Inhibition of GSH synthesis potentiates temozolomide-induced bystander effect in glioblastoma

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with poor prognosis. Current standard treatment includes chemotherapy using DNA alkylating agent temozolomide (TMZ) concomitant with surgical resection and/or irradiation. However, GBM patients exhibit various levels of the elevated expression of DNA repair enzyme, due to MGMT causing resistance to TMZ. Determination of the MGMT-positive population of primary tumor is important to evaluate the therapeutic efficacy of TMZ. Here we generated TMZ-resistant GBM cells by introducing MGMT into TMZ-sensitive GBM cell line KMG4, and established a model to assess the TMZ-induced bystander effect on TMZ-resistant cells. By mixing TMZ-resistant and -sensitive cells, GBM tumors with MGMT positivity as 50%, 10%, and 1% were generated in vivo. We could not observe any bystander effect of TMZ-induced cell death in tumor with 50% MGMT positivity. Although the bystander effect was observed within 20 days in the case of tumor with 1% MGMT positivity, final tumor size at day 28 was the same as control without sensitive cells. This bystander effect was observed in vitro using conditioned medium of TMZ-damaged GBM cells, and PCR array analysis indicated that the conditioned medium stimulated stress and toxicity pathway and upregulated anti-oxidants genes expression such as catalase and SOD2 in TMZ-resistant cells. In addition, the reduction of the activity of anti-stress mechanism by using inhibitor of GSH synthesis potentiated TMZ-induced bystander effect. These results suggest that GSH inhibitor might be one of the candidates for combination therapy with TMZ for TMZ-resistant GBM patients.

► The TMZ-induced bystander effect lasted only for a limited number of days.
► Only 1 % of TMZ-resistant cells could outgrow in the circumstance where the other 99 % cells were dead.
► Conditioned medium of TMZ-damaged GBM cells stimulated anti-oxidants genes expression in TMZ-resistant cells.
► An inhibitor of GSH significantly potentiated the TMZ-induced bystander effect.