TSN (purity of approximately 99%) was purchased from Chroma Biotechnology Company (Chengdu, China). TSN was dissolved in dimethyl sulfoxide (DMSO), which was diluted with intact cell culture medium to achieve the concentration required for the experiment. The final concentration of DMSO was < 0.1% to avoid side effects.
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher (Waltham, MA, USA). Penicillin/streptomycin (100×) and phosphate-buffered saline (PBS) were purchased from Shanghai Basal Media Technologies Co., Ltd. (Shanghai, China). Cell counting Kit-8 (CCK-8), BCA Protein Assay Kit, Annexin V-FITC Apoptosis Detection Kit, Hoechst Staining Kit, Cell Cycle and Apoptosis Analysis Kit, electrochemiluminescence (ECL) kit, and Tris-buffered saline with Tween-20 (TBS-T) were purchased from Beyotime Biotechnology Company (Shanghai, China). 740Y-P was purchased from TargetMol (Shanghai, China). Primary antibodies against cleaved caspase-3, Bax, cyclin D1, p21, matrix metalloproteinase-2 (MMP2), phosphoinositol 3-kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against PARP-1, phosphorylated PI3K (p-PI3K), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), matrix metalloproteinase-9 (MMP9), Bcl-2, cleaved caspase-9, p27, CDK-4, and CDK-6 were purchased from Abcam (Cambridge, MA, UK). Secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) were obtained from Cell Signaling Technology (Beverly, MA, USA).
Cell lines and cell culture
Human GBM cell lines U87MG, LN18, U251, LN229, and HUVECs were purchased from American Type Culture Collection (ATCC) and preserved in our laboratory at the Translational Medicine Institute of the First Hospital of Jilin University. All cell lines were cultured and maintained in DMEM supplemented with 10% FBS and antibiotics, including 1% streptomycin (100 µg/mL) and 1% penicillin (100 U/mL) in a humidified atmosphere of 5% CO2 at 37°C.
Cell viability analysis
The cell suspensions of U87MG, LN18, U251, LN229, and HUVEC cells were seeded into 96-well plates at a density of 7 × 103 cells per well. The cells were incubated overnight, which allowed adherence to the wall at 37°C. The original cell culture medium was replaced with fresh medium containing different concentrations of TSN. After 24, 48, and 72 h of drug treatment, 10 µl CCK-8 solution was added to each well (avoiding air bubbles). Incubation was extended for 1.5 h, after which the optical density (OD) value was measured and recorded at 460 nm.
Colony formation assays
U87MG and LN18 glioma cells were seeded into 6-well plates at a density of 800 cells per well, which were cultured overnight in a CO2 humidified incubator. The culture medium containing the desired drug concentrations was added to each well. After 24 h, the medium was removed, and fresh medium was added to each well every 3 d for approximately 2 w. When colony formation was visible, they were fixed with ice-cold paraformaldehyde for 20 min and then stained with crystal violet for 20 min. Colonies with > 50 cells were observed under a microscope.
Wound healing assays
U87MG and LN18 cells were inoculated into 6-well plates and cultured until the cell density reached approximately 80–90%. A sterile 200 µL was used to vertically scratch the plate surface and achieve similar width of cell wounds. Fresh serum-free medium containing different concentrations of TSN was added after discarding the original culture medium. The scratch area of each group was recorded separately by an inverted microscope at 0 and 48 h. The measurement of the wound healing area was performed using Image J software (version 1.52i, NIH, Bethesda, USA).
Cell invasion assays
The surface of the transwell upper chamber was evenly coated with the matrix and precooled serum-free solution at a ratio of 1:9 for 6 h. Then, 70 µL of serum-free medium was added to hydrate the basement membrane for 30 min. U87MG and LN18 cells treated with the indicated concentration of TSN with a density of 1 × 104 cells per well were seeded in the upper chamber using 180 µL of serum-free medium; then, 700 µL medium containing 10% FBS was added to the lower chamber. After conventional incubation for 16 h, the residual matrix glue and cells in the upper chamber were lightly removed with cotton swabs; the chamber was then fixed with methanol for 15 min. The chamber was removed, the membrane was dried in air, and then stained with 1% crystal violet for 30 min. Finally, the invasive cells were observed, photographed, and quantified in three random fields under an upright microscope (Olympus IX53/DP80, Tokyo, Japan).
Cell cycle analysis
U87MG and LN18 cells treated with TSN for 48 h were collected and fixed with 70% pre-cooled ethanol at -20°C for at least 24 h. Cells stained with propidium iodide (PI) and RNase for 20 min were detected by flow cytometry (Becton Dickinson, San Diego, CA, USA).
Hoechst apoptosis staining
U87MG and LN18 cells were seeded in 6-well plates at a density of 5 × 104 cells per well. After the cells were adherent to the wall, different concentrations (0, 50, 100, and 200 µM) of TSN were added to each well. After 24 h, the cells were stained with Hoechst 33342 (1 ug/ml) for 15 min under light-proof conditions and observed under a fluorescence microscope at 200 × magnification; then, the number of apoptotic cells in each field was counted and analyzed.
Annexin V-FITC/PI double staining assay
The Annexin V-FITC/PI cell apoptosis detection kit was used to observe the effect of TSN on the apoptosis of human glioma cells. U87MG and LN18 cells were seeded in a 6-well plate at a concentration of 4×105 cells/mL. After treatment with the corresponding TSN for 48 h, the cells with 1× binding buffer (100 µL) were incubated with PI (5 µL) and Annexin V-FITC (5 µL) at room temperature for 20 min. Subsequently, 1× binding buffer (400 µL) was added, and the apoptosis level of glioma cells was detected using flow cytometry. The apoptosis rate was analyzed using FlowJo software (version 10.3, TreeStar, USA).
Western blotting analysis
After glioma cells were treated with different concentrations of TSN for 48 h, the cells were lysed to extract the total protein. The protein concentration was determined using a BCA kit. A total of 30 mg of protein was loaded per lane onto an SDS-PAGE gel (8–10%). The protein in the gel was transferred to the PVDF membrane using a wet transblotting system (Bio-Rad, USA). The membrane was blocked in 5% skimmed milk powder for at least 1 h. It was left to incubate overnight with the appropriate primary antibody at 4°C on a shaker. Subsequently, the membranes were incubated with the appropriate secondary antibody at room temperature for 2 h. The exposure of chromogenic protein bands with ECL luminescent solution was photographed and analyzed using a gel imaging system.
Xenograft tumor model
5-week-old, male, specific pathogen-free (SPF)-level, athymic, immunocompromised (BALB/c nude) mice were obtained from Charles River Laboratories (Beijing, China). Nude mice were reared carefully in the SPF animal laboratory of The First Hospital of Jilin University. Under sterile conditions, a 100-µL suspension of U87MG cells (1×107 cells/mouse) was inoculated subcutaneously into the right armpit of nude mice. When the tumor size reached approximately 180 mm3, the mice were randomly divided into two groups with five mice in each group. The mice from the experimental and control groups were intraperitoneally injected with 1.0 mg/kg TSN and the same volume of saline, respectively, every day for consecutive 14 d.
The weight of each mouse was measured every 3 d, and the longest (L) and shortest diameters (S) of the tumors were recorded. The tumor volume was calculated according to the standard formula: tumor volume = 0.5 × L × S2. The following day, the nude mice were sacrificed by the cervical dislocation method. The tumor tissue was harvested and photographed, and the weight of the tumors was measured. The tumor was divided into two sections: one was used for immunochemistry, and the other was preserved using liquid nitrogen for standby use. Additionally, the heart, liver, spleen, lung, kidney, and jejunum of the mice were removed for HE staining.
Hematoxylin and eosin (H&E) and immunohistochemistry
The organs were fixed with 4% formaldehyde for 24 h, embedded in paraffin, cut into 4 pieces-µm thick sections, and dehydrated. The sections were then stained with H&E. In addition, some tumor slices were immunostained with the, Ki-67 (1:500) antibody, and p-Akt (1:100) antibody. Digital images were observed and captured under a standard optical microscope.
All experimental results were obtained from at least three independent experiments using GraphPad Prism 8.3 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the means ± standard deviation (SD). Student’s t-test was used to analyze data between groups, while one-way ANOVA was used to analyze the differences between multiple groups. Statistical significance was set at p < 0.05.