NF-κB inhibitor reverses temozolomide resistance in human glioma TR/U251 cells

Abstract

Glioblastoma multiforme (GBM) demonstrates an unsatisfactory clinical prognosis due to the intrinsic or acquired resistance to temozolomide (TMZ) exhibited by the tumors. One possible cause of TMZ resistance in GBM is the overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT), which can repair the TMZ-induced guanine damage in DNA. Additionally, excessive activated NF-κB is reported to be a component of the major inflammatory transcription pathway that is associated with TMZ resistance in GBM. However, the association between the NF-κB pathway and MGMT expression in GBM cells is unknown. Therefore, in the present study, the TMZ resistant (TR) U251 cell line (TR/U251) was successfully constructed to detect how the TR/U251 cell line and the parental U251 cell line each interact with TMZ in vitro. The TR/U251 cells were approximately five times more resistant to TMZ compared with the parental cells. Furthermore, it was found that the NF-κB inhibitor BAY 11-7082 suppressed the expression of MGMT in TR/U251 cells and enhanced TMZ-induced cytotoxicity and apoptosis, thereby indicating that the NF-κB pathway and MGMT interact to promote TMZ resistance. The inhibition of NF-κB may be a promising strategy to reverse drug resistance in TR glioma cells. The present results propose a potential mechanism for using the NF-κB inhibitor BAY 11-7082 as a potential therapy for the treatment of TR glioma. Although BAY 11-7082 is a well-known NF-κB inhibitor, the present study further investigated its underlying mechanisms through a series of new experiments.

Keywords: O6-methylguanine-DNA methyltransferase; glioblastoma multiforme; nuclear factor-κB.

Figure 1.

Survival rate of U251 and TR/U251 cells treated with various concentrations of TMZ. A dose-dependent association between the survival rate of U251 and TR/U251 cells and TMZ concentration can be observed. The survival of U251 cells was significantly lower compared with TR/U251 cells following treatment with various concentrations of TMZ. The IC₅₀ of TMZ in U251 cells is 57.58±7.60 µM, while the IC₅₀ of TMZ in TR/U251 cells is 270.78±27.29 µM. TMZ, temozolomide; TR, TMZ-resistant; IC₅₀, 50% inhibiting concentration.

Figure 2.

Expression of MGMT, Bcl-2 and p65 in the BAY 11-7082, TMZ and combination treatment groups. A high expression of MGMT was found in the TR/U251 cells, while expression was hardly detected in the U251 cells. Similarly, the levels of phosphorylated-P65 were increased in the TR/U251 cells compared with the U251 cells. No statistical difference was found in the NF-κB expression, assessed through the detection of the p65 subunit, between the two cell types. The TR/U251 cells demonstrated a ~5.4-fold higher level of phosphorylated-P65 compared with the U251 cells. MGMT was hardly detected when cells were cultured with a combination of TMZ and BAY 11-7082. A higher level of MGMT was detected in the cells treated with TMZ or BAY 11-7082 separately. The expression of Bcl-2 and Bax was reversed only in the cells treated with a combination of TMZ and BAY 11-7082. TMZ, temozolomide; TR, TMZ-resistant; MGMT, O⁶-methylguanine-DNA methyltransferase; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; p-p65, phosphorylated p65.

Figure 3.

Survival rate at various concentrations of BAY 11-7082. A dose- and time-dependent association between the survival rate of the TR/U251 cells and BAY 11-7082 concentration. A longer incubation time with BAY 11-7082 significantly decreased the survival rate of TR/U251 cells, and the same was found for treatment with a higher concentration of BAY 11-7082. TMZ, temozolomide.

Figure 4.

The survival rate of TR/U251 cells in each group. The survival rate of the TR/U251 cells slowly decreased between 97.6±1.4 and 78.3±3.6% when exposed to 80 µM TMZ for 24–96 h. While exposed to 50 µM BAY 11-7082, the viability of the cells decreased between 97.9±2.3 and 64.2±4.4%. The survival rate markedly decreased between 91.6±1.9 and 27.8±5.7% when the cells were exposed to 80 µM TMZ in combination with 50 µM BAY 11-7082 (P<0.01). The data are presented as the mean±standard deviation. TMZ, temozolomide; TR, TMZ-resistant; DMSO, dimethyl sulphoxide.

Figure 5.

Changes in the morphology of cells treated as a control, or with TMZ, BAY 11-7082 or a combination of the two, as shown by immunity fluorescence. This revealed that the cells in the control group demonstrated a stable morphology, while in the BAY 11-7082 + TMZ-treated group, the cells were completely lysed; the cells had burst and the normal shape was lost. TMZ, temozolomide.