Enhanced MGMT expression contributes to temozolomide resistance in glioma stem-like cells

Abstract

O6-methylguanine DNA methyltransferase (MGMT) can remove DNA alkylation adducts, thereby repairing damaged DNA and contributing to the drug resistance of gliomas to alkylating agents. In addition, glioma stem-like cells (GSCs) have been demonstrated to be involved in the recurrence and treatment resistance of gliomas. In this study, we aimed to investigate MGMT expression and regulatory mechanisms in GSCs and the association of MGMT with temozolomide (TMZ) sensitivity. GSCs were enriched from one MGMT-positive cell line (SF-767) and 7 MGMT-negative cell lines (U251, SKMG-4, SKMG-1, SF295, U87, MGR1, and MGR2) through serum-free clone culture. GSCs from the U251G, SKMG-4G, SF295G, and SKMG-1G cell lines became MGMT-positive, but those from the U87G, MGR1G, and MGR2G cell lines remained MGMT-negative. However, all the GSCs and their parental glioma cell lines were positive for nuclear factor-κB (NF-κB). In addition, GSCs were more resistant to TMZ than their parental glioma cell lines (P < 0.05). However, there was no significant difference in the 50% inhibition concentration (IC50) of TMZ between MGMT-positive and MGMT-negative GSCs (P > 0.05). When we treated the MGMT-positive GSCs with TMZ plus MG-132 (an NF-κB inhibitor), the antitumor activity was significantly enhanced compared to that of GSCs treated with TMZ alone (P <0.05). Furthermore, we found that MGMT expression decreased through the down-regulation of NF-κB expression by MG-132. Our results show that MG-132 may inhibit NF-κB expression and further decrease MGMT expression, resulting in a synergistic effect on MGMT-positive GSCs. These results indicate that enhanced MGMT expression contributes to TMZ resistance in MGMT-positive GSCs.

Figure 1.. Characteristics of glioma stem-like cells (GSCs).

A, GSCs grow as neurosphere-like gliomaspheres in stem cell medium (original magnification × 20). B, GSCs adhered to poly-lysine-coated plates when cultured in 10% FBS/DMEM/F12 medium (original magnification × 100). C, the expression of neuronal class III beta-tubulin-1 (TUJ-1), a neuronal marker on the GSC sphere (original magnification × 200). D, the expression of glial fibrillary acidic protein (GFAP), an astrocytic marker in the GSC sphere (original magnification × 200). E-G, immunostaining of tumor cells for neural stem cell markers, Nestin (E), CD133 (F), and Sox-2 (G), in the GSC sphere (original magnification × 200).

Figure 2.. O⁶-methylguanine DNA methyltransferase (MGMT) and nuclear factor-κB (NF-κB) expression in the GSC lines and the parental glioma cell lines.

A, reverse transcription-polymerase chain reaction (RT-PCR) shows that the glioma cell lines U251, SKMG-4, SF295, SKMG-1, U87, MGR1, and MGR2 are MGMT-negative, whereas the SF767 cell line is MGMT-positive. However, the GSC lines U251G, SKMG-4G, SF295G, SKMG-1G, and SF767G become MGMT-positive, whereas the U87G, MGR1G, and MGR2G lines remain MGMT-negative. Meanwhile, all GSC lines and their parental glioma cell lines displayed high NF-κB expression. B, the Western blotting data are consistent with the RT-PCR results. C, methylation-specific polymerase chain reaction (MSP) shows MGMT promoter methylation in all GSC lines and their parental glioma cell lines except for the SF767 and SF767G lines. In addition, unmethylated MGMT promoters exist in the glioma cell line SF767 and the GSC lines SF767G, U251G, SKMG-4G, SKMG-1G, and SF295G; H₂O was used as the blank control.

Figure 3.. The relationship between MGMT expression and resistance to temozolomide (TMZ ) in GSCs.

A, the 50% inhibition concentrations (IC₅₀) of TMZ for the GSC lines are significantly higher than those for the parental glioma cell lines (**P < 0.05). B, the mean IC₅₀ of TMZ for all GSC lines is significantly higher than that for all parental glioma cell lines (**P < 0.05). C, the IC₅₀ of TMZ for MGMT-positive and MGMT-negative GSC lines are similar (P > 0.05).

Figure 4.. Inhibiting NF-κB expression results in the down-regulation of MGMT expression in the GSC line U251G.

RT-PCR (upper panel) and Western blotting (lower panel) data show that TMZ treatment slightly down-regulated MGMT expression but did not affect NF-κB expression. MG-132 alone or in combination with TMZ down-regulated both NF-κB and MGMT expression. T+M, TMZ plus MG-132.