Treatment with Bortezomib Sensitizes Glioblastoma Cells to Temozolomide
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Glioblastoma Multiforme (GBM) is the most frequent and aggressive primary brain cancer in adults. Despite multimodal treatment, prognosis remains dismal because efficacy is negligible in 50% of the patients with active, unmethylated DNA repair O6 methyl guanine DNA methytransferase (MGMT) gene promoter. There is an unmet urgent need of effective therapy for GBM patients. The current study investigated the in vitro effect of Bortezomib (BTZ) administered in monotherapy or in combination with Temozolomide (TMZ) chemotherapy in n=4 GBM cell lines and 1 normal human astrocyte (NHA) cell line. Bortezomib, a proteasome inhibitor has shown therapeutic potential in several cancers, however, the mechanism of action in GBM is not fully explored. Only one study demonstrated that Bortezomib downregulates MGMT expression, but its potential in sensitizing treatment resistant GBMs to TMZ chemotherapy has not been explored. We therefore hypothesized that BTZ might sensitize GBM cells to TMZ by inhibiting MGMT mediated DNA repair. MGMT promoter methylation status was investigated by methylation specific PCR and subsequently MTS cytotoxicity and clonogenic survival assays were undertaken. Finally, treatment response was evaluated by western blotting focusing on DNA damage signaling, autophagy and apoptosis cascades. The major findings of this work are that 1) BTZ sensitized GBM cells to TMZ chemotherapy; 2) improved efficacy by reducing the TMZ IC50 dose required and 3) depleted MGMT protein level to induce early DNA damage response and cell cycle arrest that was proceeded by caspase dependent apoptosis. This study provides careful dose and time course analysis of the treatment regimen that was critical in pinpointing induction of cell cycle arrest coinciding with autophagy in response to TMZ induced DNA damage at 48 hours. This was subsequently proceeded by caspase dependent apoptosis at 72 hours that concurred with MGMT depletion. Pre-treatment with Bortezomib potently sensitized the GBM cells to Temozolomide indicated by reduction of IC50 doses, rapid and stronger induction of DNA damage response, compared to monotherapy. These findings contribute substantially to the field by providing new mechanistic knowledge that warrants further investigation in vivo.
PublisherThe University of Bergen
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