Fig 2: Proposed model for mechanism of matrine activity on human GBM cells. Matrine suppresses GBM cell growth and invasion by inhibiting PI3K/AKT/p27 signaling and inducing senescence. Decreased IGF1 in matrine‐induced senescent cells enhances response to matrine

Fig 3: Matrine inhibits growth and induces cellular senescence of U251 GBM cells in vivo. A, U251 cells expressing luciferase were orthotopically implanted into athymic nude mice. Tumor growth was monitored using the IVIS‐200 imaging system for detection of bioluminescence. Bioluminescent signals were measured at days 7, 14, 21, and 28 after implantation. B, Bioluminescence values plotted as a function of time in days to assess tumor growth (days 7, 14, 21, and 28). Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group. C, Overall survival as determined by Kaplan‐Meier survival curves. A log‐rank test was used to assess the statistical significance of the differences (P < 0.05). D, P3 cells expressing luciferase were orthotopically implanted into athymic nude mice, and tumor growth was monitored using the IVIS‐200 imaging system for detection of bioluminescence. Bioluminescent signals were measured at days 7, 14, 21, and 28 after implantation. E, Bioluminescence values plotted as a function of time in days to assess tumor growth (days 7, 14, 21, and 28). Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group. F, Overall survival as determined by Kaplan‐Meier survival curves. A log‐rank test was used to assess the statistical significance of the differences (P < 0.01). G, Graphic representation of percentage of Ki67, H2AX, and p27‐positive cells in control and matrine‐treated U251 xenografts as determined with immunohistochemistry. Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group. H, Graphic representation of percentage of Ki67, H2AX, and p27‐positive cells in control and matrine‐treated P3 xenografts as determined with immunohistochemistry. All data are expressed as the mean ± the SD of values from experiments performed in triplicate. Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group

Fig 4: Matrine inhibits the PI3K/AKT/p27 signaling pathway. A, Western blot analysis for PI3K, AKT, pAKT, and GAPDH protein levels in protein lysates (20 μg) prepared from U251 and U87 cells treated with matrine for the number of days indicated. B, Western blot analysis to detect levels of AKT, pAKT, P27, and GAPDH in lysates prepared from U251 and U87 cells treated with matrine (0.2 mmol/L), AKT activator SC79 (5 μg/mL), or PI3K inhibitor LY294002 (1 mmol/L) 72 h. C, In situ SA‐β‐gal assay to detect senescent cells. Cells were treated with 0.2 mmol/L matrine and AKT activator SC79 for 72 h. Scale bars = 20 μm. D, Graphic representation of the percentage of SA‐β‐gal‐positive cells determined in four random fields per sample. All data are expressed as the mean ± SD of values from experiments performed in triplicate. Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group or between two treated groups

Fig 5: Matrine induces cellular senescence by upregulating p27. A, Western blot analysis for cell cycle‐associated proteins in lysates prepared from U251 and U87 cells treated with matrine for the indicated number of days. The immunoblots are representative of at least two independent experiments with GAPDH serving as a protein loading control. B, Western blot analysis of p27, CDK4, and CDK6 expression in U251 and U87 cells transfected with p27 siRNA for 48 h. C, In situ SA‐β‐gal assay to detect senescent cells. Cells were treated with 0.2 mmol/L matrine or p27 siRNA for 72 h and compared to controls. Scale bars = 20 μm. D, Graphic representation of the percentage of SA‐β‐gal‐positive cells determined in four random fields per sample. All data are expressed as the mean ± the SD of values from experiments performed in triplicate. Statistical analyses were performed using permutation test. P‐value came from the comparison between treated group and control group or between two treated groups