MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma.

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.

The Radiosensitizing Effect of AZD0530 in Glioblastoma and Glioblastoma Stem-Like Cells.

AZD0530, a potent small-molecule inhibitor of the Src kinase family, is an anticancer drug used in the treatment of various cancers. In the case of glioblastoma (GBM), where resistance to radiotherapy frequently occurs, Src kinase is known as one of the molecules responsible for imparting radioresistance to GBM. Thus, we evaluated the effect of AZD0530 on the radiosensitivity of human GBM cells and human glioblastoma stem-like cells (GSCs). We show that Src activity of GBM and GSC is increased by radiation and inhibited by AZD0530, and using clonogenic assays, AZD0530 enhances the radiosensitivity of GBM and GSCs. Also, AZD0530 induced a prolongation of radiation-induced γH2AX without specific cell cycle and mitotic index changes, suggesting that AZD0530-induced radiosensitization in GBM cells and GSCs results from the inhibition of DNA repair. In addition, AZD0530 was shown to inhibit the radiation-induced EGFR/PI3K/AKT pathway, which is known to promote and regulate radioresistance and survival of GBM cells by radiation. Finally, mice bearing orthotopic xenografts initiated from GBM cells were then used to evaluate the in vivo response to AZD0530 and radiation. The combination of AZD0530 and radiation showed the longest median survival compared with any single modality. Thus, these results show that AZD0530 enhances the radiosensitivity of GBM cells and GSCs and suggest the possibility of AZD0530 as a clinical radiosensitizer for treatment of GBM.

Combination radiotherapy in an orthotopic mouse brain tumor model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 µl every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.