Inhibition of glioblastoma growth and invasion by 125I brachytherapy in rat glioma model

Abstract

Development of the novel targeted therapies for glioblastoma multiforme is very important. Brachytherapy has been proven to provide a good alternative to surgical removal of the prostate, breast and cervix with reduced risk of certain long-term side effects. Thus, ¹²⁵I brachytherapy was used to effect on the growth and invasion of glioma cells in vitro and in vivo. The inhibitory effect of ¹²⁵I seeds on C6 cells proliferation was determined by MTT assay. A rat intracranial glioma model was established and the ¹²⁵I seeds were implanted into the glioma area. CD31 expression was determined by immunohistochemical method to evaluate the angiogenesis. The ΔΨm detection and cell invasion assays were performed to detect the mitochondrial-induced apoptosis and invasion signaling in tumor cells. ¹²⁵I brachytherapy could significantly inhibit C6 rat glioma cells growth and reduce cell viability in vitro. The seeds implantation also inhibited tumor growth in the rat glioma model and improved survival rate. Analysis revealed that ROS production and the intrinsic mitochondrial pathway of apoptosis was activated by ¹²⁵I brachytherapy. The HE staining results revealed that the rat glioma model treated with ¹²⁵I seeds exhibited better defined tumor margins and fewer invasive cells to the lateral striatum compared with the untreated group. The comparison of expression of CD31 in treated or untreated groups was performed to show that the ¹²⁵I brachytherapy has potential antiangiogenic activity. Meanwhile, ¹²⁵I brachytherapy can inhibit the growth and invasion of glioma cells via decreasing the expression of MMP-2 and MMP-9.

Keywords: 125I brachytherapy; apoptosis; glioblastoma; invasion; mitochondrion.

Figure 1

The implantation of ¹²⁵I seeds induced inhibition of cell growth and reduction of cellular viability in vitro. A: Morphologic change of the cells observed under an inverted light microscope (× 400). B: The treatment of ¹²⁵I seeds reduced cellular viability of rat C6 glioma cells in vitro. Viability of cells was assessed using the MTT assay. C: Long-term colony formation assays of C6 glioma cells. Cells were grown in the presence of drugs ¹²⁵I seeds for 7 days. For each cell line, all dishes were fixed at the same time, stained, and photographed. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.

Figure 2

¹²⁵I implantation effect on the tumor in the rat glioma model. A: CT verification of the positions of ¹²⁵I seeds and the area of the tumor. B: A magnification of the whole cross-section of the brain by HE staining. ¹²⁵I seeds-treated animals showed significantly smaller tumor area compared to untreated animals. C: Tumor volumes in mm³ of treated and untreated group. D: The survival rate of the animals treated with ¹²⁵I seeds compared with untreated animals. Data are presented as mean ± S.E.M. of five animals per group. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.

Figure 3

The implantation of ¹²⁵I seeds induced the mitochondria-dependent apoptotic pathway compared with the control. A: Western Blotting analysis of Bax, Bcl-2, pro-Caspase-3, Caspase-3, pro-Caspase-8, cleaved-Caspase-8, PARP, P53, β-Actin expression. B: The relative expression of Bax, Bcl-2, pro-Caspase-3, Caspase-3, pro-Caspase-8, cleaved-Caspase-8, PARP and P53. C: Apoptosis inducing property of the measured samples by Annexin V-FITC/PI staining of cancer cells. The Y-axis shows the PI-labeled population and the X-axis shows FITC-labeled Annexin V-positive cells. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.

Figure 4

Effects of ¹²⁵I brachytherapy on the mitochondria, ROS production and H₂O₂ level. A: Effects of ¹²⁵I brachytherapy on the mitochondrial membrane potential. C6 and U87 glioma cells were treated for 24 with ¹²⁵I seeds and stained with TMRM (10 nM). The fluorescence was estimated at 490 nm (excitation) and 590 nm (emission). B: ROS production in C6 and U87 glioma cells. Cells were pre-incubated in PBS/10 mM glucose medium for 20 min at 37°C in the presence of 10 mM CM-H₂DCFDA and then treated with ¹²⁵I seeds. The fluorescence of DCF was measured at 485 nm (excitation) and 527 nm (emission). C: Cells were exposed to ¹²⁵I seeds for 24 h, then H₂O₂ level was measured. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.

Figure 5

¹²⁵I brachytherapy induced inhibition of tumor invasion. A: Effect of ¹²⁵I brachytherapy on invasion of C6 and U87 glioma cells. B: Effect of ¹²⁵I brachytherapy on cell migration. C: The implantation of ¹²⁵I seeds reduced MMP-2, MMP-9 expression in treated groups. D: A magnification of the striatum with a representative part of the tumor by HE staining. The group treated with ¹²⁵I seeds exhibited better defined tumor margins and fewer invasive cells to the meninges compared with the untreated. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.

Figure 6

¹²⁵I brachytherapy suppressed the tumor angiogenesis. A: The expression of CD31 in control and treated groups was shown by immunohistochemical method (× 200). B: Microvessel counts (MVCs) were assessed to demonstrate the extent of the vascularization in the control and treated groups. Data are presented as the mean ± S.E.M., determined from three separate experiments. Error bars indicate SD. *P < 0.05; **P < 0.01.