Kevin A. Harvey, Zhidong Xu, M. Reza Saaddatzadeh, Haiyan Wang, Karen Pollok, Aaron A. Cohen-Gadol and Rafat A. Siddiqui
Glioblastoma is a rapidly infiltrating tumor that consistently rematerializes despite various forms of aggressive treatment. Brain tumors are commonly treated with alkylating drugs, such as lomustine, which are chemotherapeutic agents. Use of these drugs, however, is associated with serious side effects. To reduce the side effects, one approach is to combine lower doses of chemotherapeutic drugs with other nontoxic anticancer agents. In this study, using glioblastoma cell lines, the authors investigated the anticancer effects of lomustine, alone and in combination with docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid normally abundant in the brain and known for its anticancer potential.
Cells were cultured from 3 human-derived tumor cell lines (U87-MG, DB029, and MHBT161) and supplemented with either DHA or lomustine to determine the growth inhibitory potential using WST-1, a mitochondrial functional indicator. Human-derived cerebral cortex microvascular endothelial cells served as a normal phenotypic control. Cellular incorporation of DHA was analyzed by gas chromatography. Using flow cytometric analysis, the DHA and/or lomustine effect on induction of apoptosis and/or necrosis was quantified; subsequently, the DHA and lomustine effect on cell cycle progression was also assessed. Western blot analysis confirmed the role of downstream cellular targets.
U87-MG growth was inhibited with the supplementation of either DHA (ED50 68.3 μM) or lomustine (ED50 68.1 μM); however, growth inhibition was enhanced when U87-MG cells were administered equimolar doses of each compound, resulting in nearly total growth inhibition at 50 μM. Gas chromatography analysis of the fatty acid profile in DHA-supplemented U87-MG cells resulted in a linear dose-dependent increase in DHA incorporation (< 60 μM). The combination of DHA and lomustine potently induced U87-MG apoptosis and necrosis as indicated by flow cytometric analysis. Activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) was evident in lomustine-treated U87-MG cells, although this activation did not appear to be dependent on DHA supplementation. Additionally, lomustine-treated cells' growth arrested in the G2/M cell cycle stage, regardless of the presence of DHA. Similar to the U87-MG observations, the combination of DHA and lomustine resulted in growth inhibition of 2 additional human-derived glioblastoma cell lines, DB029 and MHBT161. Importantly, in primary human-derived cerebral cortex endothelial cells, this combination was only growth inhibitory (40.8%) at the highest dose screened (100 μM), which indicates a certain degree of selectivity toward glioblastoma.
Taken together, these data suggest a potential role for a combination therapy of lomustine and DHA for the treatment of glioblastomas.
Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Haiyan Wang, Angie Nguyen, Malgorzata M. Kamocka, Wenjing Cai, Aaron A. Cohen-Gadol, Stacey L. Halum, Jann N. Sarkaria, Karen E. Pollok and Ahmad R. Safa
Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)—a constituent of cruciferous vegetables with a multitargeted effect—as a therapeutic agent for GBM.
The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models.
SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell–induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10.
These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.
Xiang Gao, Haiyan Wang, Shanbao Cai, M. Reza Saadatzadeh, Helmut Hanenberg, Karen E. Pollok, Aaron A. Cohen-Gadol and Jinhui Chen
Peritumoral seizures are an early symptom of a glioma. To gain a better understanding of the molecular mechanism underlying tumor-induced epileptogenesis, the authors studied modulation of the N-methyl-d-aspartate (NMDA) receptor in peritumoral tissue.
To study the possible etiology of peritumoral seizures, NMDA receptor expression, posttranslational modification, and function were analyzed in an orthotopic mouse model of human gliomas and primary patient glioma tissue in which the peritumoral border (tumor-brain interface) was preserved in a tissue block during surgery.
The authors found that the NMDA receptor containing the 2B subunit (NR2B), a predominantly extrasynaptic receptor, is highly phosphorylated at S1013 in the neurons located in the periglioma area of the mouse brain. NR2B is also highly phosphorylated at S1013 in the neurons located in the peritumoral area from human brain tissue containing a glioma. The phosphorylation of the extrasynaptic NMDA receptor increases its permeability for Ca2+ influx and subsequently mediates neuronal overexcitation and seizure activity.
These data suggest that overexcitation of the extrasynaptic NMDA receptors in the peritumoral neurons may contribute to the development of peritumoral seizures and that the phosphorylated NR2B may be a therapeutic target for blocking primary brain tumor–induced peritumoral seizures.
Haiyan Wang, Shanbao Cai, Barbara J. Bailey, M. Reza Saadatzadeh, Jixin Ding, Eva Tonsing-Carter, Taxiarchis M. Georgiadis, T. Zachary Gunter, Eric C. Long, Robert E. Minto, Kevin R. Gordon, Stephanie E. Sen, Wenjing Cai, Jacob A. Eitel, David L. Waning, Lauren R. Bringman, Clark D. Wells, Mary E. Murray, Jann N. Sarkaria, Lawrence M. Gelbert, David R. Jones, Aaron A. Cohen-Gadol, Lindsey D. Mayo, Harlan E. Shannon and Karen E. Pollok
Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM.
The combination of TMZ with the MDM2 protein–protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM.
In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy.
Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein–protein interactions.