Temporary blood–brain barrier disruption by low intensity pulsed ultrasound increases carboplatin delivery and efficacy in preclinical models of glioblastoma

Journal of Neuro-Oncology - Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults. Few cytotoxic chemotherapies have been shown to be effective against GBM, due in...     

Examination of blood–brain barrier (BBB) integrity in a mouse brain tumor model

The present study evaluates, both functionally and biochemically, brain tumor-induced alterations in brain capillary endothelial cells. Brain tumors were induced in Balb/c mice via intracranial injection of Lewis Lung carcinoma cells into the right hemisphere of the mouse brain using stereotaxic apparatus. Blood–brain barrier (BBB) permeability was assessed at various stages of tumor development, using both radiolabeled tracer permeability and magnetic resonance imaging with gadolinium diethylene-triamine-pentaacetate contrast enhancement (Gad-DTPA). The expression of the drug efflux transporter, P-glycoprotein (P-gp), in the BBB at various stages of tumor development was also evaluated by Western blot and immunohistochemistry. Median mouse survival following tumor cell injection was 17 days. The permeability of the BBB to ³H-mannitol was similar in both brain hemispheres at 7 and 10 days post-injection. By day 15, there was a twofold increase in ³H-mannitol permeability in the tumor bearing hemispheres compared to the non-tumor hemispheres. Examination of BBB permeability with Gad-DTPA contrast enhanced MRI indicated cerebral vascular permeability changes were confined to the tumor area. The permeability increase observed at the later stages of tumor development correlated with an increase in cerebral vascular volume suggesting angiogenesis within the tumor bearing hemisphere. Furthermore, the Gad-DPTA enhancement observed within the tumor area was significantly less than Gad-DPTA enhancement within the circumventricular organs not protected by the BBB. Expression of P-gp in both the tumor bearing and non-tumor bearing portions of the brain appeared similar at all time points examined. These studies suggest that although BBB integrity is altered within the tumor site at later stages of development, the BBB is still functional and limiting in terms of solute and drug permeability in and around the tumor.     

Delivery of chemotherapy and antibodies across the blood–brain barrier and the role of chemoprotection, in primary and metastatic brain tumors: report of the eleventh annual blood–brain barrier consortium meeting

Although knowledge of molecular biology and cellular physiology has advanced at a rapid pace, much remains to be learned about delivering chemotherapy and antibodies across the blood–brain barrier (BBB) for the diagnosis and treatment of central nervous system (CNS) disease. A meeting, partially funded by an NIH R13 grant, was convened to discuss the state of the science, current knowledge gaps, and future directions in the delivery of drugs and proteins to the CNS, for the treatment of primary and metastatic brain tumors. Meeting topics included CNS metastases and the BBB, and chemoprotection and chemoenhancement in CNS disorders. The discussions regarding CNS metastases generated possibilities of chemoprotection as a means not only to decrease treatment-related toxicity but also to increase chemotherapy dose intensity. The increasing incidence of sanctuary brain metastasis from breast cancer, in part due to the difficulty of monoclonal antibodies (mAbs) such as herceptin to cross the BBB, was one of the most salient “take home” messages of the meeting. OHSU, Portland Veterans Affairs Medical Center (PVAMC) and the Department of Veterans Affairs have a significant financial interest in Adherex, a company that may have a commercial interest in the results of this research and technology. This potential conflict of interest was reviewed and managed by the OHSU Integrity Program Oversight Council and the PVAMC Conflict of Interest in Research Committee.     

Blood–brain barrier and blood–cerebrospinal fluid barrier in normal and pathological conditions

Blood-borne substances can invade into the extracellular spaces of the brain via endothelial cells in sites without the blood–brain barrier (BBB), and can travel through the interstitial fluid (ISF) of the brain parenchyma adjacent to non-BBB sites. It has been shown that cerebrospinal fluid (CSF) drains directly into the blood via the arachnoid villi and also into lymph nodes via the subarachnoid spaces of the brain, while ISF drains into the cervical lymph nodes through perivascular drainage pathways. In addition, the glymphatic pathway of fluids, characterized by para-arterial pathways, aquaporin4-dependent passage through astroglial cytoplasm, interstitial spaces, and paravenous routes, has been established. Meningeal lymphatic vessels along the superior sagittal sinus were very recently discovered. It is known that, in mice, blood-borne substances can be transferred to areas with intact BBB function, such as the medial regions of the hippocampus, presumably through leaky vessels in non-BBB sites. In the present paper, we review the clearance mechanisms of interstitial substances, such as amyloid-β peptides, as well as summarize models of BBB deterioration in response to different types of insults, including acute ischemia followed by reperfusion, hypertension, and chronic hypoperfusion. Lastly, we discuss the relationship between perivascular clearance and brain disorders.     

Strategies to improve delivery of anticancer drugs across the blood–brain barrier to treat glioblastoma

Glioblastoma (GBM) is a lethal and aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. Molecularly targeted agents hold great promise in treating these genetically heterogeneous tumors, yet have produced disappointing results. One reason for the clinical failure of these novel therapies can be the inability of the drugs to achieve effective concentrations in the invasive regions beyond the bulk tumor. In this review, we describe the influence of the blood–brain barrier on the distribution of anticancer drugs to both the tumor core and infiltrative regions of GBM. We further describe potential strategies to overcome these drug delivery limitations. Understanding the key factors that limit drug delivery into brain tumors will guide future development of approaches for enhanced delivery of effective drugs to GBM.     

The PPARγ agonist pioglitazone crosses the blood–brain barrier and reduces tumor growth in a human xenograft model

Purpose

The peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear hormone receptor family, represents a target in glioma therapy due to its antineoplastic effects in vitro on human glioma cell lines. We investigate the antineoplastic effects of the PPARγ agonist pioglitazone (pio) in a human glioma xenograft model to define the minimal required dose to induce antineoplastic effects. Additionally, we assess the ability of pio to cross the blood–brain barrier by measuring brain parenchymal concentration after oral administration.

Methods

Human LN-229 cells were injected into the striatum of Balb/cJHanHsd-Prkdc-scid mice. Tumor volumes, invasion, proliferation and parenchymal pio concentrations were measured in this xenograft model after continuous intracerebral drug administration through an osmotic pump or after oral administration.

Results

Continuous intracerebral or oral administration of pio reduced tumor volumes, invasion, and proliferation in vivo. To achieve a significant antineoplastic effect, pio needed to be dosed at 240 PPM in the oral group and >1 μM when delivered intracerebrally. After oral pio administration, the drug reached >1 nM levels in brain parenchyma.

Conclusions

These data indicate that pioglitazone crosses the blood–brain barrier and has antineoplastic effects in this glioma xenograft model and may be of potential use in treatment of malignant gliomas.     

The feasibility of real-time in vivo optical detection of blood–brain barrier disruption with indocyanine green

Osmotic disruption of the blood–brain barrier (BBB) by intraarterial mannitol injection is sometimes required for the delivery of chemotherapeutic drugs to brain tissue. Osmotic disruption is affected by a number of factors, and there is a significant variability in the degree and distribution of BBB disruption in clinical and experimental settings. Brain tissue concentrations of indocyanine green (ICG) can be measured by optical techniques. The aim of this experiment was to determine whether the disruption of the BBB significantly altered the regional pharmacokinetics of ICG. We were able to track in vivo brain tissue concentrations of ICG in 13 New Zealand white rabbits by employing a novel optical approach. Evan’s blue was used to assess the distribution of BBB disruption on post mortem examination. BBB disruption by intraarterial mannitol injection was found to be highly variable, and only five of the 13 animals demonstrated the disruption at the site of optical measurements. In these animals, we observed a ninefold increase in ICG concentrations and fourfold increase in the area under the concentration-time curve, compared to those without BBB disruption at the site of measurement. This study shows the feasibility of optical monitoring of BBB disruption with intravenous (IV) ICG injections. Virtual real-time optical monitoring of the BBB disruption could help improve intraarterial delivery of chemotherapeutic drugs.     

Down-regulation of Wilms’ tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53

The Wilms’ tumor 1 (WT1) gene is overexpressed in human glioblastoma and correlates with wild-type p53 status. In other cell types, WT1 inhibits p53-mediated apoptosis in response to DNA damaging agents. However, neither this interaction nor the relationship between WT1 and radiosensitivity has been studied in glioblastoma. To study this interaction, we generated LN-229 glioma cell lines (p53 mutant) stably expressing WT1 isoforms and induced apoptosis by transfecting with different doses of wild-type p53 plasmid expression vector. Constitutive expression of WT1 did not protect against exogenous p53-mediated apoptosis. Likewise, WT1 expression did not protect against endogenous p53-mediated cell death induced by radiotherapy in U87MG cells, which contain functional wild-type p53. We then tested the efficacy of WT1 siRNA in inhibiting WT1 expression and its effect on radiosensitivity. In T98G and LN-18 glioma cells, which possess p53 mutations, WT1 siRNA decreased WT1 protein to almost undetectable levels by 96-h post-transfection. Furthermore, WT1 siRNA transfection caused a significantly larger decrease in viability following irradiation than was seen in untransfected cells in both cell lines after treatment with ED₅₀ of ionizing radiation. In conclusion, WT1 overexpression did not protect against p53-mediated apoptosis or ionizing radiation induced cell death. WT1 siRNA increased the radiosensitivity of two human glioma cell lines independently of p53. Anti-WT1 strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.     

Dual kinin B1 and B2 receptor activation provides enhanced blood–brain barrier permeability and anticancer drug delivery into brain tumors

The low permeability of the BBB is largely responsible for the lack of effective systemic chemotherapy against primary and metastatic brain tumors. Kinin B1R and B2R have been shown to mediate reversible tumor-selective BBB disruption in preclinical animal models. We investigated whether co-administration of two novel potent kinin B1R and B2R agonists offers an advantage over administering each agonist alone for enhancing BBB permeability and tumor targeting of drugs in the malignant F98 glioma rat model. A new covalent kinin heterodimer that equally stimulates B1R and B2R was also constructed for the purpose of our study. We found that co-administration of B1R and B2R agonists, or alternatively administration of the kinin heterodimer more effectively delivered the MRI contrast agent Gd-DTPA and the anticancer drug carboplatin to brain tumors and surrounding tissues than the agonists alone (determined by MRI and ICP-MS methods). Importantly, the efficient delivery of carboplatin by the dual kinin receptor targeting on the BBB translated into increased survival of glioma-bearing rats. Thus, this report describes a potential strategy for maximizing the brain bioavailability and therapeutic efficacy of chemotherapeutic drugs.     

Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.     

Therapeutic effects of the Sp1 inhibitor mithramycin A in glioblastoma

Mithramycin A (MitA) is a chemotherapeutic compound which has been used in the therapy of several types of cancer. For experimental cancer it has been shown that MitA mediates the expression of genes involved in tumor progression such as genes involved in immunosurveillance, cell motility or cell death. MitA works synergistically with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and with antiangiogenic agents. We were therefore interested in analyzing whether MitA might be a suitable agent for glioma therapy. We demonstrate herein that the cell death sensitizing effects of MitA are cell line specific, independent of the endogenous status of the tumor suppressor p53 as well as of the endogenous expression of X-linked inhibitor of apoptosis (XIAP) or basal sensitivity towards death ligand-induced cell death. In glioma cells, MitA reduced the secretion and activity of the migration-involved matrix metalloproteinases (MMP), diminished vascular endothelial growth factor (VEGF), and increased recepteur d’origine nantais (RON) kinase messenger RNA (mRNA), paralleled by a significant reduction of glioma cell migration. In contrast to other cancer types, in glioma cells MitA did not alter the expression of the immunorelevant genes major histocompatibility complex I class related (MIC)-A, MIC-B or UL16 binding proteins (ULBP). We conclude that, whereas MitA-mediated reduction of XIAP expression and sensitization to Apo2L/TRAIL are cell line specific, its antimigratory effects are more general and might be the result of altered expression of MMP, VEGF, and/or RON kinase. Therefore, MitA might be a potential agent to reduce glioma cell migration.     

Expression of 19 microRNAs in glioblastoma and comparison with other brain neoplasia of grades I–III

Several biomarkers have been proposed as useful parameters to better specify the prognosis or to delineate new target therapy strategies for glioblastoma patients. MicroRNAs could represent putative target molecules, considering their role in tumorigenesis, cancer progression and their specific tissue expression. Although several studies have tried to identify microRNA signature for glioblastoma, a microRNA profile is still far from being well‐defined.In this work the expression of 19 microRNAs (miR‐7, miR‐9, miR‐9∗, miR‐10a, miR‐10b, miR‐17, miR‐20a, miR‐21, miR‐26a, miR‐27a, miR‐31, miR‐34a, miR‐101, miR‐137, miR‐182, miR‐221, miR‐222, miR‐330, miR‐519d) was evaluated in sixty formalin‐fixed and paraffin‐embedded glioblastoma samples using a locked nucleic acid real‐time PCR. Moreover, a comparison of miRNA expressions was performed between primary brain neoplasias of different grades (grades IV–I).The analysis of 14 validated miRNA expression in the 60 glioblastomas, using three different non‐neoplastic references as controls, revealed a putative miRNA signature: mir‐10b and miR‐21 were up‐regulated, while miR‐7, miR‐31, miR‐101, miR‐137, miR‐222 and miR‐330 were down‐regulated in glioblastomas. Comparing miRNA expression between glioblastoma group and gliomas of grades I–III, 3 miRNAs (miR‐10b, mir‐34a and miR‐101) showed different regulation statuses between high‐grade and low‐grade tumors. miR‐10b was up‐regulated in high grade and significantly down‐regulated in low‐grade gliomas, suggesting that could be a candidate for a GBM target therapy.This study provides further data for the identification of a miRNA profile for glioblastoma and suggests that different‐grade neoplasia could be characterized by different expression of specific miRNAs.     

Local convection-enhanced delivery of chemotherapeutic agent transiently opens blood–brain barrier and improves efficacy of systemic chemotherapy in intracranial xenograft tumor model

Recently, local chemotherapy proved its efficacy against malignant gliomas. Under the hypothesis that local delivery of chemotherapeutic agents into the brain parenchyma induce opening of the blood–brain barrier (BBB), we evaluated the opening of BBB after convection-enhanced delivery of nimustine hydrochloride into the brain parenchyma. Local convection-enhanced delivery of nimustine hydrochloride transiently opened the BBB from about 7–12 days after delivery in normal rodent brain. Systemic chemotherapy during this period of BBB disruption had synergistic effects resulting in prolonged survival of tumor-bearing rats. The present strategy may provide a new approach for glioma chemotherapy.     

A combination of IFN-β and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation

Purpose Methylation of the O⁶-methyguanine-DNA methyltransferase (MGMT) gene promoter in gliomas has been reported to be a useful predictor of the responsiveness to temozolomide (TMZ). In our previous experiments, we observed that IFN-β sensitized TMZ-resistant glioma cells with the unmethylated MGMT promoter and that the mechanism of action was possibly due to attenuation of MGMT expression via induction of TP53. In this study, (1) we explored the synergistic effect of IFN-β and TMZ in the animal model, and (2) clarified the role of IFN-β induced TP53 in the human MGMT promoter. Methods (1) Nude mice with either subcutaneous T98 (TMZ-resistant) or U251SP (TMZ-sensitive) tumor were treated with IFN-β/TMZ for 5 consecutive days. (2) The MGMT promoter activity was assayed by a luciferase reporter system in Saos2 (p53-null) cells transduced with a p53-adenoviral vector, and T98 glioma cells treated with IFN-β. Results (1) A combination of IFN-β/TMZ had significant synergistic antitumor activity on the growth of both T98 and U251SP tumors. (2) MGMT promoter activity was suppressed by either adenovirally transduced p53 or IFN-β. Conclusions It would be appealing to consider a prospective clinical trial in which genetic markers are used for personalized drug selection, eliciting other forms of treatment or inhibition of MGMT for those with MGMT expression. In this context, IFN-β inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ.     

Thymoquinone overcomes chemoresistance and enhances the anticancer effects of bortezomib through abrogation of NF-κB regulated gene products in multiple myeloma xenograft mouse model

Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow. With the advent of novel targeted agents, the median survival rate has increased to 5−7 years. However, majority of patients with myeloma suffer relapse or develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of MM. Thus in the present study, we investigated whether thymoquinone (TQ), a bioactive constituent of black seed oil, could suppress the proliferation and induce chemosensitization in human myeloma cells and xenograft mouse model. Our results show that TQ inhibited the proliferation of MM cells irrespective of their sensitivity to doxorubicin, melphalan or bortezomib. Interestingly, TQ treatment also resulted in a significant inhibition in the proliferation of CD138+ cells isolated from MM patient samples in a concentration dependent manner. TQ also potentiated the apoptotic effects of bortezomib in various MM cell lines through the activation of caspase-3, resulting in the cleavage of PARP. TQ treatment also inhibited chemotaxis and invasion induced by CXCL12 in MM cells. Furthermore, in a xenograft mouse model, TQ potentiated the antitumor effects of bortezomib (p < 0.05, vehicle versus bortezomib + TQ; p < 0.05, bortezomib versus bortezomib + TQ), and this correlated with modulation of various markers for survival and angiogenesis, such as Ki-67, vascular endothelial growth factor (VEGF), Bcl-2 and p65 expression. Overall, our results demonstrate that TQ can enhance the anticancer activity of bortezomib in vitro and in vivo and may have a substantial potential in the treatment of MM.     

Mechanisms of the increase in the permeability of the blood–tumor barrier obtained by combining low-frequency ultrasound irradiation with small-dose bradykinin

The research was conducted to study the characteristics of the noninvasive, reversible, targeted opening of the blood–brain barrier (BBB) by use of low-frequency ultrasound (LFU) irradiation and the selective opening of the blood–tumor barrier (BTB) by intracarotid infusion of bradykinin (BK) in small-dose, with the objective of exploring maximum opening of the BTB by combining LFU irradiation with BK infusion. Thus, it provides new therapeutic strategies for targeted transport of macromolecular or granular drugs to the brain. By using the rat C6 glioma model it was shown that extravasation of Evans blue (EB) through the BTB was significantly increased by combining LFU irradiation (frequency = 1.0 MHz, power = 12 mW, duration = 20 s) with intracarotid small-dose BK infusion, compared with utilizing the two methods separately. By transmission electron microscopy (TEM) we observed that this combination significantly increased the number of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB. An even more significant increase was observed by using RT-PCR, western blot, immunohistochemistry, and immunofluorescence to detect mRNA and changes of expression of the caveolae structure proteins caveolin-1 and caveolin-2 of BMECs. In summary, this research concludes that LFU irradiation and small-dose BK together selectively enhance the permeability of the BTB and increase the number of pinocytic vesicles of BMECs to a maximum. Significant up-regulation of the level of expression of caveolae structure proteins caveolin-1 and caveolin-2 might be the molecular mechanism of the co-enhanced endocytotic transport by BMECs. Thus, this research provides new therapeutic strategies for targeted transport of macromolecular drugs and the design of drugs. Chun-yi Xia and Zhen Zhang contributed equally to this work.     

p53 gene mutations and p21 protein expression induced independently of p53, by TGF-β and γ-rays in squamous cell carcinoma cells

p53 gene mutation and the influence of TGF-β and γ-rays on p21 promoter activity, p21 mRNA and protein expression were investigated in nine cell lines (OSC-1 to -9) established from metastatic squamous cell carcinomas (SCC) of the cervical lymph nodes. The direct DNA sequence analysis of exons 2 to 11 of the p53 gene revealed 16 point mutations in all cell lines, but neither deletions nor additions were observed. TGF-β upregulated p21 promoter activity by approximately 2-fold of the control and concurrently increased p21 mRNA expression, except in OSC-8 and -9. However, γ-rays suppressed p21 promoter activity, although p21 mRNA expression in irradiated cells was increased except for OSC-8 and -9. In parallel with the messenger expression, p21 protein expression was strongly increased by TGF-β, but only weakly increased by γ-rays. These results indicate that point mutation of the p53 gene is frequent in metastatic SCC cells and p21 mRNA and its protein expression is p53-independently induced by both TGF-β and γ-rays, although the mechanism of induction by TGF-β and γ-rays is different.