Blood-brain Tumor Barrier Permeability Research Articles

Lnc00462717 regulates the permeability of the blood-brain tumor barrier through interaction with PTBP1 to inhibit the miR-186-5p/Occludin signaling pathway.

Blood-brain tumor barrier (BTB) severely restricts the efficient delivery of chemotherapeutic drugs into brain tumor tissue, which is a critical obstacle for glioma treatment. Recently, long noncoding RNAs (lncRNAs) have shown as regulation factors of numerous biological processes. In this study, we identified that Lnc00462717 was upregulated in glioma endothelial cells (GECs), and that knockdown of Lnc00462717 significantly increased the BTB permeability. Both bioinformatics and RNA immunoprecipitation (RIP) results revealed that Lnc00462717 interacts with polypyrimidine tract binding protein (PTBP1). Moreover, overexpression of PTBP1 significantly reversed the increase in BTB permeability caused by siLnc00462717. Furthermore, the binding sites between miR-186 and PTBP1 as well as between miR-186 and 3'UTR of Occludin mRNA were confirmed by RIP and luciferase assays, respectively. And the interaction of Lnc00462717 and PTBP1 significantly facilitated the binding of PTBP1 to 3'UTR of Occludin mRNA and then blocked the miR-186-5p-induced downregulation of Occludin. In addition, we identified that knockdown of Lnc00462717 or overexpression of miR-186-5p increased the accumulation of doxorubicin (Dox) in brain glioma via the ultrafast liquid chromatography-mass spectrometry system (UFLC-MS/MS system) and decreased the intracranial glioma volume in BALB/c nude mice. Taken together, these results show a novel molecular pathway in BTB that may provide a potential innovative strategy for glioma therapy.

Long non-coding RNA MALAT1 as a key target in pathogenesis of glioblastoma. Janus faces or Achilles’ heal?

Glioblastomas (GBMs) are primary brain tumors with extremely bad prognosis and therefore; discovery of novel regulators of their pathology is of immense importance. LncRNAs (long noncoding RNAs) regulate nuclear structure, embryonic pluripotency, cell differentiation, development and carcinogenesis. Many lncRNAs have weak evolutionary conservation; however, a nuclear lncRNA, MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), is exceptionally conserved and is among the most abundant lncRNAs in benign tissues. The majority of cell culture studies and clinico-epidemiological studies demonstrated that MALAT1 acts a tumor promoter in GBMs and inhibition of MALAT1 suppressed tumor growth in various preclinical models of GBM. MALAT1 involves in stemness of GBM cells by regulating SOX2, nestin and members of WNT pathway. MALAT1 induces protective autophagy and suppresses apoptosis in GBM cells via sponging miRNA-101 and increases temozolomide chemoresistance via enhancing epithelial-mesenchymal transition, suppressing miR-203 and promoting thymidilate synthase. Moreover, knockdown of MALAT1 expression enhances blood-brain tumor barrier permeability via miR-140, which may provide a double benefit of MALAT1 suppression by increasing the delivery of chemotherapy agents into the GBM tissues. On the other hand, there also exist some cell culture and animal studies showing that MALAT1 acts as a tumor suppressor in GBMs by suppression of ERK/MAPK and MMP2 signaling and by repression of miR-155 with subsequent increase of FBXW7. Whether protective or detrimental, MALAT1 seems to be an important component of GBM pathogenesis and hence; novels studies are needed in versatile models, including many different primary GBM cultures, orthotopic and xenogreft in vivo models and transgenic mice.

MiR‐132‐3p boosts caveolae‐mediated transcellular transport in glioma endothelial cells by targeting PTEN/PI3K/PKB/Src/Cav‐1 signaling pathway

Blood-brain tumor barrier (BTB) impedes the transportation of antitumor therapeutic drugs into brain tumors. Its mechanism is still unknown, but learning how to improve the BTB permeability is critical for drug intervention. Recently, microRNAs (miRNAs) have appeared as regulation factors of numerous biologic processes and therapeutic targets of diverse diseases. In this study, we have identified that miR-132-3p is an essential miRNA by increasing the transcellular transport through the BTB. We found that miR-132-3p expression was significantly up-regulated in glioma endothelial cells (GECs). Furthermore we showed that miR132-3p+ greatly induced the endocytosis of cholera toxin subunit B and FITC-bovine serum albumin and up-regulated the expression of p-PKB, p-Src and Tyr14 phosphorylation of caveolin-1 (p-Cav-1), while phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression was markedly down-regulated in GECs. Our results identify PTEN as a direct and functional downstream target of miR-132-3p, which is involved in the regulation of p-PKB, p-Src, and p-Cav-1. The inhibitors for PI3K and Src significantly reversed the increase of p-Cav-1 induced by miR-132-3p. Moreover, overexpression of PTEN greatly reduced the endocytosis of cholera toxin subunit B and the up-regulation of p-Cav-1 induced by agomiR132-3p, suggesting that miR132-3p+ increases the endothelial permeability by inhibition of PTEN expression. In addition, miR132-3p+ significantly increased the delivery of doxorubicin across the BTB in vitro and contributed to the accumulation of doxorubicin within the brain tumor tissue. Our results show that miR-132-3p contributes to the increased permeability of BTB by targeting PTEN/PI3K/PKB/Src/Cav-1, thereby revealing a novel drug target for the treatment of brain gliomas.-Gu, Y., Cai, R., Zhang, C., Xue, Y., Pan, Y., Wang, J., Zhang, Z. miR-132-3p boosts caveolae-mediated transcellular transport in glioma endothelial cells by targeting PTEN/PI3K/PKB/Src/Cav-1 signaling pathway.

Dual-modified natural high density lipoprotein particles for systemic glioma-targeting drug delivery

Therapeutic outcome for the treatment of glioma was often limited due to the two barriers involved: the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). Therefore, the development of nanocarriers that possess both BBB and BBTB permeability and glioma-targeting ability is of great importance for the chemotherapy of glioma. New frontiers in nanomedicine are advancing the research of new biomaterials. Here we constructed a natural high-density lipoprotein particle (HDL)-based drug delivery system with the dual-modification of T7 and dA7R peptide ligand (T7/dA7R-HDL) to achieve the above goals. HDL, the smallest lipoprotein, plays a biological role and is highly suitable as a platform for delivering imaging and therapeutic agents. T7 is a seven-peptide ligand of transferrin receptors (TfR) capable of circumventing the BBB and then targeting glioma. dA7R is a d-peptide ligand of vascular endothelial growth factor receptor 2 (VEGFR 2) overexpressed on angiogenesis, presenting excellent glioma-homing property. 10-Hydroxycamptothecin (HCPT), a hydrophobic anti-cancer drug, was used as the model drug in this study. By combining the dual-targeting delivery effect, the dual-modified HDL displayed higher glioma localization than that of single ligand-modified HDL or free HCPT. After loading with HCPT, T7/dA7R-HDL showed the most favorable anti-glioma effect in vivo. These results demonstrated that the dual-targeting natural nanocarriers strategy provides a potential method for improving brain drug delivery and anti-glioma treatment efficacy.

Increasing of Blood-Brain Tumor Barrier Permeability through Transcellular and Paracellular Pathways by Microbubble-Enhanced Diagnostic Ultrasound in a C6 Glioma Model.

Most of the anticancer agents cannot be efficiently delivered into the brain tumor because of the existence of blood-brain tumor barrier (BTB). The objective of this study was to explore the effect of microbubble-enhanced diagnostic ultrasound (MEUS) on the BTB permeability and the possible mechanism. Glioma-bearing rats were randomized into three groups as follows: the microbubble-enhanced continued diagnostic ultrasound (MECUS) group; the microbubble-enhanced intermittent diagnostic ultrasound (MEIUS) group and the control group. The gliomas were insonicated through the skull with a diagnostic ultrasound and injected with microbubbles through the tail veins. Evans Blue (EB) and dynamic contrast-enhanced-MRI were used to test changes in the BTB permeability. Confocal laser scanning microscopy was used to observe the deposition of the EB in the tumor tissues. The distribution and expression of junctional adhesion molecule-A (JAM-A) and calcium-activated potassium channels (KCa channels) were detected by a Western blot, qRT-PCR, and immunohistochemical staining. In the MEUS groups, the EB extravasation (11.0 ± 2.2 μg/g in MECUS group and 17.9 ± 2.3 μg/g in MEIUS group) exhibited a significant increase compared with the control group (5.3 ± 0.9 μg/g). The MEIUS group had more EB extravasation than the MECUS group. The Ktrans value of the dynamic contrast-enhanced-MRI in the MEUS groups was higher than that of the control group and correlated strongly with the EB extravasation in the tumor (R2 = 0.97). This showed that the Ktrans value might be a non-invasive method to evaluate the BTB permeability in rat glioma after microbubble-enhanced ultrasound treatment.Western blot, qRT-PCR and immunohistochemical staining revealed that MEUS increased the KCa channels expression and reduced JAM-A expression in glioma. This change was more obvious in the MEIUS group than in the MECUS group. The results demonstrated that MEUS effectively increased the BTB permeability in glioma. The mechanisms might involve the up-regulation of KCa channels expression and affecting the formation of tight junctions in the BTB by a reduction of JAM-A expression. These findings might provide some new guidance for glioma drug therapy.

Minoxidil sulfate induced the increase in blood–brain tumor barrier permeability through ROS/RhoA/PI3K/PKB signaling pathway

Adenosine 5′-triphosphate-sensitive potassium channel (KATP channel) activator, minoxidil sulfate (MS), can selectively increase the permeability of the blood–tumor barrier (BTB); however, the mechanism by which this occurs is still under investigation. Using a rat brain glioma (C6) model, we first examined the expression levels of occludin and claudin-5 at different time points after intracarotid infusion of MS (30 μg/kg/min) by western blotting. Compared to MS treatment for 0 min group, the protein expression levels of occludin and claudin-5 in brain tumor tissue of rats showed no changes within 1 h and began to decrease significantly after 2 h of MS infusion. Based on these findings, we then used an in vitro BTB model and selective inhibitors of diverse signaling pathways to investigate whether reactive oxygen species (ROS)/RhoA/PI3K/PKB pathway play a key role in the process of the increase of BTB permeability induced by MS. The inhibitor of ROS or RhoA or PI3K or PKB significantly attenuated the expression of tight junction (TJ) protein and the increase of the BTB permeability after 2 h of MS treatment. In addition, the significant increases in RhoA activity and PKB phosphorylation after MS administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine (MPG) or C3 exoenzyme or LY294002 pretreatment. The present study indicates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in BTB was required for the increase of BTB permeability induced by MS. Taken together, all of these results suggested that MS might increase BTB permeability in a time-dependent manner by down-regulating TJ protein expression and this effect could be related to ROS/RhoA/PI3K/PKB signal pathway.

Targeting potassium channels for increasing delivery of imaging agents and therapeutics to brain tumors

Every year in the US, 20,000 new primary and nearly 200,000 metastatic brain tumor cases are reported. The cerebral microvessels/capillaries that form the blood–brain barrier not only protect the brain from toxic agents in the blood but also pose a significant hindrance to the delivery of small and large therapeutic molecules. Different strategies have been employed to circumvent the physiological barrier posed by blood–brain tumor barrier (BTB). Studies in our laboratory have identified significant differences in the expression levels of certain genes and proteins between normal and brain tumor capillary endothelial cells (ECs). In this study, we validated the non-invasive and clinically relevant dynamic contrast enhancing-magnetic resonance imaging (DCE-MRI) method with invasive, clinically irrelevant but highly accurate quantitative autoradiography method using rat glioma model. We also showed that DCE-MRI metric of tissue vessel perfusion-permeability is sensitive to changes in blood vessel permeability following administration of calcium-activated potassium (BKCa) channel activator NS-1619. Our results show that human gliomas and brain tumor ECs that overexpress BKCa channels can be targeted for increased BTB permeability for MRI enhancing agents to brain tumors. We conclude that monitoring the outcome of increased MRI enhancing agents’ delivery to microsatellites and leading tumor edges in glioma patients would lead to beneficial clinical outcome.

Induction of Selective Blood-Tumor Barrier Permeability and Macromolecular Transport by a Biostable Kinin B1 Receptor Agonist in a Glioma Rat Model

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[dPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T 1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.

Adenosine 5′-Triphosphate-Sensitive Potassium Channel Activator Induces the Up-Regulation of Caveolin-1 Expression in a Rat Brain Tumor Model

This study was performed to determine whether minoxidil sulfate (MS), a selective Adenosine 5'-triphosphate-sensitive potassium channel (K (ATP) channel) activator, has an effect on the expression of caveolin-1 in the rat's brain tumor tissue. Using a rat brain glioma (C6) model, we found that the expression of caveolin-1 protein at tumor sites was greatly increased after intracarotid infusion of MS at a dose of 30 μg/kg/min for 15, 30, and 60 min via Western blot analysis. And the peak value of the caveolin-1 expression was observed in rats with glioma after 15 min of MS perfusion, which was significantly attenuated by reactive oxygen species (ROS) scavenger (N-2-mercaptopropionyl glycine, MPG). In addition, MPG also significantly inhibited the increase of blood-brain tumor barrier (BTB) permeability which was induced by MS. This led to the conclusion that the MS-induced BTB permeability increase may be related to the accelerated formation of caveolin-1 protein, and could be mediated by ROS.

The modulation of protein kinase A and heat shock protein 70 is involved in the reversible increase of blood–brain tumor barrier permeability induced by papaverine

Intra-arterial administration of papaverine has been revealed to cause an increase in the blood–brain tumor barrier (BTB) permeability. The exact mechanism of papaverine opening the BTB in chemotherapy of malignant cerebral tumors, however, has not been well described. We used a rat brain glioma (C6) model for studying how papaverine modulates the permeability of BTB by monitoring the activities of the tight junction (TJ)-associated protein occludin, claudin-5 and cytoskeletal protein filamentous actin (F-actin) and whether protein kinase A (PKA) and heat shock protein 70 (HSP70) were involved in the regulation of this biological process. The levels of occludin, claudin-5 and F-actin protein in the tumor tissues were down-regulated by papaverine via immunohistochemistry, immunofluorescence assays and Western blot, corresponding to the time-dependent change of the BTB permeability. The most obvious attenuation of occludin, claudin-5 and F-actin protein was observed at 1 h after papaverine perfusion, companied by a significant decrease in expression levels of PKA protein. The expression level of HSP70 in the tumor tissues was also progressively increased after papaverine perfusion and reached the maximum at 3 h. The results demonstrate that the reversible openning of BTB mediated by papaverine may be associated with the functional combination between PKA and HSP70. That is, BTB opening may be attributable to the down-regulation of occludin, claudin-5 and F-actin, and cAMP/PKA signaling pathway might be involved in this process. HSP70 is likely responsible for the BTB closing, which helping the repairment of injured TJ protein and the rebuilding of the BTB.

Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models

BackgroundChemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.Methodology/Principal FindingsWe examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).Conclusions/SignificanceThese findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.

Abstract 1447: PDE5 inhibitors increase trastuzumab transport for treatment of HER2-positive metastatic brain tumors

Abstract Objective: Chemotherapeutic drugs and newly developed therapeutic antibodies can be adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment. Our previous publication showed that phosphodiesterase type (PDE) 5 inhibitors enhance brain tumor permeability and efficacy of chemotherapy in a rat brain tumor model. Here, we further examined the effect of PDE 5 inhibitors on drug delivery to intracranial human lung and breast tumors in nude mice as a model of brain metastasis, and the underlying mechanisms of drug transport. Methods: In the in vitro setting, a Corning transwell system seeded with mouse brain endothelial cells (MBEC) was used for cellular [14C]dextran transport study as a model of blood brain barrier (BBB). For in vitro uptake of a therapeutic monoclonal antibody to HER2/neu, trastuzumab (Herceptin®), MBEC were treated with labeled drug with or without pre-incubation with vardenafil. To study the underlying mechanisms of transport, inhibitors of caveolae and macropinocytosis endocytic pathways were used to block the effect of PDE5 inhibitors. In the in vivo study, vardenafil was given at an oral dose of 10 mg/kg for transport of [14C]dextran and labeled trastuzumab to mice with human lung tumor growing in the brain. For the survival experiments, HER2/neu-positive human lung or breast intracranial tumor-bearing mice were randomly divided into four groups treated as follows: (1) control saline; (2) vardenafil (10 mg/kg, po); (3) trastuzumab (10 mg/kg, iv); and (4) vardenafil (10 mg/kg, P.O.) + trastuzumab (10 mg/kg, I.V.). Results: In vitro assay demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab by cultured MBECs. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced trastuzumab uptake. Oral administration of PDE5 inhibitor vardenafil to mice with intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to trastuzumab (2-fold increase). Survival of intracranial lung cancer-bearing mice treated with trastuzumab in combination with vardenafil significantly increased as compared to the untreated, vardenafil alone- or trastuzumab alone-treated mice (p&amp;lt;0.01). In mice bearing intracranial breast tumors, log-rank analysis of survival curves also showed a significant survival increase (p&amp;lt;0.02) in the group treated with trastuzumab plus vardenafil as compared to other groups. Conclusion: These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability and enhance drug delivery, and may increase the efficacy of therapeutic monoclonal antibodies in hard-to-treat brain metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1447.

Dexamethasone enhances calcium-activated potassium channel expression in blood-brain tumor barrier in a rat brain tumor model

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels ( K Ca channels) in blood-brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of K Ca channels protein was significantly increased in brain tumor tissue. And bradykinin-induced increase of K Ca channels protein was further enhanced after DEX pretreatment for 3 days. In addition, DEX pretreatment enhanced bradykinin-mediated up-regulation of the density of I KCa in the rat brain C6 cells in vitro BTB. Bradykinin markedly increased BTB permeability independent of DEX pretreatment. All of these results strongly suggest that DEX could regulate the target in the transcellular pathway of BTB- K Ca channels.

The molecular mechanism of dexamethasone-mediated effect on the blood–brain tumor barrier permeability in a rat brain tumor model

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (KCa channels) and Occludin protein in blood–brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of KCa channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of IKCa in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.

Increase in Brain Tumor Permeability in Glioma-Bearing Rats with Nitric Oxide Donors

The blood-brain tumor barrier (BTB) significantly limits the delivery of chemotherapeutics to brain tumors. Nitric oxide (NO) is involved in the regulation of cerebral vascular permeability. We investigated the effects of NO donors, L-arginine and hydroxyurea, on BTB permeability in 9L gliosarcoma-bearing Fischer rats. The rats implanted with 9L gliosarcoma were dosed orally with hydroxyurea and L-arginine. BTB permeability, defined by the unidirectional transport constant, Ki, for [14C]sucrose was measured. The expression of neural and endothelial NO synthase (NOS) in tumors and normal brain tissue was examined. Further, the levels of NO, L-citrulline, and cGMP in the tumor and normal brain tissue were measured. Oral administration of l-arginine or hydroxyurea significantly increased BTB permeability when compared with the nontreated control. The selective effects were abolished by iberiotoxin, an antagonist of calcium-dependent potassium (KCa) channel that is a cGMP pathway effector. The expression of endothelial NOS, but not neural NOS, was higher in tumor vessels than in those of normal brain. Moreover, the levels of NO, L-citrulline, a byproduct of NO formation from L-arginine, and cGMP were enhanced in the tumor tissue by oral administration of L-arginine and/or hydroxyurea. Oral administration of L-arginine or hydroxyurea selectively increased tumor permeability, which is likely mediated by alteration in cGMP levels. The findings suggest that use of oral NO donors may be a strategy to enhance the delivery of chemotherapeutics to malignant brain tumors.

Calcium-activated potassium channels mediated blood-brain tumor barrier opening in a rat metastatic brain tumor model

BackgroundThe blood-brain tumor barrier (BTB) impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases.ResultsIn this study, we examined the function and regulation of calcium-activated potassium (KCa) channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R) expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain), human brain microvessel endothelial cells (HBMEC) and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening.ConclusionThese findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.

Bradykinin-induced blood–brain tumor barrier permeability increase is mediated by adenosine 5′-triphosphate-sensitive potassium channel

Bradykinin has been shown to selectively transiently increase the permeability of the blood–brain barrier (BBB). This study was performed to determine whether ATP-sensitive potassium ( K ATP) channels mediate the increase in permeability of brain tumor microvessels induced by BK. Using a rat brain glioma (C6) model, we found increased expression of K ATP channels at tumor sites via Western blot analysis, after intracarotid infusion of bradykinin at a dose of 10 μg/kg/min for 15 min. A significant increase (73.58%) of the integrated density value (IDV) of the K ATP channel Kir6.2 subunit was observed in rats with glioma after 10 min of bradykinin perfusion. The over-expression of K ATP channels with bradykinin was significantly attenuated by the K ATP channel antagonist glibenclamide. Immunohistochemistry and immunolocalization experiments showed that the over-expression of K ATP channels was more obvious near tumor capillaries of 10 μm in diameter. I KATP modulation by bradykinin in cultured C6 cells was also studied using the patch-clamp technique in a whole-cell configuration. Administration of bradykinin led to a significant opening of K ATP channels in a time-dependent manner. This led to the conclusion that the bradykinin-mediated BBB permeability increase is due to accelerated formation of K ATP channels, which are thus as an important target in the biochemical regulation of this process.

Modulation of brain tumor capillaries for enhanced drug delivery selectively to brain tumor.

The blood-brain tumor barrier (BTB) significantly impedes delivery of most hydrophilic molecules to brain tumors. Several promising strategies, however, have been developed to overcome this problem. We discuss several drug delivery methods to brain tumor, including intracerebroventricular, convection enhanced delivery, BBB/BTB disruption, and BTB permeability modulation, which was developed in our laboratory. Using immunolocalization, immunoblotting, and potentiometric studies, we found that brain tumor capillary endothelial cells overexpress certain unique protein markers that are absent or barely detectable in normal capillary endothelial cells. We biochemically modulated these markers to sustain and enhance drug delivery, including molecules of varying sizes, selectively to tumors in rat syngeneic and xenograft brain tumor models. We also demonstrated that the cellular mechanism for vasomodulator-mediated BTB permeability increase is due to accelerated formation of pinocytotic vesicles that transport therapeutic molecules across the BTB. Other methods to deliver drugs across the BTB are effective but have severe drawbacks. Our strategy targets BTB-specific proteins to increase antineoplastic drug delivery selectively to brain tumors with few or no side effects, thus increasing the possibility of improving brain tumor treatment.

Calcium-dependent potassium channels as a target protein for modulation of the blood-brain tumor barrier.

Even though the blood-brain tumor barrier (BTB) is more permeable than the blood-brain barrier (BBB), the BTB still significantly restricts the delivery of anticancer drugs to brain tumors. Brain tumor capillaries that form the BTB, however, express certain unique protein markers that are absent or barely detectable in normal brain capillaries. We were able to biochemically modulate one such protein marker, the calcium-dependent potassium (K(Ca)) channel, by using a specific K(Ca) channel agonist, NS-1619, to obtain sustained enhancement of selective drug delivery, including molecules of varying sizes, to tumors in rat syngeneic and xenograft brain tumor models. Immunolocalization and potentiometric studies showed increased K(Ca) channel distribution on tumor cells compared with normal cells, suggesting that tumor cell-specific signals might induce overexpression of K(Ca) channels in capillary endothelial cells, leading to increased BTB permeability. We also demonstrated that the cellular mechanism for K(Ca) channel-mediated BTB permeability increase is due to accelerated formation of pinocytotic vesicles, which can transport therapeutic molecules across the BTB. This concept was investigated by using NS-1619 to facilitate increased delivery of carboplatin to brain tumor leading to enhanced survival in rats with brain tumors. Additionally, we showed that K(Ca) channel modulation resulted in enhanced permeability to macromolecules, including Her-2 monoclonal antibody and green fluorescent protein-adenoviral vectors, in a human, primary brain-tumor xenograft model. Therefore, K(Ca) channels are a potential, promising target for biochemical modulation of BTB permeability to increase antineoplastic drug delivery selectively to brain tumors.

Regulation of blood-brain tumor barrier permeability by calcium-activated potassium channels.

The blood-brain tumor barrier (BTB) limits the delivery of therapeutic drugs to brain tumors. We demonstrate in a rat brain tumor (RG2) model an enhanced drug delivery to brain tumor following intracarotid infusion of bradykinin (BK), nitric oxide (NO) donors, or agonists of soluble guanylate cyclase (sGC) and calcium-dependent potassium (K(Ca)) channels. We modulated K(Ca) channels by specific agonists and agents that produce NO and cGMP in situ to obtain sustained enhancement of selective drug delivery to brain tumors. Intracarotid infusion of BK or 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) significantly enhanced BTB permeability (K(i)) to [(14)C]alpha-aminoisobutyric acid in the brain tumor area but not in normal brain tissue. The K(i) increase achieved by BK, NS-1619, NO donors, or the sGC activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazole (YC-1) was significantly attenuated when coinfused with a K(Ca) channel antagonist, iberiotoxin. Immunoblot and immunolocalization studies demonstrate overexpression of K(Ca) channels in tumor cells and capillaries compared with normal brain. The potentiometric assays demonstrate the functional activity of K(Ca) channels in rat brain endothelial and glioma cells. Additionally, we show that BK and NS-1619 significantly increased the density of transport vesicles in the cytoplasm of brain tumor capillary endothelia and tumor cells. The cleft indices and cleft area indices in rat tumor capillaries were significantly higher than in normal brain capillaries, and BK infusion did not alter these indices. These data demonstrate that the cellular mechanism for K(Ca) channel-mediated BTB permeability increase is due to accelerated formation of pinocytotic vesicles, which can transport drugs across BTB. We conclude that K(Ca) channels serve as a convergence point in the biochemical regulation of BTB permeability.