Transforming Growth Factor‐beta Promotes Glioblastoma Multiforme to Establish Radiation Resistance by Mesenchymal Differentiation

Abstract

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current standard therapy is surgery combined with radio‐therapy and/or chemo‐therapy. Due to the existence of resistance, GBM is one of the most difficult‐to‐treat tumor. It has been shown radio‐therapy may induce mesenchymal differentiation (MD). Transforming Growth Factor‐beta (TGF‐β) is a modifier of radiation response, and plays an important role in cell transformation. Therefore, we hypothesized that TGF‐β pathway might be involved in the development of radiation resistance via regulating MD.Thus, we explored whether TGF‐β regulates MD in radiation resistance in GBM. We first examined the effect of radiation doses on cell viability by Trypan blue assay, and evaluated cell viability and morphology. The cells showed morphology change were defined as transformed cells. We used the radiation dose of 50% cell viability to establish consecutive‐irradiation‐induced resistance. The radiation dose of the cell viability greater than 50% was established high‐dose‐irradiation‐induced resistance positive control. When viability of cells recovered from consecutive‐irradiation exposures which were higher than positive control, the cells were defined as consecutive‐irradiation‐induced resistance. To develop radiation resistance, 3.5Gy exposure for 6 times were required for 1306MG, and 2Gy exposure for 4 times were needed for U87MG. The numbers of transformed cells increased in radiation resistant cells population. Moreover, immunocytochemistry staining showed expression of certain MD markers on transformed cells were different from on non‐transformed cells. Immunoblotting results showed increased mesenchymal marker N‐cadherin and decreased epithelial marker beta‐catenin in radiation resistance. Finally, we used TGF‐β and TGF‐β receptor inhibitor LY364947 to determine if TGF‐β/Smad pathway regulates radiation‐induced resistance. The immunoblotting results revealed that inhibition of TGF‐β pathway reversed MD in consecutive‐irradiation‐induced resistant cell lines via increasing the expression of β‐catenin and reducing the expression of N‐cadherin and Fibronectin. We concluded that TGF‐β promotes GBM to establish radiation resistance by MD.