(Z)-Butylidenephthalide Restores Temozolomide Sensitivity to Temozolomide-Resistant Malignant Glioma Cells by Downregulating Expression of the DNA Repair Enzyme MGMT

Temozolomide, an alkylating agent against high-grade astrocytomas such as GBM (Glioblastoma Multiforme), is the leading compound in a new class of antitumor drugs. Altered methylation by MGMT (O6-methylguanine-DNA-methyltransferase) leads to temozolomide-resistant GBM. Thus, inhibition of MGMT activity is necessary for temozolomide effectiveness. Here we show a synergistic cytotoxic effect on the GBM cell line 8901 with a low dose of (Z)-butylidenephthalide combined with temozolomide. Using CalcuSyn software to analyze the combination index of (Z)-butylidenephthalide and temozolomide, we observed significant synergism in decreasing MGMT expression. We next investigated the effect of (Z)-butylidenephthalide on a temozolomide-resistant cell line GBM22-TMZ and examined changes in MGMT. The combination of 50 μM or 100 μM (Z)-butylidenephthalide with 1,600 μM temozolomide yielded a combination index of 0.649 or 0.581 (an index < 1 indicates synergism), indicating that increasing (Z)-butylidenephthalide enhanced the synergism. In addition, (Z)-butylidenephthalide enhanced MGMT promoter methylation and decreased MGMT expression in a dose-dependent manner. Using a GBM22-TMZ xenograft tumor model, (Z)-butylidenephthalide combined with temozolomide showed significant antitumor effects compared with a single drug. Finally, a survival study with Kaplan-Meier statistical analysis demonstrated a significant benefit of the (Z)-butylidenephthalide/temozolomide combination compared to temozolomide alone. In summary, the combination of (Z)-butylidenephthalide and temozolomide shows potential clinical relevance for treating temozolomide-resistant GBM.