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01.12.2018 | Primary research | Ausgabe 1/2018 Open Access

Cancer Cell International 1/2018

Levetiracetam enhances the temozolomide effect on glioblastoma stem cell proliferation and apoptosis

Zeitschrift:
Cancer Cell International > Ausgabe 1/2018
Autoren:
Bianca Maria Scicchitano, Silvia Sorrentino, Gabriella Proietti, Gina Lama, Gabriella Dobrowolny, Angela Catizone, Elena Binda, Luigi Maria Larocca, Gigliola Sica
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12935-018-0626-8) contains supplementary material, which is available to authorized users.

Abstract

Background

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor in which cancer cells with stem cell-like features, called cancer stem cells (CSCs), were identified. Two CSC populations have been previously identified in GBM, one derived from the GBM area called enhanced lesion (GCSCs) and the other one from the brain area adjacent to the tumor margin (PCSCs) that greatly differ in their growth properties and tumor-initiating ability. To date the most effective chemotherapy to treat GBM is represented by alkylating agents such as temozolomide (TMZ), whose activity can be regulated by histone deacetylases (HDACs) inhibitors through the modulation of O6-methylguanine-DNA methyltransferase (MGMT) expression. Levetiracetam (LEV), a relatively new antiepileptic drug, modulates HDAC levels ultimately silencing MGMT, thus increasing TMZ effectiveness. However, an improvement in the therapeutic efficacy of TMZ is needed.

Methods

Cell proliferation was investigated by BrdU cell proliferation assay and by Western Blot analysis of PCNA expression. Apoptosis was evaluated by Western Blot and Immunofluorescence analysis of the cleaved Caspase-3 expression. MGMT and HDAC4 expression was analyzed by Western Blotting and Immunofluorescence. Statistical analysis was performed using the Student’s t test and Mann–Whitney test.

Results

Here we evaluated the effect of TMZ on the proliferation rate of the IDH-wildtype GCSCs and PCSCs derived from six patients, in comparison with the effects of other drugs such as etoposide, irinotecan and carboplatin. Our results demonstrated that TMZ was less effective compared to the other agents; hence, we verified the possibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV enhances the effect of TMZ on GCSCs proliferation (being less effective on PCSCs) by decreasing MGMT expression, promoting HDAC4 nuclear translocation and activating apoptotic pathway.

Conclusions

Although further studies are needed to determine the exact mechanism by which LEV makes GBM stem cells more  sensitive to TMZ, these results suggest that the clinical therapeutic efficacy of TMZ in GBM might be enhanced by the combined treatment with LEV.
Zusatzmaterial
Additional file 1. Description (Results, Material and Methods) of additional figures S1 and S2.
Additional file 2: Figure S1. Effects of different chemotherapeutic drugs on Jurkat cell proliferation. (A) BrdU cell proliferation assay of Jurkat cells treated for 48 h with TMZ (250 µM), ETO (10 µM), IRI (10 µg/ml) and CARB (10 µg/ml). (B) Western blot analysis of total lysates from Jurkat cells, treated as described above was performed to detect PCNA expression levels; β-actin was used as a loading control. (C) Densitometric analysis of three independent experiments on PCNA expression levels. ** p< 0.01, ***p < 0001 vs control by Student’s t-test.
Additional file 3: Figure S2. Effects of different chemotherapeutic agents on apoptosis induction in Jurkat cells. Jurkat cells were treated with the same concentrations of the antineoplastic drugs described in additional Fig. 1. After 48 h, the activity of the pro-caspases-2, -8 and -9 and of the effector caspases -3 and -6 was measured by using ApoTarget Caspase Colorimetric Protease Assay. The results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***  p< 0.001 vs control by Student’s t-test.
Literatur
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