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In vitro and in vivo Study on Glioma Treatment Enhancement by Combining Temozolomide with Calycosin and Formononetin
Journal of Ethnopharmacology 242 (2019) 111699
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In vitro and in vivo Study on Glioma Treatment Enhancement by Combining Temozolomide with Calycosin and Formononetin Qi Nia, Yani Fana, Xiong Zhanga, Hongwei Fana, , Yingbin Lib, ⁎
a b
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Department of Clinical Pharmacology Lab, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu 210006, China Department of Neurosurgery, the Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210011, China
ARTICLE INFO
ABSTRACT
Keywords: Glioma Temozolomide Calycosin Formononetin Combination treatment
Ethnopharmacological relevance: Astragalina alpestris is a traditional Chinese herbal medicine with anti-inflammatory, anti-immune, anti-tumor and other pharmacological effects. Calycosin and formononetin (FMN) are two natural compounds isolated from astragalus. It has been shown that calycosin and FMN are active anti-tumor ingredient. Aim of the study: The aim of this current work was to study the therapeutic enhancement of temozolomide (TMZ) on gliomavia combining with calycosin and FMN. Materials and methods: The effect of co-administration via hematoxylin-eosin staining (HE staining) was determined by measuring cell proliferation toxicity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide (MTT) assay and sequentially observing the cell morphology. To synchronously explore the effect of migration on C6, transwell assay and wound healing assay were performed. Apoptosis was measured by Annexin V/propidium iodide (PI) staining. Meanwhile, western blot was used to investigate proteins involved in the mechanisms for migration and apoptosis. Furthermore, HE staining and immunohistochemistry were also analyzed for curative effect in vivo. Results: The efficacy of TMZ on glioma could be enhanced by combining with calycosin and FMN through inhibiting the proliferation and migration of glioma cells and promoting their apoptosis. Western blot assays indicated that expression of apoptotic proteins (Bcl-2 Associated XProtein (Bax), Cleaved Caspase-3, Cleaved Caspase-9) were up-regulated. Anti-apoptotic protein (B-cell lymphoma-2,Bcl-2) was down-regulated. The migratory proteins (Matrix metallopeptidase 2, 9, MMP-2, MMP-9) was downregulated. In vivo study, this kind of co-administration (calycosin, FMN, and TMZ) exhibited the marked therapeutic effect on glioma. Conclusions: This study has identified that calycosin and FMN can increase the treatment effect of TMZ in vitro and in vivo. These attractive features substantially broadened the application range of TMZ as a glioma treatment medicine.
1. Introduction In central nervous system, brain glioma is the most aggressive malignancies with a high prevalence, high recurrence rate and low survival rate (Safdie et al., 2012). Traditionally, glioma treatment has mainly based on surgery, followed by radiotherapy or chemotherapy (Song et al., 2014). However, its failure often occurred and the treatment effect could bring the patients with even worse prognosis. This could be attributed to the continuous proliferation of glioma residual after the treatment, eventually resulting in glioma recurrence. Overall, the short-term survival was low and median that underwent glioma diagnosis was 1–2 years (Li et al., 2013; Shi et al., 2015; Yu et al., 2015). Therefore, development of novel effective drugs is urgent for ⁎
glioma patients. Temozolomide (TMZ) (Fig. 1), one of the most effective drugs for clinical treatment of brain tumors, is an alkylating agent that contains an imidazole tetrazine ring. TMZ was recognized as significant curative effects on melanoma, lymphoma, leukemia and solid tumors (Kumar et al., 2013). TMZ could easily penetrate into the blood-brain barrier further delaying the recurrence of glioma. The alkylation of DNA exerts a cytotoxic effect, giving rise to single or double-stranded DNA, breaks to prevent DNA replication, and then produces an anti-tumor effect (Li et al., 2017). In addition, TMZ mediates apoptosis by inducing stress responses that are not dependent on DNA damage (Zhang et al., 2014; Ma et al., 2015; Zou et al., 2014). However, clinical data demonstrated that patients required a high dose of TMZ, which in turn presented
Corresponding authors. E-mail addresses: [email protected] (H. Fan), [email protected] (Y. Li).
https://doi.org/10.1016/j.jep.2019.01.023 Received 12 November 2018; Received in revised form 17 January 2019; Accepted 20 January 2019 Available online 18 April 2019 0378-8741/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Journal of Ethnopharmacology 242 (2019) 111699
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Fig. 1. (A) Chemical structure of TMZ. (B) Chemical structure of calycosin. (C) Chemical structure of FMN.
obvious toxic side effects. Therefore, major concern of reducing toxicity, elevating the sensitivity and improving the therapeutic effect of TMZ still remained. Combination therapy has displayed important clinical significance for improving the lethal effect of tumor cells. Comparing with conventional chemotherapy drugs, Traditional Chinese Medicines was more involved in pleiotropic, multi-target, multi-channel, and wide-ranging characteristics. Astragalina australis, a Traditional Chinese Medicine, contains several types of bioactive compounds, such as, various flavonoids, glycosides, and other substances, benefiting vital energy, consolidating superficies, and inducing diuresis to alleviate edema (Avunduk et al., 2008). In practice, Astragalina australis is often used in combination with other herbs, such as, poria, angelica, and ginseng in various complex prescription formulas. In recent years, a large number of studies have proved the reliable role of Astragalina australis in anti-tumor. Although Astragalina australis is usually combined with other herbs, it can be taken separately by itself (Zhou et al., 2018). In various pharmacological studies, both crude extracts and monomeric components of Astragalina australis showed activities in anti-tumor, anti-oxidation, anti-infection, cardioprotection and antivirus (Na et al., 2009., Huang et al., 2016). Calycosin (Fig. 1) and formononetin FMN (Fig. 1) are components existing in Astragalina australis as proved by High Performance Liquid Chromatography (HPLC) coupled with photodiode-array detection (DAD) and electrospray ionization (ESI-MS) Detection (Lv et al., 2011). Both calycosin and FMN possessed of anti-inflammatory, anti-immune, antioxidative, anti-viral, anti-tumor and other pharmacological effects (Zhou et al., 2017; Zhao et al., 2016; Lo and Wang, 2013). They could be used as potential drugs for clinical neuroprotection via inhibiting the proliferation and migration of cells, and promoting apoptosis (Nie et al., 2016). Some studies displayed that calycosin and FMN played key roles in the treatment of breast cancer, melanoma, and cervical cancer (Fu et al., 2017; Chen et al., 2013). The results indicated that calycosin and FMN could inhibit the growth of glioma cells and promote their apoptosis. Moreover, our previous work also suggested that combination of FMN with TMZ was attributed to synergistic therapy effect for glioma (Zhang et al., 2018b). Inspiring from the conclusion, co-treatment with more flavonoids agent may further benefit to the sensitivity of TMZ. In this work, we investigated the effect of co-treatment of the three drugs (calycosin, FMN, and TMZ) on the proliferation, apoptosis, and migration in vitro by using C6 cells. Furthermore, mouse ectopic tumor models were also established to test the in vivo effect of the combination of the two Chinese herbs and TMZ on the treatment of glioma. The experimental study here revealed a critical role of TMZ in the treatment of glioma, which may provide a new avenue for glioma research.
2. Materials and methods 2.1. Cells and reagents The C6 cell line was obtained from Dr. Zhang Xin's laboratory (China Pharmaceutical University). C6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (CAS No: KGM12800500, KeyGEN, Nanjing, China) at 37 °C supplemented with 10% fetal bovine serum (FBS, KeyGEN) and 5% CO2. Calycosin (purity ≥ 99%, CAS No: 20575-57-9) and FMN (purity ≥ 99%, CAS No: 485–72-3) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd. (Taizhou, Jiangsu, China). TMZ (CAS No: 16083115) was obtained from Jiangsu Hengrui Group Pharmaceutical Co., Ltd (Lianyungang, Jiangsu, China). TMZ was dissolved into Sterilized water (CAS No: 2016091805, Qidu, Shandong, China) for injection. Stock solution of Calycosin and FMN were made by dissolving in dimethyl sulfoxide (DMSO; CAS No: KGT5131; KeyGEN, Nanjing, China). In all the drug dissolving solvents, DMSO concentration wase limited to < 0.1% (v/v), and the vehicle controls were used at the same concentration as DMSO. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT) was purchased from KeyGEN BioTECH (CAS No: 20171019, Nanjing, Jiangsu, China). Annexin V-Alexa Fluor 647/propidium iodide (PI) (CAS No: FMSAV647-050) was purchased from Fcmacs Biotech Co., Ltd (Nanjing, Jiangsu, China). Antibodies specific for Bcl-2 (CAS No: ab182858), Bax (CAS No: ab32503), Caspase-3 (CAS No: ab32351), Caspase-9 (CAS No: ab202068), MMP-2 (CAS No: ab92536), MMP-9 (CAS No: ab38898), were purchased from purchased from Abcam corporation and used as the main antibodies in Western blot assay. Antibodies specific for GFAP (CAS No: ab7260), Bcl-2 (CAS No: MM0665-9S52), Bax (CAS No: ab32503), Caspase-3 (CAS No: EPR18297) and MMP-9 (CAS No: ab76003) were purchased from Abcam corporation. 2.2. Animals BALB/C mice were purchased from the Animal Experimental Center of Nanjing First Hospital and were acclimated for at least 1 week in the animal facility without specific pathogens prior to the experiment. They were placed in a temperature (24 ± 2 °C) and relative humidity (45%–60%) controlled chamber. The mice were similar in age and body weight in all experiments and were randomly assigned to the treatment groups. All animal experimental manipulations followed China legislation on the use and care of laboratory animals and were approved by Animal Experimental Ethics Committee of Nanjing First Hospital.
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Fig. 2. (A, B, C) The cytotoxicity of TMZ (0–2000 µM), calycosin (0–640 µM), and FMN (0–320 µM) on C6 cells with different concentrations for 48 h was assessed by MTT assay. Data represented as the mean ± SD (standard deviation) of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (D, E, F, G) Both calycosin and FMN enhanced cytotoxicity of TMZ on C6 cells. Combination of calycosin (160 µM) and TMZ (0–2000 µM) promotes apoptosis of C6 glioma cells, the IC50 of TMZ decreased. Combination of FMN (40 µM) and TMZ (0–2000 µM) promotes apoptosis of C6 glioma cells, the IC50 of TMZ decreased. The IC50 of TMZ is decreased to 125 µM significantly when causal calycosin and FMN are combined with TMZ, ***p < 0.001.
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Fig. 3. Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on morphology of C6 cells.
2.3. Cell viability assay
(160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). Then, the medium in each well of a 6-well plate was discarded, 4% paraformaldehyde was fixed for 10 min, and H &E staining was performed. The morphology of the cells was observed under an inverted microscope.
The viability of cells was determined by MTT assay. C6 cells were seeded with a density of 8 × 103 cells/well in 96-well plates and cultured at 37 °C, in DMEM with 10% FBS. C6 cells were treated separately with different concentrations of three drugs (calycosin, FMN, and TMZ). The lowest effective concentrations of calycosin and FMN were used to combine with TMZ. The treatment with TMZ was conducted at various doses levels (125, 250, 500, 1000, and 2000 μM) for 48 h. In the calycosin treatment groups, calycosin was added to culture medium at final concentrations of 20, 80, 160, 320, and 640 μM. In the FMN treatment groups, FMN was added to culture medium at the concentration range of 20, 40, 80, 160, and 320 μM. In two drug coupling groups, calycosin (160 μM) was added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM), FMN (40 μM) was added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM). In the group of TMZ combination with calycosin and FMN, calycosin (160 μM) and FMN (40 μM) were added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM). Following incubation at 37 °C for 48 h, C6 cells were exposed to MTT solution with final concentration of 50 μL/well. After incubating for 4 h, DMSO was used to dissolve crystals. Then, measuring the optical density (OD) values was performed in a microplate reader (TECAN, Switzerland) at 570 nm after 10 min. The proliferation rate that was specific for C6 cells was calculated by the formula: the OD values of treated groups/the OD values of control group× 100%.
2.5. Wound healing assay C6 cells were cultured in 6-well plates with a density of 4 × 105 cells/well. When cells grew to about 90% per well, 10 μL pipette tips was used to scratch the monolayer of cells. After washing cell debris with PBS, cells were cultured in 2% FBS medium in the absence or presence of the various concentrations in each group for 24 h: (1) control; (2) TMZ (125 μM); (3) TMZ (125 μM and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). Cell scratch spacing was valued via calculating cell mobility at 0 h and 24 h. 2.6. Transwell assay C6 Cells were seeded in triplicate on 6-well dishes. Upon reaching 80% confluence, they were exposed to vehicle or different administration for 48 h. Cells with a density of 2 × 106/ml were treated in different administration groups: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). The above cells suspension (100 μL) were seeded in the upper chamber of an 8.0-μm pore polycarbonate membrane insert (Corning Inc, USA), while adding 10% culture medium (500 μL) to the lower chamber. The non-migrated cells in the upper chamber were wiped off with a cotton swab, and the
2.4. HE staining C6 cells were cultured in 6-well plates with a density of 3 × 105 cells/well with sterile coverslips and treated with different drug groups for 48 h: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin
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Fig. 4. (A, B, C, D) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on migration of C6 cells. C6 cells were exposed to these drugs before being assayed for C6 cell migration by wound healing and transwell migrations, using Graphpad respectively to measured the wounding healing rate and the number of migration cells. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 5. (A, B) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on apoptosis of C6 cells. The detection of apoptosis rate was used by flow cytometry and analyzed by Graphpad. *p < 0.05, **p < 0.01, ***p < 0.001.
antibodies overnight at 4 °C. A GAPDH antibody served as control. The Bcl-2 (1:2000; Abcam), Bax (1:5000; Abcam), Cleaved Caspase-3 (1:1000; Abcam), Cleaved Caspase-9 (1:1000; Abcam), MMP-2 (1:5000; Abcam), MMP-9 (1:1000; Abcam) antibodies were used for each group. Then, the membranes were washed and incubated with specific secondary antibodies for 1 h. The two kinds of liquids, A and B, in the ECL chemiluminescence kit are mixed equally in volume, and were configured as a working fluid. Nitrocellulose membrane was exposed to working fluid. The western blot signal that represented protein level was qualified using Image J software.
migrated cells in the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet, Photographed under a microscope to observe cell migration. 2.7. Flow cytometric assay C6 cells were plated in 6-well plates and treated with different drug administration: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM), which were cultured at 37 °C for 48 h. The C6 cells were washed with cold phosphate-buffered saline (PBS) (KeyGEN, Nanjing, China) and resuspended in it. An aliquot of 100 μL of the cell suspension was placed in a flow tube, and 5 μL of Annexin V-Alexa Fluor 647/PI and 10 μL of a 20 μg/ml PI solution were added. The above solvent was mixed and protected from light at room temperature for 15 min. Annexin V FITC Apoptosis Detection Kit (FMSAV647-100, BD, USA) was used to detect cell apoptosis.
2.9. Tumor xenografts Glioma C6 cells (1.0 × 107cells per mouse) were subcutaneously injected into BALB/C mice (Animal Experimental Center of Nanjing First Hospital). When the tumor size reached about 50 mm3, mice were randomly divided into 6 groups (9 mice per group): (1) control; (2) TMZ (30 mg/kg); (3) TMZ (30 mg/kg) plus calycosin (40 mg/kg); (4) TMZ (30 mg/kg) plus FMN (18 mg/kg); 5) calycosin (40 mg/kg) plus FMN (18 mg/kg); and 6) TMZ (30 mg/kg) plus calycosin (40 mg/kg) and FMN (18 mg/kg). The treatment was performed every 2 days for 15 days. In the end of this experiment, mice were sacrificed by cervical dislocation. The organ tissue and tumor tissue were immersed in formalin. HE staining was used for detecting cell apoptosis. Cell morphology in tissues was observed under fluorescence microscope. Individual tumor samples were detected for expression levels of proteins by immunohistochemistry. All animal experimental manipulations were approved by the Ethics Committee for Animal Experiment of Nanjing Medical University.
2.8. Western blot assay C6 cells were exposed to different drug administration and were incubated at 37 °C for 48 h. The cells were digested with trypsin and washed twice in cold PBS. Then 200 μL of ice-cold lysis buffer was added to fully lyse the cells. The protein concentration was measured by using BCA Protein Quantitation Kit (KeyGEN, Nanjing, China). Cell lysates were added to SDS buffer for SDS-PAGE gel electrophoresis and then transferred to a Nitrocellulose membrane (KeyGEN, Nanjing, China). The membrane was washed 3 times with TBST and then blocked with 5% skim milk powder blocking solution for 90 min at room temperature. The main strips were probed with indicated primary
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Fig. 6. (A, B, C, D, E) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on major proteins of C6 cells. C6 cells were exposed to these drugs for 48 h before being analyzed for the expression of Bax, Bcl-2, Caspase-3, and Caspase-9 by Western blot. The signal representing proteins levels were qualified using Image J software. *p < 0.05, **p < 0.01, ***p < 0.001.
2.10. Statistical analysis
concentrations on C6 cells for 48 h was assessed by MTT assay. C6 cells were treated with increasing doses of TMZ for 48 h (Fig. 2A). The IC50 of TMZ was 1000 μM approximately. Treatment with 20–640 μM calycosin inhibited cells proliferation in a concentration-dependent manner in C6 cells (Fig. 2B). The minimum effective concentration of calycosin was 160 μM. Treatment with 20–320 μM FMN inhibited cells proliferation in a concentration-dependent manner in C6 cells (Fig. 2C). The minimum effective concentration of FMN was 40 μM.
All the experiments were reproduced in triplicate. Statistical analysis were performed by Student's t-test (SPSS, SPSS® Statistics V22.0, IBM, NY, USA). The results were shown as the mean ± standard deviation. P < 0.05 was recognized as a statistically significant difference. 3. Results
3.2. Effects of calycosin and FMN administered separately or in combination with TMZ on proliferation of C6 cells
3.1. Effects of calycosin, FMN and TMZ on proliferation of C6 cells
When cells were treated with the combination treatment of
The cytotoxicity of TMZ, calycosin and FMN at different
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Fig. 7. (A, B, C) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on major protein of C6 cells. C6 cells were exposed to Calycosin (160 µM) and FMN (40 µM) administered separately or in combination with TMZ (125 µM) for 48 h before being analyzed for the expression of MMP-2 and MMP-9 by Western blot. The signal representing proteins levels were qualified by Image J software. *p < 0.05, **p < 0.01, ***p < 0.001.
calycosin or FMN, which comprised a low effective concentration of calycosin (160 μM) and FMN (40 μM) and various concentrations of TMZ (125, 250, 500, 1000, and 2000 µM) for 48 h, the IC50 of cotreatment with TMZ was decreased (Fig. 2D, E). The IC50 of TMZ was decreased dramatically to 125 µM when causal calycosin and FMN were combined with TMZ (Fig. 2F, G), which indicated that calycosin and FMN could elevate sensitivity of TMZ.
calycosin and FMN. To test whether calycosin and FMN could increase the efficacy of TMZ in reducing glioma cell motility and migration, C6 cells were treated with calycosin (160 μM) and FMN (40 μM) separately or in combination with TMZ (125 μM) for 24 h, and subjected to wound healing and transwell migrations. Treated with TMZ, suppression of cell migration was weak (Fig. 4A, B, C, D). When the cells were exposed to TMZ that combined with calycosin (160 μM) or FMN (40 μM), the cell migration rate was obviously decreased. Furthermore, cells that were treated with TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM) were found to rarely migrated, compared with other groups.
3.3. Effects of calycosin and FMN administered separately or in combination with TMZ on morphology of C6 cells H&E staining assay was performed to detect the level of apoptosis and morphological changes in cells. Based on MTT, C6 cells were treated with calycosin (160 μM) and FMN (40 μM) administered separately or in combination with TMZ (125 μM) for 48 h. Treated with TMZ, apoptotic cells endowed less, slightly shrinking nuclei. When C6 cells were treated with TMZ, combined with calycosin or FMN, the number of viable cells was remarkably reduced. Larger nuclear shrinkage was observed in the sample of TMZ combined with calycosin or FMN. Furthermore, in the combination treatment of TMZ, calycosin, and FMN, C6 cells exhibited most nuclear apoptotic changes, which contained nuclear condensation and aggregation (Fig. 3).
3.5. Effects of calycosin and FMN administered separately or in combination with TMZ on apoptosis of C6 cells Due to induction of considerable apoptosis, we subsequently determined whether calycosin (160 μM) and FMN (40 μM) could improve the therapeutic effect of TMZ (125 μM) in promoting tumor apoptosis by Annexin V/PI staining with flow cytometry. The results suggested that TMZ combining with calycosin and FMN could elevated apoptotic rate well (Fig. 5). 3.6. Effects of calycosin and FMN administered separately or in combination with TMZ on major proteins of C6 cells
3.4. Effects of calycosin and FMN administered separately or in combination with TMZ on migration of C6 cells
To measure the effects of drug combination on cell proliferation and apoptosis, the current study also detected the expression level of
TMZ exerted inhibitory effects on C6 cells when combined with
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apoptosis-associated proteins (Bcl-2, Bax, Cleaved Caspase-3, and Cleaved Caspase-9). The results confirmed that comparing with control group, C6 cells treated with TMZ (125 μM) exhibited a characteristic increase in Bax, Cleaved Caspase-3, Cleaved Caspase-9 and the expression levels of Bcl-2 in C6 cells were decreased. Furthermore, the combination strategy that TMZ (125 μM) combined with calycosin (160 μM), TMZ combined with FMN (40 μM), TMZ combined with calycosin and FMN prompted increased expression of Bax, Cleaved Caspase-3, Cleaved Caspase-9 and suppressed expression of Bcl-2, compared with TMZ monotherapy. What's more, in view of TMZ combined with calycosin and FMN group, expression levels of Bax, Cleaved Caspase-3, Cleaved Caspase-9 were elevated and expression level of Bax was lower than others (Fig. 6A, B, C, D, E). To verify the effects of drug combination on cell migration, the current study also detected the level of migrated-associated proteins (MMP-2 and MMP-9) (Fig. 7A, B, C). Compared with control group, the levels of migrated-associated proteins of C6 cells treated with TMZ were decreased slightly. The levels of migrated-associated proteins of C6 cells treated with TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both were lower than that of TMZ (125 μM) monotherapy. Likewise, effect of TMZ combined with calycosin and FMN on migration was the best, compared to other treatments. These results suggested that calycosin and FMN increased TMZ-induced apoptosis and migration in C6 cells effectively. 3.7. Effects of the three drugs in glioma xenografts A C6 xenograft mouse model was employed to further detect the anticancer activity of the combination of TMZ, calycosin and FMN. No pathological changes in the tissues (heart, liver, spleen, lung, and kidney) were seen after administration of TMZ alone or in combination with calycosin, FMN, or both (Fig. 8A, B, C, D, E). These data did not show any noticeable toxicity toward all drug-administered groups. The tumor density of the drug-administered group was significantly smaller than that of the control group, and the three-drug combination group had the best effect (Fig. 9). To further verified the molecular changes in the animal model, the related proteins of tumor tissues were detected by immunohistochemistry (Fig. 10A, B, C, D, E). GFAP was specifically expressed in all groups. The expression of apoptotic proteins (Bcl-2 and Caspased-3) were up-regulated, and the expression of migratory protein (MMP-9) was down-regulated. 4. Discussion Glioma was a common malignant primary tumor in the central nervous system, accounting for about 40% intracranial tumors. Rapid proliferation and strong invasiveness are the features of glioma (Safdie al, 2012). There is no significant clinical feature in its early stage. As the degree of malignancy increases, symptoms, such as elevated intracranial pressure, dizziness, decreased vision, and nausea, are developed. Conventional surgical resection does not completely cure, leading to its poor prognosis and easy relapse. Recently, treatments for glioma was generally ranged from Chinese medicine, surgery, radiotherapy, chemotherapy to gamma knife and X knife (Song et al., 2014; Li et al., 2013). Following surgery and radiotherapy, the administration of effective anti-tumor drugs was more conducive to the recovery and prognosis of patients. TMZ was identified as a broad-spectrum antitumor drug for the treatment of glioma. Although TMZ has presented anti-tumor effect and could make contribution to improve the quality of life of patients, its effective rate of treatment for patients was only about 40%. Besides, patients’ long-term survival rate was recognized to be low. High proliferation rate and mobility are not conducive to the
Fig. 8. (A, B, C, D, E) Effects of pathological changes in the tissues (Heart, Liver, Spleen, Lung, Kidney) of each group after administration in vivo. The mice treated with TMZ in combination with calycosin, FMN, or both for 2 weeks. After anesthetizing the mice, the cervical vertebrae were sacrificed, and the heart, liver, spleen, lung and kidney were taken out for pathological analysis.
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Fig. 9. Effects of pathological changes in the tumor tissues of each group after administration in vivo. After anesthetizing the mice, the cervical vertebrae were sacrificed, and the tumor tissue were taken out for pathological analysis.
Meanwhile, migratory growth is another important cause of tumor refractory. Glioma cells grow invasively and spread easily to other tissues around them. MMP-2 and MMP-9 are the protein bio-markers that represent tumor migration and invasion (Zhang et al., 2018a). As reflection of this previous observation, we noticed in current study that the cell wound healing rate and the number of migrated cells were declined in scratch test and transwell assay. The migratory proteins (MMP-2 and MMP-9) were down-regulated in the combination group, comparing with TMZ only administered group. To further investigate the efficacy of the drugs, we implanted C6 glioma cells subcutaneously in mice. H&E assay showed that the tumor density of the co-administration group became significant sparse. To improve the therapeutic effect of glioma, we also paid attention to whether the toxicity of the drugs on the tissues and organs of the body was increased in combined medication. The results demonstrated that the drugs in each group presented little toxicity on mouse heart, liver, spleen, lung, and kidney. In the immunohistochemistry assay, GFAP was specifically expressed in each group. The expression of apoptotic proteins was up-regulated, and the expression of migratory proteins was down-regulated. These data from our study suggest that calycosin and FMN might be used as potential co-administered drugs for boosting TMZ sensitivity and efficacy.
prognosis of glioma patients. Therefore, enhancing the anti-tumor effect of TMZ according to inhibiting the proliferation and migration of tumor cells has attracted the main attention for the treatment of glioma. Previous research has revealed that FMN combined with TMZ could produce a synergistic effect in our observation (Zhang et al., 2018b). To reduce the dosage of TMZ, lower toxicity, and further improve its sensitivity, we employed the strategy of the multi-drug combination (calycosin, FMN, and TMZ) therapy for glioma. In our study, to verify the effects of calycosin, FMN and TMZ on C6 cells, we first performed MTT assay and confirmed that the three drugs significantly inhibited the proliferation of C6 cells in a dose-dependent manner. Our aim was to increase the efficacy of TMZ via combining with calycosin and FMN. we used calycosin and FMN at minimum effective concentration combined with TMZ. The results showed that coadministration of three drugs could inhibit cell proliferation in a dosedependent manner. Among them, the inhibition rate for the minimum concentration group (calycosin:160 µM, FMN:40 µM, TMZ:125 µM) was greater than 50%. Therefore, the concentrations of three drugs in this group were served as experimental concentrations. It is well known that the block of proliferation is closely associated with apoptosis. And, apoptosis plays a vital role in eliminating cancer cells. Therefore, apoptosis has become the key indicator in most cancer treatments (Jiang et al., 2017). Our previous research found that co-administration of three drugs increased the number of apoptotic cells. Meanwhile, expression of these apoptotic proteins: Bax, Cleaved Caspase-3, Cleaved Caspase-9, which are involved in apoptotic pathway, were up-regulated. Anti-apoptotic protein (Bcl-2) was down-regulated. HE staining assay showed that the number of viable C6 cells that treated with three drugs were decreased. And the greater amount of nuclear shrinkage was observed.
5. Conclusion In conclusion, co-administration of calycosin, FMN, and TMZ could substantially inhibit the growth and migration of C6 cells in vitro by regulating related proteins that are crucial in the etiology of glioma, and suppress tumor growth as seen in xenograft tumor. This research may provide a theoretical basis for the clinical treatment of glioma.
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Conflict of interests The author states that there is no conflict of interest. Contributions of the authors Fan, H. W. and Ni, Q. designed experimental scheme and prepared the manuscript. Ni, Q., Fan, Y. N. and Zhang, X. carried out the experiment. Ni, Q. and Zhang, X. wrote the paper. Fan, H. W. and Li, Y. B. revised the manuscript critically. Li, Y. B. and Ni, Q. performed experimental data analysis. References Avunduk, S., Mitaine-Offer, A.C., Alankus-Caliskan, O., Miyamoto, T., Senol, S.G., Lacaille-Dubois, M.A., 2008. Triterpene glycosides from the roots of Astragalus flavescens. J. Nat. Prod. 71 (1), 141–145. https://doi.org/10.1021/np0703500. Chen, J., Zhao, X.G., Ye, Y., Wang, Y., Tian, J., 2013. Estrogen receptor beta-mediated proliferative inhibition and apoptosis in human breast cancer by calycosin and formononetin. Cell Physiol. Biochem. 32 (6), 1790–1797. https://doi.org/10.1159/ 000356612. Fu, D.J., Zhang, L., Song, J., Mao, R.W., Zhao, R.H., Liu, Y.C., Hou, Y.H., Li, J.H., Yang, J.J., Jin, C.Y., Li, P., Zi, X.L., Liu, H.M., Zhang, S.Y., Zhang, Y.B., 2017. Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of pc-3cells via MAPK/Wnt signaling pathways. Eur. J. Med. Chem. 127, 87–99. https://doi.org/10.1016/j.ejmech.2016.12.027. Huang, W.H., Liao, W.R., Sun, R.X., 2016. Astragalus polysaccharide induces the apoptosis of human hepatocellular carcinoma cells by decreasing the expression of Notch1. Int. J. Mol. Med. 38 (2), 551–557. https://doi.org/10.3892/ijmm.2016. 2632. Jiang, G.Q., Liu, J., Ren, B.Y., Zhang, L., Owusu, L., Liu, L.K., Zhang, J., Tang, Y.W., Li, W.L., 2017. Anti-tumor and chemosensitization effects of cryptotanshinone extracted from salvia miltiorrhiza bge on ovarian cancer cells in vitro. J. Ethnopharmacol. 205, 33–40. https://doi.org/10.1016/j.jep.2017.04.026. Kumar, D.M., Patil, V., Ramachandran, B., Nila, M.V., Dharmalingam, K., Somasundaram, K., 2013. Temozolomide-modulated glioma proteome: role of interleukin-1 receptorassociated kinase-4 (IRAK4) in chemosensitivity. Proteomics 13 (14), 2113–2124. https://doi.org/10.1002/pmic.201200261. Li, F., Chen, T., Hu, S.L., Lin, J.K., Hu, R., Feng, H., 2013. Superoxide mediates direct current electric field-induced directional migration of glioma cells through the activation of AKT and ERK. PLoS One 8 (4), e61195. https://doi.org/10.1371/journal. pone.0061195. Li, Z.Y., Zhang, C., Chen, L., Chen, B.D., Li, Q.Z., Zhang, X.J., Li, W.P., 2017. Radicol, a novel trinorguaiane-type sesquiterpene, induces temozolomide-resistant glioma cell apoptosis via ER stress and Akt/mTOR pathway blockade. Phytother. Res. 31 (5), 729–739. https://doi.org/10.1002/ptr.5793. Lo, Y., Wang, W., 2013. Formononetin potentiates epirubicin-induced apoptosis via ROS production in HeLa cells in vitro. Chem. Biol. Interact. 205 (3), 188–197. https://doi. org/10.1016/j.cbi.2013.07.003. Lv, Y.W., Hu, W., Wang, Y.L., Huang, L.F., He, Y.B., Xie, X.Z., 2011. Identification and determination of flavonoids in astragali radix by high performance liquid chromatography coupled with DAD and ESI-MS detection. Molecules 16 (3), 2293–2303. https://doi.org/10.3390/molecules16032293. Ma, L., Liu, J., Zhang, X., Qi, J., Yu, W., Gu, Y., 2015. P38 MAPK-dependent Nrf2 induction enhances the resistance of glioma cells against TMZ. Med. Oncol. 32 (3), 69. https://doi.org/10.1007/s12032-015-0517-y. Na, D., Liu, F.N., Miao, Z.F., Du, Z.M., Xu, H.M., 2009. Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by anti-apoptosis. World J. Gastroenterol. 15 (5), 570–577. 〈https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2653347/〉. Nie, X.H., Jia, O.Y., Xing, Y., Li, D.Y., Liu, R.E., Xu, R.X., 2016. Calycosin inhibits migration and invasion through modulation of transforming growth factor beta-mediated mesenchymal properties in U87 and U251 cells. Drug Des. Dev. Ther. 10, 767–779. https://doi.org/10.2147/DDDT.S90457. Safdie, F., Brandhorst, S., Wei, M., Wang, W., Lee, C., Hwang, S., Conti, P.S., Chen, T.C., Longo, V.D., 2012. Fasting enhances the response of glioma to chemo- and radiotherapy. PLoS One 7 (9), e44603. https://doi.org/10.1371/journal.pone.0044603. Shi, R., Wang, P.Y., Li, X.Y., Chen, J.X., Li, Y., Zhang, X.Z., Zhang, C.G., Jiang, T., Li, W.B., Ding, W., Cheng, S.J., 2015. Exosomal levels of miRNA-21 from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients. Oncotarget 6 (29), 26971–26981. https://doi.org/10.18632/oncotarget.4699. Song, Y., Hu, Z., Long, H., Peng, Y., Zhang, X., Que, T., Zheng, S., Li, Z., Wang, G., Yi, L., Liu, Z., Fang, W., Qi, S., 2014. A complex mechanism for HDGF-mediated cell growth, migration, invasion, and TMZ chemosensitivity in glioma. J. Neurooncol. 119 (2), 285–295. https://doi.org/10.1007/s11060-014-1512-4. Yu, Z., Xie, G., Zhou, G., Cheng, Y., Zhang, G., Yao, G., Chen, Y., Li, Y., Zhao, G., 2015. NVP-BEZ235, a novel dual PI3K-mTOR inhibitor displays anti-glioma activity and reduces chemoresistance to temozolomide in human glioma cells. Cancer Lett. 367 (1), 58–68. https://doi.org/10.1016/j.canlet.2015.07.007. Zhang, J., Liu, L., Wang, J., Ren, B., Zhang, L., Li, W., 2018a. Formononetin, an isoflavone from astragalus membranaceus, inhibits proliferation and metastasis of ovarian
Fig. 10. (A, B, C, D, E) To further verify the molecular changes in the animal model, the related proteins of tumor tissues were detected by immunohistochemistry.
Acknowledgment This work was supported by a grant from the "Twelfth Five-Year Plan" Nanjing Health Young Talents Training Project (QRX11032).
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Q. Ni, et al. cancer cells. J. Ethnopharmacol. 221, 91–99. https://doi.org/10.1016/j.jep.2018.04. 014. Zhang, X., Guo, M., Shen, L., Hu, S., 2014. Combination of photodynamic therapy and temozolomide on glioma in a rat c6 glioma model. Photodiagnosis Photodyn. Ther. 11 (4), 603–612. https://doi.org/10.1016/j.pdpdt.2014.10.007. Zhang, X., Ni, Q., Wang, Y., Fan, H.W., Li, Y.B., 2018b. Synergistic anticancer effects of formononetin and temozolomide on glioma C6 cells. Biol. Pharm. Bull. 41 (8), 1194–1202. https://doi.org/10.1248/bpb.b18-00002. Zhao, X., Li, X., Ren, Q., Tian, J., Chen, J., 2016. Calycosin induces apoptosis in colorectal cancer cells, through modulating the ERβ/MiR-95 and IGF-1R, PI3K/Akt signaling pathways. Gene 591 (1), 123–128. https://doi.org/10.1016/j.gene.2016.07.012. Zhou, L., Wu, Y., Guo, Y., Li, Y., Li, N., Yang, Y., Qin, X., 2017. Calycosin enhances some chemotherapeutic drugs inhibition of akt signaling pathway in gastric cells. Cancer Investig. 35 (5), 289–300. https://doi.org/10.1080/07357907.2016.1278226. Zhou, R., Chen, H., Chen, J., Chen, X., Wen, Y., Xu, L., 2018. Extract from astragalus membranaceus inhibit breast cancer cells proliferation via PI3K/AKT/mTOR signaling pathway. BMC Complement. Altern. Med. 18 (1), 83. https://doi.org/10. 1186/s12906-018-2148-2.
Zou, Y., Wang, Q., Li, B., Xie, B., Wang, W., 2014. Temozolomide induces autophagy via ATM-AMPK-ULK1 pathways in glioma. Mol. Med. Rep. 10 (1), 411–416. https://doi. org/10.3892/mmr.2014.2151.
Glossary C6: a kind of rat glioma cell line MTT: trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI: propidium iodide OD: the optical density IC50: the concentration leading to 50% inhibition of viability in MTT assay DMSO: dimethylsulfoxide FMN: formononetin DMEM: Dulbecco's modified Eagle's medium FBS: Fetal bovine serum
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In vitro and in vivo Study on Glioma Treatment Enhancement by Combining Temozolomide with Calycosin and Formononetin Qi Nia, Yani Fana, Xiong Zhanga, Hongwei Fana, , Yingbin Lib, ⁎
a b
T
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Department of Clinical Pharmacology Lab, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu 210006, China Department of Neurosurgery, the Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210011, China
ARTICLE INFO
ABSTRACT
Keywords: Glioma Temozolomide Calycosin Formononetin Combination treatment
Ethnopharmacological relevance: Astragalina alpestris is a traditional Chinese herbal medicine with anti-inflammatory, anti-immune, anti-tumor and other pharmacological effects. Calycosin and formononetin (FMN) are two natural compounds isolated from astragalus. It has been shown that calycosin and FMN are active anti-tumor ingredient. Aim of the study: The aim of this current work was to study the therapeutic enhancement of temozolomide (TMZ) on gliomavia combining with calycosin and FMN. Materials and methods: The effect of co-administration via hematoxylin-eosin staining (HE staining) was determined by measuring cell proliferation toxicity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide (MTT) assay and sequentially observing the cell morphology. To synchronously explore the effect of migration on C6, transwell assay and wound healing assay were performed. Apoptosis was measured by Annexin V/propidium iodide (PI) staining. Meanwhile, western blot was used to investigate proteins involved in the mechanisms for migration and apoptosis. Furthermore, HE staining and immunohistochemistry were also analyzed for curative effect in vivo. Results: The efficacy of TMZ on glioma could be enhanced by combining with calycosin and FMN through inhibiting the proliferation and migration of glioma cells and promoting their apoptosis. Western blot assays indicated that expression of apoptotic proteins (Bcl-2 Associated XProtein (Bax), Cleaved Caspase-3, Cleaved Caspase-9) were up-regulated. Anti-apoptotic protein (B-cell lymphoma-2,Bcl-2) was down-regulated. The migratory proteins (Matrix metallopeptidase 2, 9, MMP-2, MMP-9) was downregulated. In vivo study, this kind of co-administration (calycosin, FMN, and TMZ) exhibited the marked therapeutic effect on glioma. Conclusions: This study has identified that calycosin and FMN can increase the treatment effect of TMZ in vitro and in vivo. These attractive features substantially broadened the application range of TMZ as a glioma treatment medicine.
1. Introduction In central nervous system, brain glioma is the most aggressive malignancies with a high prevalence, high recurrence rate and low survival rate (Safdie et al., 2012). Traditionally, glioma treatment has mainly based on surgery, followed by radiotherapy or chemotherapy (Song et al., 2014). However, its failure often occurred and the treatment effect could bring the patients with even worse prognosis. This could be attributed to the continuous proliferation of glioma residual after the treatment, eventually resulting in glioma recurrence. Overall, the short-term survival was low and median that underwent glioma diagnosis was 1–2 years (Li et al., 2013; Shi et al., 2015; Yu et al., 2015). Therefore, development of novel effective drugs is urgent for ⁎
glioma patients. Temozolomide (TMZ) (Fig. 1), one of the most effective drugs for clinical treatment of brain tumors, is an alkylating agent that contains an imidazole tetrazine ring. TMZ was recognized as significant curative effects on melanoma, lymphoma, leukemia and solid tumors (Kumar et al., 2013). TMZ could easily penetrate into the blood-brain barrier further delaying the recurrence of glioma. The alkylation of DNA exerts a cytotoxic effect, giving rise to single or double-stranded DNA, breaks to prevent DNA replication, and then produces an anti-tumor effect (Li et al., 2017). In addition, TMZ mediates apoptosis by inducing stress responses that are not dependent on DNA damage (Zhang et al., 2014; Ma et al., 2015; Zou et al., 2014). However, clinical data demonstrated that patients required a high dose of TMZ, which in turn presented
Corresponding authors. E-mail addresses: [email protected] (H. Fan), [email protected] (Y. Li).
https://doi.org/10.1016/j.jep.2019.01.023 Received 12 November 2018; Received in revised form 17 January 2019; Accepted 20 January 2019 Available online 18 April 2019 0378-8741/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Fig. 1. (A) Chemical structure of TMZ. (B) Chemical structure of calycosin. (C) Chemical structure of FMN.
obvious toxic side effects. Therefore, major concern of reducing toxicity, elevating the sensitivity and improving the therapeutic effect of TMZ still remained. Combination therapy has displayed important clinical significance for improving the lethal effect of tumor cells. Comparing with conventional chemotherapy drugs, Traditional Chinese Medicines was more involved in pleiotropic, multi-target, multi-channel, and wide-ranging characteristics. Astragalina australis, a Traditional Chinese Medicine, contains several types of bioactive compounds, such as, various flavonoids, glycosides, and other substances, benefiting vital energy, consolidating superficies, and inducing diuresis to alleviate edema (Avunduk et al., 2008). In practice, Astragalina australis is often used in combination with other herbs, such as, poria, angelica, and ginseng in various complex prescription formulas. In recent years, a large number of studies have proved the reliable role of Astragalina australis in anti-tumor. Although Astragalina australis is usually combined with other herbs, it can be taken separately by itself (Zhou et al., 2018). In various pharmacological studies, both crude extracts and monomeric components of Astragalina australis showed activities in anti-tumor, anti-oxidation, anti-infection, cardioprotection and antivirus (Na et al., 2009., Huang et al., 2016). Calycosin (Fig. 1) and formononetin FMN (Fig. 1) are components existing in Astragalina australis as proved by High Performance Liquid Chromatography (HPLC) coupled with photodiode-array detection (DAD) and electrospray ionization (ESI-MS) Detection (Lv et al., 2011). Both calycosin and FMN possessed of anti-inflammatory, anti-immune, antioxidative, anti-viral, anti-tumor and other pharmacological effects (Zhou et al., 2017; Zhao et al., 2016; Lo and Wang, 2013). They could be used as potential drugs for clinical neuroprotection via inhibiting the proliferation and migration of cells, and promoting apoptosis (Nie et al., 2016). Some studies displayed that calycosin and FMN played key roles in the treatment of breast cancer, melanoma, and cervical cancer (Fu et al., 2017; Chen et al., 2013). The results indicated that calycosin and FMN could inhibit the growth of glioma cells and promote their apoptosis. Moreover, our previous work also suggested that combination of FMN with TMZ was attributed to synergistic therapy effect for glioma (Zhang et al., 2018b). Inspiring from the conclusion, co-treatment with more flavonoids agent may further benefit to the sensitivity of TMZ. In this work, we investigated the effect of co-treatment of the three drugs (calycosin, FMN, and TMZ) on the proliferation, apoptosis, and migration in vitro by using C6 cells. Furthermore, mouse ectopic tumor models were also established to test the in vivo effect of the combination of the two Chinese herbs and TMZ on the treatment of glioma. The experimental study here revealed a critical role of TMZ in the treatment of glioma, which may provide a new avenue for glioma research.
2. Materials and methods 2.1. Cells and reagents The C6 cell line was obtained from Dr. Zhang Xin's laboratory (China Pharmaceutical University). C6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (CAS No: KGM12800500, KeyGEN, Nanjing, China) at 37 °C supplemented with 10% fetal bovine serum (FBS, KeyGEN) and 5% CO2. Calycosin (purity ≥ 99%, CAS No: 20575-57-9) and FMN (purity ≥ 99%, CAS No: 485–72-3) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd. (Taizhou, Jiangsu, China). TMZ (CAS No: 16083115) was obtained from Jiangsu Hengrui Group Pharmaceutical Co., Ltd (Lianyungang, Jiangsu, China). TMZ was dissolved into Sterilized water (CAS No: 2016091805, Qidu, Shandong, China) for injection. Stock solution of Calycosin and FMN were made by dissolving in dimethyl sulfoxide (DMSO; CAS No: KGT5131; KeyGEN, Nanjing, China). In all the drug dissolving solvents, DMSO concentration wase limited to < 0.1% (v/v), and the vehicle controls were used at the same concentration as DMSO. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT) was purchased from KeyGEN BioTECH (CAS No: 20171019, Nanjing, Jiangsu, China). Annexin V-Alexa Fluor 647/propidium iodide (PI) (CAS No: FMSAV647-050) was purchased from Fcmacs Biotech Co., Ltd (Nanjing, Jiangsu, China). Antibodies specific for Bcl-2 (CAS No: ab182858), Bax (CAS No: ab32503), Caspase-3 (CAS No: ab32351), Caspase-9 (CAS No: ab202068), MMP-2 (CAS No: ab92536), MMP-9 (CAS No: ab38898), were purchased from purchased from Abcam corporation and used as the main antibodies in Western blot assay. Antibodies specific for GFAP (CAS No: ab7260), Bcl-2 (CAS No: MM0665-9S52), Bax (CAS No: ab32503), Caspase-3 (CAS No: EPR18297) and MMP-9 (CAS No: ab76003) were purchased from Abcam corporation. 2.2. Animals BALB/C mice were purchased from the Animal Experimental Center of Nanjing First Hospital and were acclimated for at least 1 week in the animal facility without specific pathogens prior to the experiment. They were placed in a temperature (24 ± 2 °C) and relative humidity (45%–60%) controlled chamber. The mice were similar in age and body weight in all experiments and were randomly assigned to the treatment groups. All animal experimental manipulations followed China legislation on the use and care of laboratory animals and were approved by Animal Experimental Ethics Committee of Nanjing First Hospital.
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Fig. 2. (A, B, C) The cytotoxicity of TMZ (0–2000 µM), calycosin (0–640 µM), and FMN (0–320 µM) on C6 cells with different concentrations for 48 h was assessed by MTT assay. Data represented as the mean ± SD (standard deviation) of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (D, E, F, G) Both calycosin and FMN enhanced cytotoxicity of TMZ on C6 cells. Combination of calycosin (160 µM) and TMZ (0–2000 µM) promotes apoptosis of C6 glioma cells, the IC50 of TMZ decreased. Combination of FMN (40 µM) and TMZ (0–2000 µM) promotes apoptosis of C6 glioma cells, the IC50 of TMZ decreased. The IC50 of TMZ is decreased to 125 µM significantly when causal calycosin and FMN are combined with TMZ, ***p < 0.001.
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Fig. 3. Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on morphology of C6 cells.
2.3. Cell viability assay
(160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). Then, the medium in each well of a 6-well plate was discarded, 4% paraformaldehyde was fixed for 10 min, and H &E staining was performed. The morphology of the cells was observed under an inverted microscope.
The viability of cells was determined by MTT assay. C6 cells were seeded with a density of 8 × 103 cells/well in 96-well plates and cultured at 37 °C, in DMEM with 10% FBS. C6 cells were treated separately with different concentrations of three drugs (calycosin, FMN, and TMZ). The lowest effective concentrations of calycosin and FMN were used to combine with TMZ. The treatment with TMZ was conducted at various doses levels (125, 250, 500, 1000, and 2000 μM) for 48 h. In the calycosin treatment groups, calycosin was added to culture medium at final concentrations of 20, 80, 160, 320, and 640 μM. In the FMN treatment groups, FMN was added to culture medium at the concentration range of 20, 40, 80, 160, and 320 μM. In two drug coupling groups, calycosin (160 μM) was added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM), FMN (40 μM) was added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM). In the group of TMZ combination with calycosin and FMN, calycosin (160 μM) and FMN (40 μM) were added to culture mediums in combination with various concentrations of TMZ (125, 250, 500, 1000, and 2000 μM). Following incubation at 37 °C for 48 h, C6 cells were exposed to MTT solution with final concentration of 50 μL/well. After incubating for 4 h, DMSO was used to dissolve crystals. Then, measuring the optical density (OD) values was performed in a microplate reader (TECAN, Switzerland) at 570 nm after 10 min. The proliferation rate that was specific for C6 cells was calculated by the formula: the OD values of treated groups/the OD values of control group× 100%.
2.5. Wound healing assay C6 cells were cultured in 6-well plates with a density of 4 × 105 cells/well. When cells grew to about 90% per well, 10 μL pipette tips was used to scratch the monolayer of cells. After washing cell debris with PBS, cells were cultured in 2% FBS medium in the absence or presence of the various concentrations in each group for 24 h: (1) control; (2) TMZ (125 μM); (3) TMZ (125 μM and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). Cell scratch spacing was valued via calculating cell mobility at 0 h and 24 h. 2.6. Transwell assay C6 Cells were seeded in triplicate on 6-well dishes. Upon reaching 80% confluence, they were exposed to vehicle or different administration for 48 h. Cells with a density of 2 × 106/ml were treated in different administration groups: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM). The above cells suspension (100 μL) were seeded in the upper chamber of an 8.0-μm pore polycarbonate membrane insert (Corning Inc, USA), while adding 10% culture medium (500 μL) to the lower chamber. The non-migrated cells in the upper chamber were wiped off with a cotton swab, and the
2.4. HE staining C6 cells were cultured in 6-well plates with a density of 3 × 105 cells/well with sterile coverslips and treated with different drug groups for 48 h: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin
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Fig. 4. (A, B, C, D) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on migration of C6 cells. C6 cells were exposed to these drugs before being assayed for C6 cell migration by wound healing and transwell migrations, using Graphpad respectively to measured the wounding healing rate and the number of migration cells. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 5. (A, B) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on apoptosis of C6 cells. The detection of apoptosis rate was used by flow cytometry and analyzed by Graphpad. *p < 0.05, **p < 0.01, ***p < 0.001.
antibodies overnight at 4 °C. A GAPDH antibody served as control. The Bcl-2 (1:2000; Abcam), Bax (1:5000; Abcam), Cleaved Caspase-3 (1:1000; Abcam), Cleaved Caspase-9 (1:1000; Abcam), MMP-2 (1:5000; Abcam), MMP-9 (1:1000; Abcam) antibodies were used for each group. Then, the membranes were washed and incubated with specific secondary antibodies for 1 h. The two kinds of liquids, A and B, in the ECL chemiluminescence kit are mixed equally in volume, and were configured as a working fluid. Nitrocellulose membrane was exposed to working fluid. The western blot signal that represented protein level was qualified using Image J software.
migrated cells in the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet, Photographed under a microscope to observe cell migration. 2.7. Flow cytometric assay C6 cells were plated in 6-well plates and treated with different drug administration: (1) control; (2) TMZ(125 μM); (3) TMZ (125 μM) and calycosin (160 μM); (4) TMZ (125 μM) and FMN (40 μM); (5) calycosin (160 μM) and FMN (40 μM); and (6) TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM), which were cultured at 37 °C for 48 h. The C6 cells were washed with cold phosphate-buffered saline (PBS) (KeyGEN, Nanjing, China) and resuspended in it. An aliquot of 100 μL of the cell suspension was placed in a flow tube, and 5 μL of Annexin V-Alexa Fluor 647/PI and 10 μL of a 20 μg/ml PI solution were added. The above solvent was mixed and protected from light at room temperature for 15 min. Annexin V FITC Apoptosis Detection Kit (FMSAV647-100, BD, USA) was used to detect cell apoptosis.
2.9. Tumor xenografts Glioma C6 cells (1.0 × 107cells per mouse) were subcutaneously injected into BALB/C mice (Animal Experimental Center of Nanjing First Hospital). When the tumor size reached about 50 mm3, mice were randomly divided into 6 groups (9 mice per group): (1) control; (2) TMZ (30 mg/kg); (3) TMZ (30 mg/kg) plus calycosin (40 mg/kg); (4) TMZ (30 mg/kg) plus FMN (18 mg/kg); 5) calycosin (40 mg/kg) plus FMN (18 mg/kg); and 6) TMZ (30 mg/kg) plus calycosin (40 mg/kg) and FMN (18 mg/kg). The treatment was performed every 2 days for 15 days. In the end of this experiment, mice were sacrificed by cervical dislocation. The organ tissue and tumor tissue were immersed in formalin. HE staining was used for detecting cell apoptosis. Cell morphology in tissues was observed under fluorescence microscope. Individual tumor samples were detected for expression levels of proteins by immunohistochemistry. All animal experimental manipulations were approved by the Ethics Committee for Animal Experiment of Nanjing Medical University.
2.8. Western blot assay C6 cells were exposed to different drug administration and were incubated at 37 °C for 48 h. The cells were digested with trypsin and washed twice in cold PBS. Then 200 μL of ice-cold lysis buffer was added to fully lyse the cells. The protein concentration was measured by using BCA Protein Quantitation Kit (KeyGEN, Nanjing, China). Cell lysates were added to SDS buffer for SDS-PAGE gel electrophoresis and then transferred to a Nitrocellulose membrane (KeyGEN, Nanjing, China). The membrane was washed 3 times with TBST and then blocked with 5% skim milk powder blocking solution for 90 min at room temperature. The main strips were probed with indicated primary
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Fig. 6. (A, B, C, D, E) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on major proteins of C6 cells. C6 cells were exposed to these drugs for 48 h before being analyzed for the expression of Bax, Bcl-2, Caspase-3, and Caspase-9 by Western blot. The signal representing proteins levels were qualified using Image J software. *p < 0.05, **p < 0.01, ***p < 0.001.
2.10. Statistical analysis
concentrations on C6 cells for 48 h was assessed by MTT assay. C6 cells were treated with increasing doses of TMZ for 48 h (Fig. 2A). The IC50 of TMZ was 1000 μM approximately. Treatment with 20–640 μM calycosin inhibited cells proliferation in a concentration-dependent manner in C6 cells (Fig. 2B). The minimum effective concentration of calycosin was 160 μM. Treatment with 20–320 μM FMN inhibited cells proliferation in a concentration-dependent manner in C6 cells (Fig. 2C). The minimum effective concentration of FMN was 40 μM.
All the experiments were reproduced in triplicate. Statistical analysis were performed by Student's t-test (SPSS, SPSS® Statistics V22.0, IBM, NY, USA). The results were shown as the mean ± standard deviation. P < 0.05 was recognized as a statistically significant difference. 3. Results
3.2. Effects of calycosin and FMN administered separately or in combination with TMZ on proliferation of C6 cells
3.1. Effects of calycosin, FMN and TMZ on proliferation of C6 cells
When cells were treated with the combination treatment of
The cytotoxicity of TMZ, calycosin and FMN at different
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Fig. 7. (A, B, C) Effects of TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both on major protein of C6 cells. C6 cells were exposed to Calycosin (160 µM) and FMN (40 µM) administered separately or in combination with TMZ (125 µM) for 48 h before being analyzed for the expression of MMP-2 and MMP-9 by Western blot. The signal representing proteins levels were qualified by Image J software. *p < 0.05, **p < 0.01, ***p < 0.001.
calycosin or FMN, which comprised a low effective concentration of calycosin (160 μM) and FMN (40 μM) and various concentrations of TMZ (125, 250, 500, 1000, and 2000 µM) for 48 h, the IC50 of cotreatment with TMZ was decreased (Fig. 2D, E). The IC50 of TMZ was decreased dramatically to 125 µM when causal calycosin and FMN were combined with TMZ (Fig. 2F, G), which indicated that calycosin and FMN could elevate sensitivity of TMZ.
calycosin and FMN. To test whether calycosin and FMN could increase the efficacy of TMZ in reducing glioma cell motility and migration, C6 cells were treated with calycosin (160 μM) and FMN (40 μM) separately or in combination with TMZ (125 μM) for 24 h, and subjected to wound healing and transwell migrations. Treated with TMZ, suppression of cell migration was weak (Fig. 4A, B, C, D). When the cells were exposed to TMZ that combined with calycosin (160 μM) or FMN (40 μM), the cell migration rate was obviously decreased. Furthermore, cells that were treated with TMZ (125 μM) combined with calycosin (160 μM) and FMN (40 μM) were found to rarely migrated, compared with other groups.
3.3. Effects of calycosin and FMN administered separately or in combination with TMZ on morphology of C6 cells H&E staining assay was performed to detect the level of apoptosis and morphological changes in cells. Based on MTT, C6 cells were treated with calycosin (160 μM) and FMN (40 μM) administered separately or in combination with TMZ (125 μM) for 48 h. Treated with TMZ, apoptotic cells endowed less, slightly shrinking nuclei. When C6 cells were treated with TMZ, combined with calycosin or FMN, the number of viable cells was remarkably reduced. Larger nuclear shrinkage was observed in the sample of TMZ combined with calycosin or FMN. Furthermore, in the combination treatment of TMZ, calycosin, and FMN, C6 cells exhibited most nuclear apoptotic changes, which contained nuclear condensation and aggregation (Fig. 3).
3.5. Effects of calycosin and FMN administered separately or in combination with TMZ on apoptosis of C6 cells Due to induction of considerable apoptosis, we subsequently determined whether calycosin (160 μM) and FMN (40 μM) could improve the therapeutic effect of TMZ (125 μM) in promoting tumor apoptosis by Annexin V/PI staining with flow cytometry. The results suggested that TMZ combining with calycosin and FMN could elevated apoptotic rate well (Fig. 5). 3.6. Effects of calycosin and FMN administered separately or in combination with TMZ on major proteins of C6 cells
3.4. Effects of calycosin and FMN administered separately or in combination with TMZ on migration of C6 cells
To measure the effects of drug combination on cell proliferation and apoptosis, the current study also detected the expression level of
TMZ exerted inhibitory effects on C6 cells when combined with
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apoptosis-associated proteins (Bcl-2, Bax, Cleaved Caspase-3, and Cleaved Caspase-9). The results confirmed that comparing with control group, C6 cells treated with TMZ (125 μM) exhibited a characteristic increase in Bax, Cleaved Caspase-3, Cleaved Caspase-9 and the expression levels of Bcl-2 in C6 cells were decreased. Furthermore, the combination strategy that TMZ (125 μM) combined with calycosin (160 μM), TMZ combined with FMN (40 μM), TMZ combined with calycosin and FMN prompted increased expression of Bax, Cleaved Caspase-3, Cleaved Caspase-9 and suppressed expression of Bcl-2, compared with TMZ monotherapy. What's more, in view of TMZ combined with calycosin and FMN group, expression levels of Bax, Cleaved Caspase-3, Cleaved Caspase-9 were elevated and expression level of Bax was lower than others (Fig. 6A, B, C, D, E). To verify the effects of drug combination on cell migration, the current study also detected the level of migrated-associated proteins (MMP-2 and MMP-9) (Fig. 7A, B, C). Compared with control group, the levels of migrated-associated proteins of C6 cells treated with TMZ were decreased slightly. The levels of migrated-associated proteins of C6 cells treated with TMZ (125 μM) in combination with calycosin (160 μM), FMN (40 μM), or both were lower than that of TMZ (125 μM) monotherapy. Likewise, effect of TMZ combined with calycosin and FMN on migration was the best, compared to other treatments. These results suggested that calycosin and FMN increased TMZ-induced apoptosis and migration in C6 cells effectively. 3.7. Effects of the three drugs in glioma xenografts A C6 xenograft mouse model was employed to further detect the anticancer activity of the combination of TMZ, calycosin and FMN. No pathological changes in the tissues (heart, liver, spleen, lung, and kidney) were seen after administration of TMZ alone or in combination with calycosin, FMN, or both (Fig. 8A, B, C, D, E). These data did not show any noticeable toxicity toward all drug-administered groups. The tumor density of the drug-administered group was significantly smaller than that of the control group, and the three-drug combination group had the best effect (Fig. 9). To further verified the molecular changes in the animal model, the related proteins of tumor tissues were detected by immunohistochemistry (Fig. 10A, B, C, D, E). GFAP was specifically expressed in all groups. The expression of apoptotic proteins (Bcl-2 and Caspased-3) were up-regulated, and the expression of migratory protein (MMP-9) was down-regulated. 4. Discussion Glioma was a common malignant primary tumor in the central nervous system, accounting for about 40% intracranial tumors. Rapid proliferation and strong invasiveness are the features of glioma (Safdie al, 2012). There is no significant clinical feature in its early stage. As the degree of malignancy increases, symptoms, such as elevated intracranial pressure, dizziness, decreased vision, and nausea, are developed. Conventional surgical resection does not completely cure, leading to its poor prognosis and easy relapse. Recently, treatments for glioma was generally ranged from Chinese medicine, surgery, radiotherapy, chemotherapy to gamma knife and X knife (Song et al., 2014; Li et al., 2013). Following surgery and radiotherapy, the administration of effective anti-tumor drugs was more conducive to the recovery and prognosis of patients. TMZ was identified as a broad-spectrum antitumor drug for the treatment of glioma. Although TMZ has presented anti-tumor effect and could make contribution to improve the quality of life of patients, its effective rate of treatment for patients was only about 40%. Besides, patients’ long-term survival rate was recognized to be low. High proliferation rate and mobility are not conducive to the
Fig. 8. (A, B, C, D, E) Effects of pathological changes in the tissues (Heart, Liver, Spleen, Lung, Kidney) of each group after administration in vivo. The mice treated with TMZ in combination with calycosin, FMN, or both for 2 weeks. After anesthetizing the mice, the cervical vertebrae were sacrificed, and the heart, liver, spleen, lung and kidney were taken out for pathological analysis.
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Fig. 9. Effects of pathological changes in the tumor tissues of each group after administration in vivo. After anesthetizing the mice, the cervical vertebrae were sacrificed, and the tumor tissue were taken out for pathological analysis.
Meanwhile, migratory growth is another important cause of tumor refractory. Glioma cells grow invasively and spread easily to other tissues around them. MMP-2 and MMP-9 are the protein bio-markers that represent tumor migration and invasion (Zhang et al., 2018a). As reflection of this previous observation, we noticed in current study that the cell wound healing rate and the number of migrated cells were declined in scratch test and transwell assay. The migratory proteins (MMP-2 and MMP-9) were down-regulated in the combination group, comparing with TMZ only administered group. To further investigate the efficacy of the drugs, we implanted C6 glioma cells subcutaneously in mice. H&E assay showed that the tumor density of the co-administration group became significant sparse. To improve the therapeutic effect of glioma, we also paid attention to whether the toxicity of the drugs on the tissues and organs of the body was increased in combined medication. The results demonstrated that the drugs in each group presented little toxicity on mouse heart, liver, spleen, lung, and kidney. In the immunohistochemistry assay, GFAP was specifically expressed in each group. The expression of apoptotic proteins was up-regulated, and the expression of migratory proteins was down-regulated. These data from our study suggest that calycosin and FMN might be used as potential co-administered drugs for boosting TMZ sensitivity and efficacy.
prognosis of glioma patients. Therefore, enhancing the anti-tumor effect of TMZ according to inhibiting the proliferation and migration of tumor cells has attracted the main attention for the treatment of glioma. Previous research has revealed that FMN combined with TMZ could produce a synergistic effect in our observation (Zhang et al., 2018b). To reduce the dosage of TMZ, lower toxicity, and further improve its sensitivity, we employed the strategy of the multi-drug combination (calycosin, FMN, and TMZ) therapy for glioma. In our study, to verify the effects of calycosin, FMN and TMZ on C6 cells, we first performed MTT assay and confirmed that the three drugs significantly inhibited the proliferation of C6 cells in a dose-dependent manner. Our aim was to increase the efficacy of TMZ via combining with calycosin and FMN. we used calycosin and FMN at minimum effective concentration combined with TMZ. The results showed that coadministration of three drugs could inhibit cell proliferation in a dosedependent manner. Among them, the inhibition rate for the minimum concentration group (calycosin:160 µM, FMN:40 µM, TMZ:125 µM) was greater than 50%. Therefore, the concentrations of three drugs in this group were served as experimental concentrations. It is well known that the block of proliferation is closely associated with apoptosis. And, apoptosis plays a vital role in eliminating cancer cells. Therefore, apoptosis has become the key indicator in most cancer treatments (Jiang et al., 2017). Our previous research found that co-administration of three drugs increased the number of apoptotic cells. Meanwhile, expression of these apoptotic proteins: Bax, Cleaved Caspase-3, Cleaved Caspase-9, which are involved in apoptotic pathway, were up-regulated. Anti-apoptotic protein (Bcl-2) was down-regulated. HE staining assay showed that the number of viable C6 cells that treated with three drugs were decreased. And the greater amount of nuclear shrinkage was observed.
5. Conclusion In conclusion, co-administration of calycosin, FMN, and TMZ could substantially inhibit the growth and migration of C6 cells in vitro by regulating related proteins that are crucial in the etiology of glioma, and suppress tumor growth as seen in xenograft tumor. This research may provide a theoretical basis for the clinical treatment of glioma.
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Conflict of interests The author states that there is no conflict of interest. Contributions of the authors Fan, H. W. and Ni, Q. designed experimental scheme and prepared the manuscript. Ni, Q., Fan, Y. N. and Zhang, X. carried out the experiment. Ni, Q. and Zhang, X. wrote the paper. Fan, H. W. and Li, Y. B. revised the manuscript critically. Li, Y. B. and Ni, Q. performed experimental data analysis. References Avunduk, S., Mitaine-Offer, A.C., Alankus-Caliskan, O., Miyamoto, T., Senol, S.G., Lacaille-Dubois, M.A., 2008. Triterpene glycosides from the roots of Astragalus flavescens. J. Nat. Prod. 71 (1), 141–145. https://doi.org/10.1021/np0703500. Chen, J., Zhao, X.G., Ye, Y., Wang, Y., Tian, J., 2013. Estrogen receptor beta-mediated proliferative inhibition and apoptosis in human breast cancer by calycosin and formononetin. Cell Physiol. Biochem. 32 (6), 1790–1797. https://doi.org/10.1159/ 000356612. 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Fig. 10. (A, B, C, D, E) To further verify the molecular changes in the animal model, the related proteins of tumor tissues were detected by immunohistochemistry.
Acknowledgment This work was supported by a grant from the "Twelfth Five-Year Plan" Nanjing Health Young Talents Training Project (QRX11032).
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Glossary C6: a kind of rat glioma cell line MTT: trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI: propidium iodide OD: the optical density IC50: the concentration leading to 50% inhibition of viability in MTT assay DMSO: dimethylsulfoxide FMN: formononetin DMEM: Dulbecco's modified Eagle's medium FBS: Fetal bovine serum
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