can induce cell apoptosis, abnormal chromosome, dysfunctional telomere and cell cycle arrests in various cancer cells [15, 22, 23]. Based on the preclinical studies of pharmacodynamics, pharmacokinetics and toxicology, LDM has entered the clinical trial in China [24]. Since TMZ has limited efficacy as single agent, there have been many investigations on its combination with additional agents to improve the therapeutic efficacy. However, little attention has ever been paid to the interaction of TMZ with LDM or other enediyne agents. The previously encouraging data from TMZ combination therapies motivated us to question: what responses and accompanied molecular mechanisms would happen to LDM plus TMZ on glioblastoma cell in vitro and tumor growth in vivo? The present work investigated the synergistic efficacy of LDM plus TMZ on glioma growth delay and apoptosis induction in vitro and in vivo. MTT assay, Annexin V-FITC/ PI staining and TdT-mediated dUTP Nick End Labeling (TUNEL) assay were performed to investigate the effects of LDM plus TMZ on glioma cell proliferation and apoptosis induction. Meanwhile, we also examined the synergistic antiglioma effects of LDM plus TMZ with U87 glioma xenograft model and rat C6 glioma orthotopic implantation model. This report on the combinational effects of LDM and TMZ reveals a promising strategy for glioma treatment.
Materials and methods Chemicals, cell lines and animals Lidamycin was kindly provided by Prof. Jin Lianfang of Chinese Academy of Medical Science, China. TMZ,
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis MO, USA). TMZ was dissolved in DMSO as a stock solution, and the stock solution was diluted with medium before experiment. The final DMSO concentration did not exceed 0.1 %, and in the following experiments, 0.1 % DMSO was added as control treatment. The rat glioma C6 cells and human glioma U87 cells were obtained from the Cell Center, Peking Union Medical College, China; The specific-pathogen-free (SPF) male athymic mice (18–22 g) and Sprague–Dawley (SD) rats (180–220 g) purchased from Vitalriver (Beijing, China) were housed under pathogen-free conditions. All animal studies were performed according to the protocol approved by the Animal Care and Use Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College. Cell proliferation assay Based on the differential chemo-sensitivity of C6 cells and U87 cells to LDM or TMZ (data not shown and not published), different concentrations of LDM and TMZ were used in the present and the following experiments. The rat glioma C6 cells (1 9 103 cells/well), and human glioma U87 cells (1.5 9 103 cells/well) were respectively plated in 96-well plates. After incubation for 24 h, the cells were treated with appropriate concentrations of LDM (10-10– 10-14 M) and TMZ (60, 90, 120, and 150 lM) or the mixture of LDM and TMZ for further 72 h incubation. A standard MTT assay was performed. The culture medium was removed and 20 ll MTT (5 mg/ml) were added. Then, the formazan was dissolved with DMSO and measured at 570 nm using Multiskan MK3 microplate reader (Thermo Labsystem, USA). Above experiments were carried out in triplicate. Annexin V-FITC/PI double staining assay Cell apoptosis was determined with commercial Annexin V-FITC/PI kit as manufacture’s instruction. In brief, the cells were seeded into 6-wells plates for 24 h, then the cells were grouped and treated with the following agents alone or in combination: Control group, 0.1 %DMSO; TMZ group, 300 lM for C6 cells or 210 lM for U87 cells; LDM group, 0.1 nM for C6 cells or 0.05 nM for U87 cells; Combination group, TMZ ? LDM. After 72 h incubation, all the cells were harvested. The samples were double stained with fluorescein isothiocyanate (FITC)–conjugated Annexin V and propidium iodide (PI) according to the manufacturer’s instruction. The stained cells were analyzed by flow cytometry. The resulting apoptosis was presented as rectangular quadrant, in which the total apoptosis
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Fig. 1 MTT assay of the C6 cells and U87 cells treated with various dosing regimens for 72 h. LDM and TMZ showed potently inhibitory effects on the proliferation of C6 cells and U87 cells (a, b). When treated with combinations of LDM (10-10–10-14 M) with TMZ (60, 90, 120, 150 lM), the survival ratios of C6 cells and U87 cells were
determined (c, d). The CIs of LDM with TMZ were calculated as ‘materials and methods’. The CIs plot showed synergistic effects on the cell proliferations (e, f). CI [ 1, =1 and \1 indicate antagonistic, additive and significant synergistic effect, respectively. The dashed line means CI = 1
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glioma cells in vitro and in vivo. a the representative images of the apoptosis assays with Annexin V-FITC/PI of C6 cells and U87 cells were shown. b the apoptosis ratio was calculated and plotted, and the graph displayed that the combinations of the two drugs enhanced apoptosis ratios of the indicated cells. c the TUNEL assays for subcutaneous U87 tumor section were performed, and the representative images were shown. The apoptosis indexes of three independent sections were calculated and plotted (d). For all the graphs, **p \ 0.01, and ***p \ 0.001 between the indicated groups
ratio = the ratio of the right upper ? the ratio of the right lower. Above experiments were carried out in triplicate. Western blot analysis The C6 cells and U87 cells were plated in flasks and treated with different dose regimens for 72 h, then homogenized with RIPA lysis buffer [25 mM Tris (pH 7.8), 2 mM EDTA, 20 % glycerol, 0.1 % Nonidet P-40 (NP-40), 1 mM dithiothreitol] and protease inhibitors. After the cell lysates were centrifuged, the protein concentrations of the supernatants were determined with the BCA protein assay. The proteins (30 lg/lane) were electrophoresed by SDS-PAGE gel and transferred to PVDF membrane. Then the membranes were probed with primary antibodies, including anti-p53, anti-bax, anti-procaspase 3, anti-bcl-2 and antiactin (1:1,000, Cell Signaling Technology, Danvers MA, USA). The membranes were developed with horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000) and enhanced chemiluminescence blot detection system as the manufacturer’s instructions; in addition, the densitometry of protein expression from three independent experiments were performed with Gel pro Analyzer 4.0 (Media cybernetics, Inc., MD, USA). Animal model procedure and drugs administration The U87 glioma cells were inoculated subcutaneously (s.c.) into the flank of the Balb/c (nu/nu) athymic mice (5 9 107 cells/200 ll PBS/mouse). After the tumor reached a size of approximately 2,000 mm3, the animals were sacrificed, and the tumor was cut into 1–2 mm3 pieces, then the tumor pieces were implanted s.c. to the right flank of athymic mice [25]. When the tumors reached 100–150 mm3, tumor-bearing animals were randomly divided into 4 groups (n = 6 per group). Group 1: Control group; group 2: TMZ group, 0.5 mg/kg TMZ (i.p), 6 times per week for 2 weeks; group 3: LDM group, 25 lg/kg (tail vein, i,v.), once a week for 2 weeks; group 4: TMZ 0.5 mg/ kg ? LDM 25 lg/kg. During the experiment, the tumor volumes and body weights were recorded at the indicated time. Tumor volume was calculated using the following formula: V = a 9 b2/2, where a and b are the maximal
length and width of the tumor, respectively. After 30 days, the tumors were harvested, weighed and examined. Tumor specimens were fixed in liquid nitrogen, embedded in optimal cutting medium, and frozen for cryosection. Serial sections (6 lm) were cut on a cryotome and stored at 80 °C until stained. To establish C6 brain orthotopic model, the rats were anesthetized and placed in a stereotactic apparatus; then a small drill hole was made at the site of 3 mm left lateral and 1 mm anterior to the bregma. For Kaplan–Meier assay, C6 cells (2 9 105 cells/10 ll) were orthotopically implanted into the white matter of the brain at a depth of 4 mm through the hole [26]. The tumor-bearing rats were randomly assigned into 4 groups (n = 10 per group), Control group, TMZ group (5 mg/kg, 6 times per week for 2 weeks), LDM group (25 lg/kg, once a week for 2 weeks), and combination group (TMZ 5 mg/kg ? LDM 25 lg/kg with drugs administrated as above, respectively). During the experiments, the survival times of every rat were recorded for Kaplan–Meier assay until all rats died. For tumor size assay, C6 cells (1 9 105 cells/10 ll) were implanted into the white matter of the brain in another 40 rats. After grouped and treated as the above-mentioned procedure, the rats were sacrificed at the 30th day and the brains were fixed with 10 % formalin. Then the fixed brain specimens were embedded in paraffin and sectioned with microtome. The brain slices were subjected to routine hematoxylin and eosin (H&E) staining and photographed at 209 for histological examination. The maximal length (a) and width (b) of the tumor slices were recorded with computer-assistant program, and then the tumor sizes (V) were calculated with the above-mentioned formula. In situ TUNEL assay of tissue section The procedure was performed as the manufacture’s instruction. Frozen tissue sections (6 lm) were fixed in ice-cold acetone and permeabilized in Protein K solution (20 lg/ml), then rinsed twice with 0.1 M PBS (pH = 7.4). After incubation with 50 ll TUNEL reaction mixture in a humidified atmosphere for 60 min at 37 °C in the dark, the FITC-labeled slides were washed, dried, and counterstained with PI (1 lg/ ml). Images were captured by fluorescence microscopy at 4009 magnification and analyzed with Leica Qwin Pro 3.0 for counting apoptotic cells. The apoptotic index (AI %) was expressed as follows [27, 28]: AI % = A 9 100/(A ? C), where A = the number of TUNEL-positive cells, and C = the number of counterstained unlabeled cells. Evaluation of the drug combination The combination index (CI) was estimated based on the previous description [29, 30]. For in vitro assay, the CI of
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LDM with TMZ is calculated as CI = AB/A 9 B, where A and B are the survival ratio of respective single agent, AB is the survival ratio of the combination group. For in vivo experiment, the combination evaluation is performed with fractional tumor volume (FTV). The FTV is calculated as FTV = (the mean value of experiment group)/(the mean value of control). The in vivo CI is calculated as the formula: CI = observed FTV of the combination/expected FTV of the combination, where expected FTV = (FTV of LDM) 9 (FTV of TMZ). CI \ 1 indicates a synergism, CI = 1 indicates an additive effect and CI [ 1 indicates an antagonistic effect. Statistical analysis Descriptive statistics were generated for all quantitative data with Mean ± S.D. The levels of significance were tested by one-way ANOVA using the SPSS software for comparison of multiple groups. For survival analysis, Kaplan–Meier assay with log-rank test was used. The significance was accepted at p \ 0.05.
Results Synergy of LDM and TMZ in cytotoxicity to glioma cells MTT assay showed that the IC50 values of LDM to glioma C6 cells and U87 cells were 5.86 9 10-11 and 2.05 9 10-11 M (Fig. 1a), respectively. The data indicated that LDM displayed highly potent anti-proliferation effects on glioma cells. The IC50 values of TMZ to glioma C6 cells and U87 cells were 214.24 and 147.36 lM (Fig. 1b). In addition, the C6 cells and U87 cells were exposed to various concentrations of LDM (10-10, 10-11, 10-12, 10-13, 10-14M) and TMZ (60, 90, 120, 150 lM), and their combination for 72 h; After the cell survivals were determined (Fig. 1c, d), the CI values of the two drugs at different combination regimens were calculated and plotted in Fig. 1e, f. The CI values plot suggested synergistic effects of LDM and TMZ on inhibiting C6 and U87 cell proliferation. Synergy of LDM and TMZ in inducing apoptosis of glioma cells in vitro and in vivo For in vitro apoptosis assay, the treated cells were analyzed by Annexin V-FITC/PI double-staining method. As shown, enhanced inductions of apoptosis by drug combination were found both in C6 cells and in U87 cells (Fig. 2a). Evidently, the combination of LDM with TMZ resulted in
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Fig. 3 Western blot analysis of apoptosis-related proteins in glioma cells. The apoptosis signal pathway was regulated by LDM plus TMZ in C6 cells (a) and U87 cells (b). As shown, p53 and bax were upregulated, while Bcl-2 and procaspase 3 were sharply down-regulated
a higher apoptotic percentage than either single agent, with significant differences (Fig. 2b). For in vivo apoptosis assay, the results of TUNEL assay showed that LDM, TMZ or combination of the two drugs induced the cell apoptosis efficiently. In particular, the combination of LDM with TMZ obtained greater apoptotic index than any single agent groups (Fig. 2c, d, p \ 0.001, vs LDM and TMZ, respectively), indicating a synergistic effect of LDM and TMZ on cell apoptosis in vivo. Synergistic action on the regulations of apoptotic signal pathways by LDM plus TMZ To confirm the pro-apoptosis potentials and gain further insight into the effects of combination of LDM with TMZ, the regulation of apoptotic signaling pathway was analyzed by Western blot. As shown, the expressions of pro-apoptotic
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Synergy of LDM and TMZ in the growth suppression of U87 xenograft and C6 orthotopic implants
Fig. 4 The synergism of LDM with TMZ on tumor growth inhibition of U87 xenograft. Combination therapy of LDM and TMZ was more effective than any drug alone against tumor growth in a subcutaneous U87 xenograft model (n = 6 per group). a When reached 100–150 mm3, the tumor volumes were measured at the indicated times. Tumor growth curves were plotted. b Body weights of mice were plotted. c The mice were sacrificed at 30 days after treatments, then the U87 xenograft tumors were excised and weighted, the mean values of every group were calculated and plotted. *p \ 0.05, and ***p \ 0.001 between indicated groups
and anti-apoptotic proteins were regulated significantly by the combination (Fig. 3, Supplemental Fig. 1). For C6 cells, the expression levels of p53 and bax were significantly upregulated, whereas the expressions of procaspase 3 and bcl-2 were dramatically down-regulated by the combination, compared with that by the single drug (Fig. 3a, Supplemental Fig. 1). For U87 cells, similar effects of the combination on the expressions of apoptosis-related proteins were found (Fig. 3b, Supplemental Fig. 1).
The human glioma U87 xenograft was used to investigate the inhibitory effects of LDM (25 lg/kg), TMZ (0.5 mg/ kg), and the combination on glioma growth. As indicated by the tumor volume curve, the combination of LDM with TMZ exerted more significant anti-tumor effects than the respective agent given alone (Fig. 4a). There was no statistically significant difference in body weight of animals between treated animals and the controls, indicating that the dosing regimens were well tolerated (Fig. 4b). Similar results were showed by tumor weights histogram. The single treatment with LDM induced tumor weight decrease (Fig. 4c, p \ 0.05, vs control), and the combination therapy of LDM and TMZ exerted much more significant inhibition than either single drug treatment (Fig. 4c, p \ 0.001, vs LDM and TMZ, respectively). The CI value is 0.83 (Supplement Table 1), indicating a synergism of the two drugs on U87 glioma growth inhibition. In addition, the effects of LDM, TMZ and their combination on C6 brain orthotopic implants were also investigated. As the survival days of individual rat concerned with Kaplan–Meier analysis, the overall survival days of each group were determined. The overall survivals treated with LDM (25 lg/kg) and TMZ (5 mg/kg) were significantly different to control group, and the two significance values are p \ 0.001 (Fig. 5a). The median survival time of mice treated with LDM (25 lg/kg) and TMZ (5 mg/kg) were 20 and 18.5 days, respectively, significantly longer than that 14 days of control; Moreover, the combination of two drugs significantly prolonged the survival time of rat at 26 days with the differences presented as p \ 0.001 vs LDM and p \ 0.001 vs TMZ (Fig. 5a). As depicted by H & E staining of brain tumor section, the combination of LDM with TMZ significantly improved the anti-tumor effects (Fig. 5b). The statistical results of estimated tumor volumes indicated that LDM given alone inhibited the tumor growth with p \ 0.001 vs control (Fig. 5c). TMZ treatment also exhibited significant growth inhibition (Fig. 5c, p \ 0.001, vs control). The combinations of LDM with TMZ displayed more significant anti-tumor effects than either agent given alone (Fig. 5c, p \ 0.001 vs LDM and p \ 0.001 vs TMZ), the synergistic effect on C6 tumor growth inhibition was showed with CI value of 0.41 (Supplement Table 1), which further proved the synergism on U87 xenograft growth inhibition.
Discussion Despite recent advances in the glioblastoma therapies, the overall survival of glioblastoma patients remains poor.
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Fig. 5 Synergistic inhibition by LDM plus TMZ on C6 brain orthotopic implant. Combined therapy with LDM and TMZ was more effective than any drugs alone against tumor growth in a C6 glioma brain orthotopic model (n = 10 per group). a Kaplan–Meier method was used for survival study. According to the survival days of glioma-bearing rats, the results showed significant differences in survival days between the indicated groups (p \ 0.001, by Log-rank test). b After the indicated treatments, the rats were decapitated at 30 days, and then the brain were fixed and sectioned. The slices were photographed with a microscope. The representative coronal sections are shown. c Tumors volumes were measured with computer-assistant method. The mean values of every group were calculated and plotted. ***p \ 0.001 between indicated groups. Each Column represents the mean ± S.D. The scale bar = 2 mm
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Meanwhile, the standard clinic cares of glioma patients, including maximal resection, chemotherapies and radiotherapy, have resulted in many disadvantages [2, 4, 5]. As reported, the clinic applications of TMZ have obtained encouraging anti-glioma effects [31, 32], and the experimental studies and clinic practices also confirmed the potential role of TMZ combined with other agents in glioma growth delay [33–35]. Nevertheless, TMZ applications still have some limitations for glioma therapy [8]. To circumvent the limitations and improve the effectiveness of present glioma treatments, it is of importance to explore novel therapeutic strategies, including new drugs and drug combinations; in particular, the TMZ-based combinations. The members of the enediyne antibiotic family share similarly unique molecular architecture and intricate mechanisms of actions with much more potent cytotoxicity against cancer cells than widely used chemotherapeutic drugs [13]. Little is known about the anti-glioma effects of enediyne antibiotics. Our previously work has depicted the anti-glioma effects of LDM as single agent [17], in which only the inhibitory effects on U87 cells in culture and the subcutaneous xenograft were observed. In the present study, a profound investigation of LDM on cell growth inhibition and apoptosis induction in both rat glioma C6 cells and human glioma U87 cells were performed in vitro and in vivo, and the in vivo growth suppression were also determined with U87 xenograft and C6 orthotopic implantation. In particular, the widely reports of TMZ combination therapies motivate us to investigate whether or not the combination of LDM with TMZ can lead to synergistic effects on glioma cell growth delay and apoptosis induction. As shown, LDM displayed extremely potent efficacy on cell proliferation inhibition. When concurrently administrated with TMZ, the combination exerted synergistic inhibition of proliferations in both C6 and U87 cells. In addition, previous reports showed that LDM could induce apoptosis in human multiple myeloma cells and human colorectal cancer cells via mitochondrial pathway, which could be reversed by anti-apoptotic agents [36, 37]. Meanwhile, the anti-apoptotic pretreatments could block TMZ-induced apoptosis in glioma cells [38]. The present study demonstrated that the combination of LDM with TMZ could enhance apoptosis induction in C6 and U87 cells in vitro and in vivo; furthermore, Western blot analysis revealed that the synergistic regulations of P53, bax, caspase 3 and bcl-2 may involve in the potentiated apoptosis induction. Subcutaneous human glioma xenograft and intracerebral orthotopic implants are recognized as reasonable models for evaluating the antitumor effects of a specific drug or
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drug combination [39–41]. The concurrently performance of human glioma U87 subcutaneous xenograft and rat glioma C6 intracerebral orthotopic implant in the present research may be sufficient for interpreting the anti-glioma effects of the LDM and TMZ combination. As shown, in U87 glioma xenograft and C6 orthotopic models, LDM and TMZ significantly inhibited the tumor growth when administrated separately; notably, the combination of the two drugs exerted much higher efficacy in suppressing tumor growth and prolonging survival time. Therefore, the in vivo experiments confirmed and highlighted the synergistic anti-glioma efficacy of LDM plus TMZ described in our in vitro results. The major finding of the present study is that the combination of LDM and TMZ shows synergistic efficacy in vitro and in vivo, in which the synergistic enhancements of apoptosis may involved. To our knowledge, synergy of enediyne agents combined with TMZ has not been reported previously. Hence, our data strongly suggest that LDM plus TMZ has the potential to be a new therapeutic strategy for the treatment of glioma. Acknowledgments The work was supported by ‘Significant new drug development’ Science and Technology Major Projects of China (No. 2012ZX09301002), National Natural Science Foundation of China (No. 81202559) and Doctor Initial Foundation of Heilongjiang Bayi Agricultural University (No. B 2010-6). Conflict of interest of interest.
The authors declare that they have no conflict
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