Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier

Abstract

Background: The proteasome plays a vital role in the physiology of glioblastoma (GBM), and proteasome inhibition can be used as a strategy for treating GBM. Marizomib is a second-generation, irreversible proteasome inhibitor with a more lipophilic structure that suggests the potential for penetrating the blood-brain barrier. While bortezomib and carfilzomib, the 2 proteasome inhibitors approved for treatment of multiple myeloma, have little activity against malignant gliomas in vivo, marizomib could be a novel therapeutic strategy for primary brain tumors.

Methods: The in-vitro antitumor activity of marizomib was studied in glioma cell lines U-251 and D-54. The ability of marizomib to cross the blood-brain barrier and regulate proteasome activities was evaluated in cynomolgus monkeys and rats. The antitumor effect of marizomib in vivo was tested in an orthotopic xenograft model of human GBM.

Results: Marizomib inhibited the proteasome activity, proliferation, and invasion of glioma cells. Meanwhile, free radical production and apoptosis induced by marizomib could be blocked by antioxidant N-acetyl cysteine. In animal studies, marizomib distributed into the brain at 30% of blood levels in rats and significantly inhibited (>30%) baseline chymotrypsin-like proteasome activity in brain tissue of monkeys. Encouragingly, the immunocompromised mice, intracranially implanted with glioma xenografts, survived significantly longer than the control animals (P < .05) when treated with marizomib.

Conclusions: These preclinical studies demonstrated that marizomib can cross the blood-brain barrier and inhibit proteasome activity in rodent and nonhuman primate brain and elicit a significant antitumor effect in a rodent intracranial model of malignant glioma.

Keywords: blood-brain barrier; chymotrypsin-like; malignant glioma; marizomib; proteasome inhibition.

© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Figures

Fig. 1.

Marizomib inhibits the proteasome activity, proliferation, and invasion of glioma cells. (A) Proteasome activity was measured in glioma cell lines D-54 and U-251 after treatment with marizomib (60 nM). The chymotrypsin-like activity of treated cells was presented as a percentage of untreated cells. (B) Marizomib treatment for 72 hours effectively reduced survival of D-54 and U-251 cells in a dose-dependent manner. The IC50s were shown in a lower panel. (C) Proteasome activity was measured in neural stem cells and glioblastoma-derived glioma stem cells at baseline and 2 hours after treatment with marizomib (60 nM). (D) Marizomib showed different proliferation inhibition on diverse primary cell cultures after treatment for 72 hours. (E) Invasion capability of cells treated or untreated with 60 nM marizomib for 24 hours was analyzed using Matrigel invasion chambers. The average of invaded cells for each counting grid was shown in the lower panel. ***P < .001.

Fig. 2.

Marizomib induces apoptosis and caspase-3 activation in glioma cells. (A) FITC Annexin V apoptosis assay kit was used to detect apoptotic cell death induced by marizomib treatment for 24 hours in D-54 cells. (B) DAPI staining was performed to observe apoptotic cells indicated by small, condensed nuclei in D-54 cells treated by marizomib (60 nM) for 24 hours. (C) The caspase-3 activity of D-54 cells was measured using Apopcyto Caspase-3 Fluorometric assay kit 24 hours after treatment with marizomib. An 80% increase in caspase-3 activity was found in presence of 60 nM marizomib. **P < .01, ***P < .001. (D) D-54 and U-251 cells were treated with 60 nM marizomib for indicated time points. Western blot was used to detect cleaved caspase-3 and PARP. Actin was the internal control.

Fig. 3.

Marizomib increases reactive oxygen species (ROS) generation, but N-acetyl cysteine (NAC) quenches ROS induction, blocks caspase-3 activation, and improves the survival of D-54 cells. (A) After treatment with marizomib (60 nM) for 12 hours, ROS generation was measured as the number of DCF fluorescent cells by direct fluorescent microscopy. The percentage of ROS-positive cells is shown in the lower panel. (B) Carboxy-H2DCFDA detectable ROS were measured by fluorescence spectroscopy. Values represent the mean ± SD of 3 experiments. (C) The addition of N-acetyl cysteine (NAC) (10 mM) blocked the marizomib-induced ROS activation. (D) NAC pretreatment was able to rescue the D-54 cells from marizomib-induced cell death. (2-way ANOVA analysis, P < .0001). (E) The caspase-3 activation triggered by marizomib was abolished by NAC pretreatment (2-way ANOVA analysis, P < .0001). (F) D-54 cells were treated with 20 or 60 nM marizomib in the presence or absence of 10 mM NAC for indicated time points. Western blot was used to detect cleaved caspase-3 and PARP. Actin was used as the internal control. **P < .01, ***P < .001, ns = not significant.

Fig. 4.

Effect of marizomib regiments on the proteasome activity of prefrontal cortex in cynomolgus monkeys. (A) The chymotrypsin-like, C-L and T-L activities were assessed in control monkey prefrontal cortex in vivo, and all 3 proteasome activities were present at levels well distinguished from baseline. (B) Marizomib administered orally resulted in a differential inhibition of proteasome activities in monkey prefrontal cortex. The degree of inhibition observed was very similar for the twice-weekly and once-weekly dosing schedules.

Fig. 5.

Marizomib prolonged animal survival in an orthotopic mouse glioma xenograft model. (A) Control vehicle or marizomib was administered twice weekly for 2.5 weeks (on days 1, 4, 8, 11, and 15) into the tail vein of athymic BALB/c nu/nu mice. There was no statistical difference in the weight of animals among the vehicle control, maximum tolerated dose group (MTD: 200 µg/kg) and lower dose group (MTD-1: 150 µg/kg). (B) Kaplan-Meier survival probability plots of tumor-bearing mice in vehicle or marizomib treatment groups (n = 6–8), using the log-rank method to test for a difference between groups.