Antitumor effect of fibrin glue containing temozolomide against malignant glioma

Abstract

Temozolomide (TMZ), used to treat glioblastoma and malignant glioma, induces autophagy, apoptosis and senescence in cancer cells. We investigated fibrin glue (FG) as a drug delivery system for the local administration of high-concentration TMZ aimed at preventing glioma recurrence. Our high-power liquid chromatography studies indicated that FG containing TMZ (TMZ-FG) manifested a sustained drug release potential. We prepared a subcutaneous tumor model by injecting groups of mice with three malignant glioma cell lines and examined the antitumor effect of TMZ-FG. We estimated the tumor volume and performed immunostaining and immunoblotting using antibodies to Ki-67, cleaved caspase 3, LC3 and p16. When FG sheets containing TMZ (TMZ-FGS) were inserted beneath the tumors, their growth was significantly suppressed. In mice treated with peroral TMZ plus TMZ-FGS the tumors tended to be smaller than in mice whose tumors were treated with TMZ-FGS or peroral TMZ alone. The TMZ-FGS induced autophagy, apoptosis and senescence in subcutaneous glioma tumor cells. To assess the safety of TMZ-FG for normal brain, we placed it directly on the brain of living mice and stained tissue sections obtained in the acute and chronic phase immunohistochemically. In both phases, TMZ-FG failed to severely damage normal brain tissue. TMZ-FG may represent a safe new drug delivery system with sustained drug release potential to treat malignant glioma.

Keywords: Drug delivery system; fibrin glue; glioblastoma; malignant glioma; temozolomide.

Figure 1

Dose-dependent effects of temozolomide (TMZ) on the growth of glioma cell lines. (a) Cell proliferation assay (n = 6). (▀), Control; (▵), 0.1 mM TMZ; (⋄), 0.4 mM TMZ; (•), 0.8 mM TMZ; (×), 1 mM TMZ. (b) Morphology of glioma cells incubated for 72 h with 1 mM TMZ and of the controls. Bar, 500 μm. K-GIC, Kumamoto-glioma-initiating cell.

Figure 2

High-performance liquid chromatography (HPLC) of the temozolomide (TMZ) released from fibrin glue (FG). (a) HPLC of the calibration curve standards. (b) HPLC of PBS incubated for 24 h with FG sheets containing TMZ (TMZ-FGS). (c) Time-course of the total amount of TMZ released from FG (n = 3). (d) Ratio of TMZ released from FG in the course of 168 h (n = 3). For analysis we used the unpaired two-tailed t-test. *P < 0.05. **P < 0.001. NS, not significant.

Figure 3

Growth curve and plots of the relative subcutaneous tumor volume. (●), Sham (group 1); (□), fibrin glue sheets (FGS) without temozolomide (TMZ) (FGS; group 2); (○), FGS containing TMZ (TMZ-FGS; group 3); (△), distilled water perorally (DW-PO; group 4); (♦), TMZ perorally (TMZ-PO; group 5); (×), TMZ-FGS plus TMZ perorally (TMZ-FGS-PO; group 6). (a) Growth curve (n = 6). (▽), FGS placement; (↓), oral administration of TMZ or distilled water; (▼), killed. (b) Plots of relative tumor volume in the three different TMZ-treated groups at the time the mice were killed (n = 6). Data were analyzed using anova. A P < 0.05 was considered statistically significant. *P < 0.05. **P < 0.001. (c) Images of mice bearing subcutaneous tumors induced with U87MG cells and the tumors removed at the time the mice were killed.

Figure 4

(On the previous page) Immunohistochemical findings on the subcutaneous tumors. (a) Immunohistochemical staining of subcutaneous tumors treated for 7 days with fibrin glue sheets (FGS) that did not (control) and did contain temozolomide (TMZ-FGS). Original magnification, ×400; bar, 50 μm. (b) Positivity ratios of the antibody reactions (n = 3; 10 fields per sample). Analysis was with the unpaired two-tailed t-test. A P < 0.05 was considered statistically significant. **P < 0.001. (c) H&E staining of subcutaneous tumors elicited by transplanted U87MG cells. The mice were treated for 7 days with FGS alone, peroral TMZ (TMZ-PO) or TMZ-FGS. The FGS was placed under the tumors. Similar findings were obtained when Kumamoto-glioma-initiating cell 1 (K-GIC1) or K-GIC2 cells were transplanted (data not shown). Low magnification; bar, 5 mm.

Figure 5

Western blot analysis of subcutaneous tumors. (a) Western blots of cleaved caspase 3, LC3-I, LC3-II, p16 and β-tubulin in subcutaneous tumors treated for 7 days with fibrin glue sheets (FGS) alone (control) or temozolomide (TMZ-FGS) (n = 3). (b) Graphs of the intensity of the bands. Analysis was with the unpaired two-tailed t-test. A P < 0.05 was considered statistically significant. *P < 0.05. **P < 0.001.

Figure 6

Adverse effects of fibrin glue sheets containing temozolomide (TMZ-FGS) on normal mouse brain. Effects in the acute (a) and chronic (b) phase are shown. Left, sham; middle, FGS alone; right, FGS containing 1 mM TMZ. Low magnification; bar, 5 mm.