Peimine was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Unless otherwise stated, all compounds were dissolved in DMSO. Procell Life Science & Technology Co., Ltd. provided the human GBM cell lines U87 (cat. no. CL-0238) and U251 (cat. no. CL-0237), in addition the normal human brain glial cell line HEB (cat. no. CL0130). The U87 cell line used was the U87-MG ATCC (CVCL 0022) cell line (HTB-14) and was authenticated using STR analysis. U87, U251, HEB and GL261 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.), with high glucose and high glutamine levels. All cells were cultured at 37˚C in a humidified incubator supplied with 95% air and 5% CO₂.

In total, 8x10³ U87, U251 and HEB cells were plated in 96-well plates and incubated for 24 h at 37˚C. Following incubation, AKT activator SC79 (10 µM), AKT inhibitor MK2206 (10 µM) or peimine (0 or 25 µM) were added to the wells. The treatment time was 1, 24 and 24 h at 37˚C, respectively. Subsequently, 10 µl MTT solution was added to each well and incubated for 4 h at 37˚C. To dissolve the formazan crystals, 100 µl DMSO was added. Next, the plates were vortexed at room temperature for 10 min. The Bio-Tek ELX800 Multi-Mode Reader (BioTek Instruments, Inc.) was used to measure absorbance at 490 nm.

Trypsin was used to lift U87 cells during the logarithmic growth phase. The cell suspension was then resuspended using 1 ml DMEM and counted. In a 6-well plate, 1,000 cells were then plated in each well for each experimental group. The cells were cultured for 14 days at 37˚C or until the majority of the single clones had ≥50 cells. Every 3 days, the medium was changed and the condition of the cells was routinely checked. After the 14-day growth period, cells were once again washed with PBS and imaged under a microscope. After fixing each well for 30 min with 1 ml 4% paraformaldehyde at 26˚C, the cells were washed again with PBS. The cells were then stained for 20 min with 1 ml at 26˚C crystal violet staining solution. Subsequently, the cells were washed several times with PBS at 26˚C, dried and imaged using a digital camera (16).

In 6-well plates, U87 cells in the logarithmic growth phase were trypsinized, before 3x10⁵ were resuspended and seeded into each well. Cells were cultured until the confluence reached ~95% at 37˚C, then a 200-µl pipette tip was used to create a wound in the monolayer of cells. The culture media was removed from the wells, the monolayer of cells was washed twice and then 0, 25 or 50 µM of 2 ml peimine solution was added. Images of wound closure were captured at 0 and 24 h. After 24 h at 37˚C, the culture media was aspirated, the wells were washed twice with PBS and then imaged using an X71 fluorescence microscope (Olympus Corporation).

Using the Transwell inserts (0.4-µm pore size; Corning, Inc.), the effects of peimine on the invasion and migration of U87 cells were evaluated. For migration assays, a total of 5x10³ cells per well in 200 µl serum-free media was added into the upper chamber of a Transwell insert in a 24-well plate. A total of 600 µl DMEM containing the 0, 25 or 50 µM peimine was added to the lower chamber. For invasion assays, Matrigel (Corning, Inc.) diluted 1:8 (60 µl) was first added to the upper chamber of the insert and once it had solidified on a cell culture incubator for 1 h at 37˚C, cells were added to the upper chamber and media was added to the lower chamber in the same manner as the migration assay. Cells were incubated for 24 h at 37˚C, before cells that had passed through the Transwell membrane were fixed for 20 min using 4% paraformaldehyde at 26˚C and then stained for 20 min using 1% crystal violet at 26˚C. Images were taken using an X71 fluorescent microscope (Olympus Corporation) and data was analyzed with Image J software (version 1.53e; National Institutes of Health).

The production of ROS is inevitable for aerobic organisms, which occurs at a controllable rate in healthy cells. Under oxidative stress conditions, ROS production increases sharply, causing changes in membrane lipids, proteins and nucleic acids. The oxidative damage of these biomolecules is associated with cancer. Dichloro fluorescein (DCF), the fluorescent oxidized product of 2',7'-dichlorofluorescin diacetate (DCFDA; MilliporeSigma), was used to measure intracellular ROS levels. First, 1.5x10⁵ U87 cells were plated into 6-well plates and pre-treated for 6 h at 37˚C with either DMEM or 3 mM N-acetyl-L-cysteine (MilliporeSigma). Peimine (0, 25 or 50 µM) was then added to the media and cells were treated for a further 8 h at 37˚C, after which the media was replaced with fresh complete media containing 2 µg DCFDA for 6 min at 37˚C in the dark. Using an X71 fluorescent microscope, the proportion of apoptotic cells was determined.

Membrane permeability JC-1 staining is widely used in apoptosis studies to monitor mitochondrial health. JC-1 staining reagent showed potential-dependent accumulation in mitochondria. At low concentration, it existed as monomer and produced green fluorescence at ~529 nm. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Biosharp Life Sciences) was selected as the positive control. U87 cells (2.5x10⁴) were treated with 0, 25 or 50 µM peimine for 24 h at 37˚C. Next, cells were collected, centrifuged for 5 min at 26˚C (1,400 x g) and resuspended in PBS. CCCP (10 mM) was added to the cell culture medium at a ratio of 1:1,000, diluted to 10 µM, and the cells were treated with 200 µl for 20 min at 37˚C. The cells were then centrifuged again, resuspended in binding buffer and incubated for 30 min at 37˚C in the dark with 200 µl the JC-1 probe (Biosharp Life Sciences). Cells were then resuspended in 500 µl pre-cooled PBS and rinsed twice. An X71 fluorescent microscope was used to calculate the proportion of apoptotic cells.

U87 cells (2.5x10⁴) in 24-well plates (Costar; Corning, Inc.) were treated with 0, 25 or 50 µM of peimine for 24 h at 37˚C. Cells were then harvested, centrifuged for 5 min at 26˚C (1,400 x g) and resuspended in PBS. After re-suspending in binding buffer, 1.5x10⁵ U87 cells were stained using the Annexin V-FITC/PI kit (cat. no. BL1884A; Biosharp Life Sciences) for 20 min at 37˚C. The percentage of apoptotic cells was then determined using an X71 fluorescence microscope. Staining quality [apoptotic cells were double positive (Annexin V⁺/PI⁺) by FITC and PI binding staining] was analyzed by Image J software (version 1.53e; National Institutes of Health).

Hubei University of Science and Technology Animal Ethics Committee approved the animal experiments (approval no. 2022-11-027) and all procedures adhered to national and international standards for the care and use of animals in research. GL261 GBM cells (cat. no. CTCC-400-0404; Zhejiang Meisen Cell Technology Co., Ltd.) were used to establish the mouse model. In total, 5-week-old male C57BL/6 mice (n=3/group; total, n=9) weighing 20-24 g were selected for the study. GL261 cells were subcutaneously injected into the right back of each mouse with 5x10⁶ cells in 100 µl sterile PBS. After the tumor reached a mean volume of ~100 mm³, mice were divided into the following three groups at random: Control; low-dose (20 mg/kg); and high-dose (40 mg/kg) (17). Body weights were recorded daily and tumor volumes were measured every 2 days using the following formula: Volume=a x b² x 0.52, where a is the length and b is the width in mm. Mice in the low-dose and high-dose groups were treated intragastrically every 2 days with 20 or 40 mg/kg peimine, respectively. Control mice received saline instead of peimine. The mice were sacrificed under anesthesia (intraperitoneal injection of sodium pentobarbital 100 mg/kg) after 14 days. Death was confirmed by a lack of response to a toe pinch, lack of breathing and lack of heartbeat. Samples from the tumors and livers in the animals were then collected for additional examination.

Liver tissues from mice were fixed using 4% paraformaldehyde for 24 h at 26˚C, dehydrated and embedded in paraffin. The samples were fixed in 10% formalin for 12 h at 4˚C, and then embedded in paraffin for subsequent sectioning. The 4-µm specimens were first placed in distilled water and then in an aqueous solution of hematoxylin for staining for ~10 min at room temperature. After which, the slices were placed into ammonia and acid water for several seconds, and then rinsed in running water for 1 h. The sections were placed in distilled water for several seconds, after which they were dehydrated in alcohol at concentrations of 90 and 70% for 10 min each. Subsequently, the sections were stained with eosin staining solution for 2-3 min at room temperature. After staining, the samples were dehydrated with 100% alcohol and placed in xylene. The sections were sealed and placed in an incubator for drying. H&E staining (cat. no. BL700A; Biosharp Life Sciences) was performed on the liver tissues for toxicological analysis using light microscropy.

Treated U87 cells were first collected, washed three times with ice-cold PBS and then lysed using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors (Beyotime Institute of Biotechnology) for 30 min. The resultant lysate was centrifuged and the supernatant was collected (4˚C, 15 min and 14,000 x g). A BCA assay kit (Beyotime Institute of Biotechnology) was used to measure the protein content according to the manufacturer's protocol. Loading buffer was added to the protein samples and then boiled at 100˚C for 10 min. Next, the protein samples (40 µg/lane) were loaded on 8-10% SDS gels, resolved using SDS-PAGE, transferred onto PVDF membranes and the membranes were blocked (5% skimmed milk for 1 h at 26˚C). Following blocking, the membranes were incubated with the indicated primary antibodies overnight at 4˚C. The following day, the membranes were washed, and incubated with the HRP-conjugated secondary antibodies at 26˚C. Signals were visualized using ECL plus reagent (Beyotime Institute of Biotechnology). GAPDH was used as the loading control. The following antibodies were used: p53 (1:1,000; cat. no. R380701), Bax (1:1,000; cat. no. R22708), Bcl-2 (1:1,000; cat. no. R23309), Caspase 3 (1:1,000; cat. no. R23315), Cleaved-Caspase 3 (1:1,000; cat. no. 341034) (all Chengdu Zen-Bioscience Co., Ltd.), phosphorylated (p)-PI3K (1:1,000; cat. no. AP1463), PI3K (1:1,000; cat. no. A19742), p-AKT (1:1,000; cat. no. AP1259), AKT (1:1,000; cat. no. A17909), GAPDH (1:1,000; cat. no. 390035) and HRP Goat Anti-Mouse IgG (1:1,000, cat. no. AS003) (all ABclonal Biotech Co., Ltd.). The blot densities were quantified using ImageJ software (version 1.53e; National Institutes of Health).

Experimental data were analyzed by Image J (version 1.53e) and SPSS 27.0 software (IBM Corp.). GraphPad prism 8.0 software (Dotmatics) was used for visualization. Data are presented as the mean ± SD. Data were analyzed using a one-way ANOVA followed by Tukey's post hoc test using SPSS 27.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

MTT assays were performed to assess the viability of HEB, U87 and U251 cells following treatment with various concentrations of peimine (0, 6.25, 12.5, 25, 50 or 100 µM; Fig. 1B-D). The results showed a dose-dependent reduction of GBM cell viability caused by peimine. Compared with the control group, with the increase of drug concentration, GBM cell viability was inhibited to an marked degree, but there were no obvious effects of peimine on HEB cells (Fig. 1B). In U87 and U251 cells, the IC₅₀ values were found to be 21.3 and 92.8 µM, respectively after 48 h of treatment. By contrast, the IC₅₀ value was 39.9 µM in U87 cells treated for 24 h. As the IC₅₀ of U87 cells is much lower than that of U251 cells, for subsequent experiments, 25 and 50 µM peimine was used for U87 cells based on the IC₅₀ value, with a treatment duration of 24 h.

A colony formation assay was next used to assess the proliferation of U87 cells. Treatment of U87 cells with varying concentrations of peimine resulted in a dose-dependent decrease in colony formation. Notably, significant differences in colony formation ability were observed in cells treated with 25 and 50 µM peimine compared with that in cells treated with 0 µM peimine, demonstrating that peimine had an inhibitory effect on the growth of GBM cell colonies (Fig. 1E and F). These findings suggest the inhibitory effects of peimine on GBM cell proliferation, with minimal toxicity and side effects observed in HEB cells. This suggests that peimine exhibits selectivity for cancerous cells whilst maintaining a safe profile on normal cells, highlighting it as a promising candidate for further drug development.

Results from wound healing assays showed significant inhibition of U87 cell migration following 24 h of treatment with 25 and 50 µM peimine (Fig. 2A and B), where peimine dose-dependently reduced migration compared with that in the control group.

Similarly, Transwell assay results showed significantly reduced U87 cell migration and invasion following the 24 h peimine treatment in a dose-dependent manner (Fig. 2C and D). These results were consistent with the results of the wound healing assays. Together, these results suggest the inhibitory effect of peimine on the invasion and migration of GBM cells.

The possible inhibitory effects of Peimine on GBM in vivo was assessed using a mouse xenograft model. Tumor size, volume and weight were found to be markedly reduced in both the low (20 mg/kg) and high (40 mg/kg) dose groups compared with those in the control group (Fig. 3A-C). Peimine did not considerably alter the body weight of the mice, where at therapeutic doses, no negative side effects (such as marked weight loss) were noted (Fig. 3D-F). Additionally, as shown in Fig. 3E and F, there were no marked variations in the liver/body ratio, liver morphology or the sectional tissue morphology between the peimine-treated group and the control group. These results suggest that peimine can effectively reduce GBM growth in vivo whilst maintaining favorable safety profiles, highlighting its potential as a therapeutic option for the treatment of GBM.

ROS serves a pivotal role in apoptosis, the excessive accumulation which in cells can induce apoptosis. As shown in Fig. 4A and B, based on DCFH-DA staining. Quantification of fluorescent images revealed a significant increase in cellular ROS levels induced by peimine, indicative of increased oxidative damage. The mitochondrial membrane potential is typically altered during early apoptosis. The JC-1 probe was therefore next used to measure this change (Fig. 4C). CCCP, an apoptosis inducer serving as a mitochondrial oxidative phosphorylation uncoupling agent, was used as a positive control to denote a reduction of the mitochondrial membrane potential. As the peimine concentration increased, the quantity of JC-1 monomers (green) in GBM cells was increased, whilst that of JC-1 aggregates (red) was decreased, suggesting the induction of early apoptosis. Furthermore, U87 cell apoptosis induction was assessed further using Annexin V-FITC/PI staining (Fig. 4D and E). These results collectively suggest that peimine can induce apoptosis in GBM cells.

Western blot analysis was used to assess the effect of peimine on the expression of proteins associated with apoptosis in U87 cells (Fig. 5A and B). Following 24 h treatment with 25 and 50 µM peimine, Bcl-2 expression was found to be downregulated in a dose-dependent manner, whereas p53 and Bax expression were found to be upregulated. Furthermore, with 25 and 50 µM peimine, there was an increase in Cleaved-Caspase 3 expression.

Furthermore, to elucidate the molecular mechanism underlying peimine-mediated repression of proliferation and metastasis, the PI3K/AKT signaling cascade was next investigated, also using western blot analysis. The results showed that after treating U87 cells with peimine for 24 h, the levels of PI3K and AKT phosphorylation were found to be significantly decreased, in a dose-dependent manner (Fig. 5C and D).

To determine whether peimine can mediate apoptosis through AKT signaling, AKT signaling was activated or inhibited in cells. Rescue experiments were first conducted using 10 µM SC79, an AKT agonist. The results showed that the proliferative effects induced by peimine (25 µM) over 24, 48 and 72 h were nullified following pretreatment with SC79 for 1 h (Fig. 5E); however, this nullification was not significant. To further explore the mechanism of peimine on AKT signaling in vitro, the relationship between peimine activity and AKT pathway was explored using 10 µM MK2206, an AKT inhibitor (Fig. 5F). The combination of peimine and AKT inhibitor MK2206 had a stronger effect on the proliferation of U87 cells than peimine alone. Together, these results highlight the role of the PI3K/AKT signaling cascade in mediating the anti-tumor activities in GBM cells of peimine.