Introduction: Recent progress in chemotherapy for malignant gliomas was led by DNA-methylating agent temozolomide (TMZ), however, the drug resistance is still remained. Previous studies revealed that TMZ induced prolonged G2/M arrest of human glioma cells followed by senescence-like phenomenon or mitotic catastrophe, and suggested that G2 checkpoint system might be linked to DNA repair mechanism.
Methods: We tested the effect of flavopiridol (FP) which has been known to inhibit the action of cdc2, a key protein in G2 checkpoint pathway, on TMZ-treated U87MG human glioma cells.
Results: The drug toxicity and activation of cell signalling proteins was evaluated by colony formation efficiency and immunoblotting, respectively. Furthermore, to analyze the action of cdk inhibitor in TMZ-resistant cells, we treated U87MG cells repeatedly with dose escalation to establish TMZ-resistant clones, and used U87MG cells transfected with fusion protein of myristoylated Akt and murine estrogen receptor of which Akt activity can be enhanced by exogeneous treatement with estrogen analogue. FP potentiated the cytotoxicity of TMZ at a lower concentration. This effect was clearly associated with suppression of key proteins at G2-M transition (Plk1, Aurora and Pin1), as well as with accumulation of the cells exclusively at G2 phase. Furthermore, FP enhanced both TMZ-induced g-H2AX expression in U87MG cells and cytotoxity of TMZ on U87MG-AktER cells with over-activated Akt. We established TMZ-resistant clones which showed activation of G2 checkpoint in response to TMZ, and found that FP treatment re-sensitized these clones to TMZ. In vivo, glioma cells transplanted subcutaneously into mice were markedly reduced by combination of TMZ and FP in comparison to the TMZ alone.
Conclusions: Our results suggest that TMZ resistance could be promoted by enhanced DNA repair activity in G2-M transition, and that cdk inhibitor could suppress this activity leading to potentiation of TMZ action on glioma cells.
Patient Care: Flavopiridol enhances will save the patients with temozolomide-resistant glioblastoma.
Learning Objectives: To learn about pharmacologic inhibition of glioma cells.