The human GBM cell lines U373-MG-ATCC, U251, SHG-44 and LN229 (CRL-2611) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The human GBM cell lines U251, SHG-44 and LN229 were cultured in Dulbecco's modified essential medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin + streptomycin (Gibco; Thermo Fisher Scientific, Inc.). U373-MG-ATCC cells were maintained in complete EMEM medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS and 100 U/ml penicillin + streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cell cultures were maintained at 37°C in a 5% CO₂ incubator. MTB was purchased from Selleckchem (S2219) and TMZ was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Stock solutions were made in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) and subsequently diluted in culture medium for use. Working concentrations were freshly prepared daily by diluting the stock with phosphate-buffered saline (PBS).

Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China) assay was performed to test cell toxicity of MTB and proliferation of U373-MG-ATCC, U251, SHG-44 and LN229 cells in this study. For toxicity test, we chose astrocytes as a control. U-251MG cells treated with MTB (0, 0.1, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, 50, 100 and 200 µM) for 48 h, were collected to detect the optical density (OD) 490. For proliferation determination, U-251MG cells were pretreated with NC, 2.5 µM MTB, 100 µM TMZ and MTB + TMZ for 24 h, and then cells were trypsinized and seeded into 96-well plates at a density of 4×10³ cells in 200 µl of complete medium per well. Next, the 96-well plates were placed at 37°C in a 5% CO₂ incubator. CCK-8 assay was performed according to the manufacturer's instructions provided in the CCK-8 kit for the next continuous 4 days. The value of 450 nm was measured by ELISA. This assay was repeated for 3 times.

U-251MG cells were divided into 4 groups and treated with NC (Ctrl), 2.5 µM MTB (MTB), 100 µM TMZ (TMZ) and combination treatment (2.5 µM MTB + 100 µM TMZ), separately. TMZ at a concentration of 100 µM was used as previously reported (21).

U-251MG cells were treated as described above for 48 h. Briefly, cells in each group were detached from the culture dish and resuspended in DMEM containing 2% FBS at a density of 1×10⁶ cells/ml. Then, the cells were incubated with Hoechst 33342 staining solution (Sigma-Aldrich; Merck KGaA) for 60 min at 37°C according to the manufacturer's instructions. Finally, the cells were observed under a fluorescence microscope (scale bar, 50 µm). Apoptotic cell death was determined by counting the number of cells with condensed nuclei in 6 randomly selected areas.

U-251MG cells were treated with MTB (2.5 µM) or TMZ (100 µM) for 48 h. Proteins were extracted with RIPA lysis buffer adding cocktail tablets (Sigma Aldrich; Merck KGaA) and phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich; Merck KGaA). An equivalent amount of protein (40 µg) was resolved on SDS-PAGE (6–12%) gel, transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) and probed with specific primary antibodies. The primary antibodies against cleaved caspase-9, Ki-67, PCNA, cleaved caspase-3, Beclin1, P62, LC3, phosphorylated (Tyr705) STAT3 (p-STAT3), STAT3, phosphorylated (Tyr1007/1008) JAK2 (p-JAK2), JAK2, were obtained from the Cell Signaling Technology, Inc. (Danvers, MA, USA) (Table I). After 1 h of incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Finally, the blots were visualized by ECL and detected using a ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were normalized with GAPDH as an internal control.

Immunofluorescence staining was performed to confirm the localization of p-STAT3 in U-251MG cells. U-251MG cells were treated and grouped as previously described in the manuscript. Then, the cells were collected and washed with PBS twice and fixed with 4% paraformaldehyde for 20 min at room temperature. Next, the samples were permeabilized with 0.4% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 10 min, washed with PBS and then blocked with 2% bovine serum albumin (BSA; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) in PBS for 1 h at 37°C. After this, the samples were incubated with anti-p-STAT3 in 1% BSA (Beijing Solarbio Science and Technology Co., Ltd.) in PBS overnight at 4°C. The samples were subsequently washed with PBS and incubated with fluorescein-conjugated secondary antibody in 1% BSA (Beijing Solarbio Science and Technology Co., Ltd.) in PBS for 1 h. Finally, the samples were stained with DAPI (1 µg/ml) for 10 min to stain the cell nuclei. The samples were then visualized a confocal laser scanning microscope at ×400 magnification (Leica Microsystems, GmbH, Wetzlar, Germany).

U-251MG cells were seeded in 6-well cell culture plates. After 48 h of treatment with TMZ with or without MTB, the cells were analyzed by flow cytometry (BD FACSCanto II; BD Biosciences, San Diego, CA, USA) using CycleTEST™ Plus DNA Reagent kit (BD Biosciences) according to the manufacturer's recommended protocol. The obtained results were analyzed using ModFit LT (version 4.0; Verity Software House, Topsham, ME, USA).

For testing of the efficacy of MTB combined with TMZ in vivo, orthotopic xenograft models were established. Twenty 6-week-old male BALB/c-nu/nu mice weighing 16–18 g were bred in aseptic cages, which were kept in an isolator unit with filtered air. The mice had access to water and food ad libitum. U251 cell suspension (0.1 ml) [2-5×10⁷/ml Hank's balanced salt solution (HBSS), Gibco; Thermo Fisher Scientific, Inc.] was stereotypically injected into the left striata of mice. After the tumor was established, the mice were randomized into the following groups: Ctrl; MTB (50 mg/kg/day body weight) (19); TMZ (20 mg/kg/day body weight); MTB + TMZ (50 mg/kg/day MTB + 20 mg/kg/day TMZ). All mice were administered the drug or saline daily by oral gavage 1 day after tumor cell injection (18). All animals were sacrificed at 30 days and tumors were collected. Tumor weight was measured. All animal protocols were approved by the Beijing Tiantan Hospital and Capital Medical university laws governing animal care.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining was performed to detect apoptosis according to the manufacturer's instructions (TUNEL Apoptosis Detection Kit; Upstate Biotechnology, Inc., Lake Placid, NY, USA). TUNEL-positive cells were quantified by counting brown staining within the nucleus of apoptotic cells. The extent of apoptosis was calculated with the Motic Image software (version 1.2; Micro Optical Group Co., Haimen, China) and expressed as a number of TUNEL-positive cells from the same field under an Olympus BX60 microscope and Spot camera (Olympus Optical Co., Tokyo, Japan; magnification, ×400).

Tumors were cut into 2-µm sections and paraffin-embedded tumor sections were constructed. Sections were deparaffinized and blocked with 3% H₂O₂ for 10 min at room temperature followed by incubation with anti-Ki-67 and anti-cleaved caspase-3 at 4°C overnight. Slides were rinsed with PBS and incubated at room temperature for 30 min with biotinylated goat anti-mouse immunglobulin G secondary antibody (Dako, Glostrup, Denmark). After washing in Tris-hydrochloric acid buffer (TBS), the slides were incubated with streptavidin-peroxidase reagent (Dako) and treated with 3,3′-diaminobenzidine (DAB; Sigma Aldrich; Merck KGaA) for 5 min. The slides were counterstained and dehydrated and the final percentage of positive cells was calculated with the Motic Image software (version 1.2; Micro Optical Group Co.)

Autophagy was assessed by western blot analysis and immunofluorescence staining for LC3. Detailed methods were shown in paragraph Western blot analysis and Immunofluorescence staining. All stained specimens were observed under a confocal laser scanning microscope (Leica Microsystems, GmbH). For the quantitative analysis, LC3 positive puncta were analyzed by ImageJ (version 1.50b; National Institutes of Health, Bethesda, ME, USA; http://imagej.nih.gov/ij/).

Statistically significant differences between the control and other groups were evaluated by means of analysis of variance (ANOVA). For comparisons among >2 groups, the one-way ANOVA followed by post hoc Tukey's test was applied. For the CCK-8 assay, the one-way ANOVA with Dunnett's post hoc test was used. Data are illustrated in bar graphs, values are presented as mean ± SEM from at least 3 independent experiments and post hoc test. Asterisks/pound symbols denote statistical significance in the figures. Statistically significant differences in Kaplan-Meier survival were determined by a log-rank test using GraphPad Prism. Statistical significance was considered if P<0.05 in any analysis. All the data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA).

To clarify the influence of MTB on GBM cells, we conducted a CCK-8 assay using U373-MG-ATCC, U251, SHG-44 and LN229 cells and astrocytes treated with MTB at concentrations of 0 to 200 µM for 24 h, respectively. The result (Fig. 1) showed that MTB exerted an inhibitory effect on the viability of GBM cells in a dose-dependent manner (P<0.01; compared to the astrocyte group). Particularly, U251 cells showed more sensitivity to MTB than the other cell lines. The concentration of MTB that inhibited cell viability was 2.5 µM in U251 cells. In addition, treatment with MTB at 2.5 µM did not induce significant cytotoxicity of U251 cells. These data suggest that MTB is a potential anticancer agent against GBM cells, particularly U251 cells. Thus, 2.5 µM of MTB and U251 cells were used for subsequent experiments.

A concentration of 100 µM TMZ was used as previously described (21). As shown in Fig. 2A, both MTB and TMZ promoted the apoptosis of U251 cells compared with the control (P<0.05). As shown in Fig. 2B, both MTB and TMZ suppressed the growth of U-251MG cells compared with the control (P<0.05). However, the combination of MTB and TMZ enhanced the proliferation-inhibiting effect compared with either the MTB or TMZ group (P<0.05). In addition, the combination of MTB and TMZ enhanced the apoptosis-promoting effect compared to TMZ or MTB used alone at the same dose (P<0.05). The present results were verified by western blot analysis of Ki-67, PCNA, cleaved caspase-3 and cleaved caspase-9 (Fig. 2C). The relative expression of Ki-67 and PCNA were significantly reduced by MTB and TMZ, and the combination therapy (P<0.05), while the expression of cleaved caspase-3 and cleaved caspase-9 exhibited inverse results. We also assessed the effect of MTB on cell cycle distribution (Fig. 2D). MTB and TMZ combination significantly arrested the cell cycle at the G2 phase.

To elucidate the role of MTB in autophagy, expression levels of Beclin1, LC3-II, LC3-I and p62 were detected by western blot assay. As shown in Fig. 3A, both MTB and TMZ increased the level of Beclin1 and LC3-II and decreased the level of p62 compared to the control (P<0.05). Expectedly, the combination of MTB and TMZ enhanced the autophagy-promoting effect, evidenced by higher levels of Beclin1 and LC3-II/I ratio, as well as a lower level of P62 compared with the MTB and TMZ group (P<0.05). In addition, the combination of MTB and TMZ obviously increased the number of LC3-positive puncta per cell when compared to that when TMZ or MTB was used alone at the same dose (Fig. 3B, P<0.05). 3-Methyladenine (3-MA) was used to verifiy the autophagy-promoting effect of MTB. As shown in Fig. 3C, TMZ alone significantly increased the number of LC3-positive puncta, and 3-MA totally inhibited the autophagy induced by TMZ, while the suppression was reversed by MTB.

To elucidate the mechanism of MTB in autophagy induction, expression levels of JAK2 and STAT3 were determined by western blot analysis. As shown in Fig. 4A, western blot analysis showed that the combination of MTB and TMZ decreased the phosphorylation levels of JAK2 and STAT3 compared to that following treatment with TMZ or MTB used alone at the same dose (P<0.05). In particular, MTB was observed to suppress STAT3 nuclear translocation. As shown in Fig. 4B, the nuclear level of p-STAT3 was high in the untreated U251 cells; whereas, the nuclear level of p-STAT3 was significantly reduced by MTB and TMZ. In addition, the nuclear level of p-STAT3 in the MTB + TMZ group was significantly decreased compared to that following TMZ or MTB used alone at the same dose (P<0.05).

We employed U-251MG orthotopic xenograft models to confirm the results of the combination treatment of MTB + TMZ in vivo. MTB at 50 mg/kg/day (body weight) (19) and TMZ at 20 mg/kg/day (MTB + TMZ) were administered by oral gavage. At day 30 after tumor cell implantation, mice were sacrificed for tumor weight analysis, immunohistochemistry, TUNEL and western blot assay. As shown in Fig. 5A, the combination treatment with MTB + TMZ significantly decreased xenograft tumor weight compared to that following TMZ or MTB used alone at the same dose (P<0.05). As shown in Fig. 5B, the combination treatment with MTB and TMZ significantly increased the survival rate of the mice (P<0.05). In addition, the results of the TUNEL assay combined with immunohistochemistry demonstrated that the combination treatment with MTB and TMZ significantly promoted the apoptosis of the xenograft tumor cells (Fig. 5C and D; P<0.05); immunohistochemistry of cleaved caspase-3 confirmed an elevated apoptosis rate (Fig. 5D). Meanwhile, the low expression of Ki-67 indicated suppression of proliferation following treatment with the combination of MTB and TMZ (Fig. 5D). Similarly, the combination of MTB and TMZ enhanced the autophagy-promoting effect in the xenograft tumors (Fig. 6A). Both TMZ and MTB decreased the phosphorylation of JAK2 and STAT3, while the combination of MTB and TMZ almost totally inhibited p-JAK2/p-STAT3 (Fig. 6B). The in vivo results were consistent with the in vitro findings.