Temozolomide (TMZ) is an alkylating agent commonly used as a first-line treatment for high-grade glioblastoma. However, TMZ has short half-life and frequently induces tumor resistance, which can limit its therapeutic efficiency. In the present study, it was hypothesized that combined treatment with TMZ and acteoside has synergistic effects in glioblastoma therapy. Using cell viability and wound-healing assays, it was determined that this treatment regimen reduced cell viability and migration to a greater extent than either TMZ or acteoside alone. Following previous reports that TMZ affected autophagy in glioma cells, the present study examined the effects of TMZ + acteoside combination treatment on apoptosis and autophagy. The TMZ + acteoside combination treatment increased the cleavage of caspase-3 and levels of B-cell lymphoma 2 (Bcl-2)-associated X protein and phosphorylated p53, and decreased the level of Bcl-2. The combination treatment increased microtubule-associated protein 1 light chain 3 and apoptosis-related gene expression. It was also determined that TMZ + acteoside induced apoptosis and autophagy through the mitogen-activated protein kinase signaling pathway. These findings suggest that acteoside has beneficial effects on TMZ-based glioblastoma therapy.
Glioblastoma is the most common type of malignant primary brain tumor and the most invasive and devastating primary brain tumor (
Previous studies have reported that glioma cells undergo cell death via autophagy, or type II programmed cell death, in response to TMZ (
Acteoside is a phenylethanoid glycoside that is widely distributed in several tonified traditional Chinese herbal medicines (
Therefore, evidence suggests that both acteoside and TMZ induce anticancer effects through cell death. However, their association and anticancer effects in the context of glioblastoma remain to be elucidated. Therefore, the objective of the present study was to verify the synergistic anticancer effects of acteoside combined with TMZ in glioblastoma therapy. A cell viability assay was performed to analyze the anticancer effects of the combination treatment in C6 glioblastoma cells (a rat glioblastoma cell line), and the results were compared with those following treatment with TMZ alone. To further examine the mechanism of cell death caused by cotreatment with acteoside and TMZ, apoptosis- and autophagy-related genes in C6 cells were examined.
The C6 rat glioblastoma cell line was purchased from American Type Culture Collection (Rockville, MD, USA). The cells were cultured under sterile conditions at 37°C in a humid environment with 5% of CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% antibiotics and antimycotics (all Welgene, Daegu, Korea).
To induce callus formation (
The ethylacetate fractions were concentrated
To identify the TMZ half maximal inhibitory concentration (IC50 value) against C6 cells, 1×104 cells in single cell suspensions were seeded into individual wells of 96-well plates and incubated for 24 h at 37°C prior to TMZ treatment at the indicated concentrations (1, 5, 10, and 20 mM) at 37°C for 24 h. Following determination of the TMZ IC50 value as 5 mM, the cells were treated for 24 h with 5 mM TMZ, 50
A C6 cell suspension in 1 ml was cultured in a 12-well plate and grown to confluence. The cells were treated with TMZ, acteoside or TMZ + acteoside and then scratched with a sterile 10-
The C6 cells were treated with TMZ, acteoside or TMZ + acteoside for 24 h. Cells were lysed on ice by the RIPA (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) lysis buffer supplemented with protease and phosphatase inhibitors cocktail (Roche, Basel, Switzerland) for 30 min. After centrifugation at 18,341 × g for 15 min at 4°C, supernatant was obtained. Protein concentration was determined using Bradford Protein Assays (Bio-rad, Hercules, CA, USA). Equal amounts of total proteins (30
Lysosomal-associated membrane protein 1 (LAMP1) and LC3 are markers for lysosomal and autophagy. The cells (1×105 cells/well) were prepared on sterilized glass coverslips (BD Biosciences, Franklin Lakes, NJ, USA) in triplicate. Following treatment with TMZ, acteoside or TMZ + acteoside for 24 h, the cells were fixed in 4% paraformaldehyde for 30 min, blocked with 5% normal chicken serum (S-3000; Vector Laboratories, Inc., Burlingame, CA, USA), 0.1% Triton X-100 and then incubated 1 h for permeabilization and to block non-specific protein-protein interactions. The cells were then incubated with the anti-LAMP1 (1:200; sc-19992, Santa Cruz Biotechnology, Inc.) and anti-LC3 (1:400; PM036, MBL International, Woburn, MA, USA) overnight at 4°C. The cells were then washed twice with PBS and incubated for an additional 2 h in the dark with a mixture of Alexa Fluor 488 and 594 secondary antibodies (1:200; Alexa Fluor 488 (A-11008) and 594 (A-11007), Thermo Fisher Scientific, Inc., Waltham, MA, USA) and washed again with PBS. The nuclei were stained using 4,6-diamidino-2-phenylindole (Sigma; Merck KGaA), and then washed twice with PBS. The slides were then mounted and images were captured under an LSM-700 laser confocal microscope.
The cells were analyzed for Mitotracker Green and MitoSOX by flow cytometry using a FACSCanto II flow cytometer, as indicated by the manufacturer (BD Biosciences). Following two washes with PBS, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.25% Triton X-100 in PBS for 20 min. The cells were stained with primary antibodies for overnight at 4°C (1:200) and then with secondary antibodies for 1 h on ice. Following two washes with PBS, the cells were fixed in 4% paraformaldehyde and assayed immediately. The flow cytometry data were collected using 10,000 cells and were analyzed using FlowJo 7.6.1 software (Tree Star, Inc., Ashland, OR, USA).
All data were analyzed using GraphPad Prism 5.0 and are presented as the mean ± standard error of the mean. Data were analyzed with one-way analysis of variance with Tukey's multiple-comparisons post hoc test for the comparison of mean values among multiple groups. P<0.05 was considered to indicate a statistically significant difference.
Combined treatment with TMZ and acteoside inhibited the viability of C6 cells. TMZ reduced cell viability in a dose-dependent manner (
The results of the western blot analysis revealed that the levels of cleaved caspase-3, Bax and phosphorylated p53 were higher and the levels of Bcl-2 were lower following combination treatment with TMZ and acteoside than following treatment with TMZ alone (
TMZ has been reported to induce autophagy (
Previous studies have demonstrated that TMZ regulates the MAPK pathway, including p38, JNK and ERK (
The results of the present study establish that the combined treatment with TMZ with acteoside offers therapeutic potential for glioblastoma treatment, and provide evidence that chemosensitization to a combination of TMZ and acteoside can occur through autophagy enhancement. As the mutation of glioblastoma cells can lead to apoptotic pathway inactivation, the induction of autophagy by TMZ + acteoside cotreatment may represent an alternative method for glioblastoma therapy. However, whether autophagy is the sole mechanism for glioblastoma therapy with TMZ + acteoside cotreatment remains to be elucidated.
Autophagy is an essential cellular mechanism for the degradation of proteins and cytoplasmic organelles. The catabolic advantage of induced autophagy may be important in stressful conditions; therefore, the induction of autophagy may be an adaptive mechanism for cell death prevention (
In normal autophagy, particular cytoplasmic components are isolated within autophagosomes which then fuse with a lysosome to be degraded and recycled (
Acteoside is a phenyl-ethanoid glycoside derived from plants (
This study was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIP; grant nos. 2016R1C1B1015811 and 2017R1D1A3B03036420).
All data generated or analyzed during this study are included in this published article.
TWH and JJK drafted the manuscript and performed the experiments. TWH, DHK, DBK, TWJ, and GHK performed the experiments and data interpretation. KAY contributed materials/analysis tools and helped in data analysis. MM, DEC, JHP, and JJK designed the experiments, provided critical suggestions for the manuscript, and reviewed and revised the manuscript. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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Chemical structure of acteoside.
Effects of acteoside on cell viability. (A) C6 cells were treated with various concentrations of TMZ for 24 h and cytotoxicity was assessed by cell viability with 3-(4,5-dimethylthiazol-2-yl)-,5-diphenyltetrazolium bromide. (B) C6 cell were treated with 1, 10, 50, 100 and 200
Combination treatment with TMZ and acteoside prevents cell migration in a wound-healing assay. (A) Representative microscopic images (scale bar, 500
Acteoside and TMZ affect apoptosis synergistically in C6 cells. (A) Western blot analysis revealed the levels of cleaved caspase-3, Bax, Bcl-2, p-p53, total-p53 and β-actin in C6 cells treated with TMZ (5 mM), acteoside (50
Cotreatement with TMZ + acteoside induces autophagy in C6 cells. (A) Confocal microscopy shows expression of autophagosomes with anti-LAMP1 and anti-LC3 antibodies in C6 cells treated for 24 h with TMZ (5 mM) + acteoside (50
Acteoside induces mitogen-activated protein kinase pathway gene expression in TMZ-based treatment. Western blot analysis showed the levels of p-p38, total-p38, p-JNK, total-JNK, p-ERK and total-ERK in cells treated with TMZ (5 mM) with or without acteoside (50