Abstract
BackgroundResistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ.MethodsCell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan–Meier method.ResultsPre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood–brain barrier, diminished proteasome activity and significantly prolonged animal survival.ConclusionBTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.
Highlights
Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter
We demonstrated that BTZ administered 48 h prior to this effective TMZ dose crosses the blood–brain barrier (BBB), depleted MGMT mRNA levels and attenuated proteasome activity in vivo, accounting for restored TMZ sensitivity and prolonged animal survival
GBM cells are most sensitive to BTZ We first investigated the responses of patient-derived GBM primary cells and cell lines of different genetic backgrounds (Supplementary Table I) to monotherapy with the alkylating agent TMZ
Summary
Glioblastoma (GBM) is the most frequent and lethal primary brain malignancy in adults. Been shown to reduce levels of MGMT in T98G cells in vitro, which harbour a partially unmethylated MGMT promoter and further shown to induce apoptosis in these cells when administered prior to TMZ12,13 associated with activation of NFκB, MAPK, STAT3 and HIF-1α pathways. These studies were performed using homogeneous cell lines (U87 and T98G) in short-term cell viability assays after 24 h with considerably high doses (>100 nM) of bortezomib.[12,13,14] In investigating the involvement of the NFkB/p65 pathway, these studies did not use phosphor-site-specific antibodies, challenging interpretation of activation of the pathway. Our extensive characterisation of potential toxicity through analyses of liver function, bone marrow-derived cells, coagulation profiles and animal weights proved the treatment safe and tolerated, consistent with patient clinical trials.[19,21,22,23] Our results suggest that such a sensitisation schedule may be of clinical benefit for GBM patients with unmethylated MGMT promoter
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