Rutin increases the cytotoxicity of temozolomide in glioblastoma via autophagy inhibition.

The chemotherapeutic agent temozolomide (TMZ) is widely used in the treatment of glioblastoma multiforme (GBM). Rutin, a citrus flavonoid ecglycoside found in edible plants, has neuroprotective and anticancer activities. This study aimed to investigate the efficacy and the underlying mechanisms of rutin used in combination with TMZ in GBM. In vitro cell viability assay demonstrated that rutin alone had generally low cytotoxic effect, but it enhanced the efficacy of TMZ in a dose-dependent manner. Subcutaneous and orthotopic xenograft studies also showed that tumor volumes were significantly lower in mice receiving combined TMZ/Rutin treatment as compared to TMZ or rutin alone treatment. Moreover, immunoblotting analysis showed that TMZ activated JNK activity to induce protective response autophagy, which was blocked by rutin, resulting in decreased autophagy and increased apoptosis, suggesting that rutin enhances TMZ efficacy both in vitro and in vivo via inhibiting JNK-mediated autophagy in GBM. The combination rutin with TMZ may be a potentially useful therapeutic approach for GBM patient.

IDH1 mutation-associated long non-coding RNA expression profile changes in glioma.

Isocitrate dehydrogenase 1 (IDH1) mutation is an important prognostic marker in glioma. However, its downstream effect remains incompletely understood. Long non-coding RNAs (lncRNAs) are emerging as important regulators of tumorigenesis in a number of human malignancies, including glioma. Here, we investigated whether and how lncRNA expression profiles would differ between gliomas with or without IDH1 mutation. By using our previously reported lncRNA mining approach, we performed lncRNA profiling in three public glioma microarray datasets. The differential lncRNA expression analysis was then conducted between mutant-type and wild-type IDH1 glioma samples. Comparison analysis identified 14 and 9 lncRNA probe sets that showed significantly altered expressions in astrocytic and oligodendroglial tumors, respectively (fold change ≥ 1.5, false discovery rate ≤ 0.1). Moreover, the differential expressions of these lncRNAs could be confirmed in the independent testing sets. Functional exploration of the lncRNAs by analyzing the lncRNA-protein interactions revealed that these IDH1 mutation-associated lncRNAs were involved in multiple tumor-associated cellular processes, including metabolism, cell growth and apoptosis. Our data suggest the potential roles of lncRNA in gliomagenesis, and may help to understand the pathogenesis of gliomas associated with IDH1 mutation.

Outcome of elderly patients undergoing intracranial meningioma resection--a systematic review and meta-analysis.

BACKGROUND: Intracranial meningioma is a common condition in the elderly population. Surgical resection in this group of patients may be rendered more hazardous due to the patients' ageing physiology and to multiple comorbidities. This systematic review and meta-analysis aimed to summarise outcome data of elderly patients undergoing intracranial meningioma resection. METHODS: Using Ovid Medline, longitudinal studies published from 2002 to October 2012 with patients aged ≥ 65 years that described outcomes after intracranial meningioma resection were reviewed. Outcome data included mortality, recurrence, complication rate and length of hospital stay (LoS). Grading score systems and covariates for predicting outcome were collected. Pooled estimates of mortality data were calculated in StatsDirect using a random effects method. I(2) statistic was used to assess heterogeneity. RESULTS: Thirteen eligible studies with a total of 7010 patients (mean age, 73.6 years) were included, in which 82% patients came from one study. The pooled estimates of 90-day and 1-year mortality from available data were 6.6% (95% confidence interval [CI], 4.6-9.1%; n = 735; I(2) = 32.1) and 9.6% (95% CI, 7.0-12.6%; n = 564; I(2) = 24.3), respectively. The overall complication rates ranged from 2.7% to 29.8%, and the overall incidence of complications was 20% per patient (range, 3-61%). Other outcome data were heterogeneous mainly due to incomparable study designs. CONCLUSIONS: Current evidence indicates satisfactory surgical outcomes in the elderly with intracranial meningiomas, though the risks of complications necessitate careful consideration when deciding to operate. Risk factor analysis emphasised the importance of considering pre-operative status and comorbidities during patient selection. Future research should address the causes and prevention of complications, and compare outcomes between younger and older patients using detailed stratifications of tumour characteristics.

A long non-coding RNA signature in glioblastoma multiforme predicts survival.

Long non-coding RNAs (lncRNAs) represent the leading edge of cancer research, and have been implicated in cancer biogenesis and prognosis. We aimed to identify lncRNA signatures that have prognostic values in glioblastoma multiforme (GBM). Using a lncRNA-mining approach, we performed lncRNA expression profiling in 213 GBM tumors from The Cancer Genome Atlas (TCGA), randomly divided into a training (n=107) and a testing set (n=106). We analyzed the associations between lncRNA signatures and clinical outcome in the training set, and validated the findings in the testing set. We also validated the identified lncRNA signature in another two independent GBM data sets from Gene Expression Omnibus (GEO), which contained specimens from 68 and 101 patients, respectively. We identified a set of six lncRNAs that were significantly associated with the overall survival in the training set (P≤0.01). Based on this six-lncRNA signature, the training-set patients could be classified into high-risk and low-risk subgroups with significantly different survival (HR=2.13, 95% CI=1.38-3.29; P=0.001). The prognostic value of this six-lncRNA signature was confirmed in the testing set and the two independent data sets. Further analysis revealed that the prognostic value of this signature was independent of age and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. The identification of the prognostic lncRNAs indicates the potential roles of lncRNAs in GBM pathogenesis. This six-lncRNA signature may have clinical implications in the subclassification of GBM.

Outcome comparison between younger and older patients undergoing intracranial meningioma resections.

Studies directly comparing the outcomes of intracranial meningioma resection between elderly and younger patients are currently limited. This study aimed to assess the perioperative complications, mortalities and functional outcomes in these two groups. Consecutive elderly patients (aged ≥ 65) and tumor-location-matched younger patients who underwent intracranial meningioma resections were retrospectively reviewed. Outcomes were assessed at 30-day, 90-day, 6-month and 1-year. We used a standardized classification of operative complications, and conducted subgroup analyses based on tumor location [convexity, parasagittal and falcine (CPF) as one group; skull base (SB) as another]. There were 92 patients in each group. The mean age was 74.6 ± 6.4 years in the elderly and 49.3 ± 10.1 years in the younger groups. The cumulative 30-day, 90-day and 1-year mortality rates were 0, 2.2 and 4.3 % for the elderly, respectively, and 1.1 % for all time points in the young. These differences were not statistically significant. Overall, the elderly suffered from more perioperative complications (P = 0.010), and these were mostly minor complications according to the classification of operative complications. However, these differences were observed only in the SB but not in the CPF subgroup. More elderly patients had impaired functional outcome 1-year after surgery. Significantly more elderly patients had new neurological deficits 1-year after surgery (26.1 vs. 6.6 %; P = 0.001). Comparable mortality rates were observed in elderly and younger patients. However, the elderly had more minor complications and poorer functional outcomes. Patient selection remains key to good clinical outcome.

Targeted gene expression profiling predicts meningioma outcomes and radiotherapy responses.

Surgery is the mainstay of treatment for meningioma, the most common primary intracranial tumor, but improvements in meningioma risk stratification are needed and indications for postoperative radiotherapy are controversial. Here we develop a targeted gene expression biomarker that predicts meningioma outcomes and radiotherapy responses. Using a discovery cohort of 173 meningiomas, we developed a 34-gene expression risk score and performed clinical and analytical validation of this biomarker on independent meningiomas from 12 institutions across 3 continents (N = 1,856), including 103 meningiomas from a prospective clinical trial. The gene expression biomarker improved discrimination of outcomes compared with all other systems tested (N = 9) in the clinical validation cohort for local recurrence (5-year area under the curve (AUC) 0.81) and overall survival (5-year AUC 0.80). The increase in AUC compared with the standard of care, World Health Organization 2021 grade, was 0.11 for local recurrence (95% confidence interval 0.07 to 0.17, P < 0.001). The gene expression biomarker identified meningiomas benefiting from postoperative radiotherapy (hazard ratio 0.54, 95% confidence interval 0.37 to 0.78, P = 0.0001) and suggested postoperative management could be refined for 29.8% of patients. In sum, our results identify a targeted gene expression biomarker that improves discrimination of meningioma outcomes, including prediction of postoperative radiotherapy responses.

Long non-coding RNA expression profiles predict clinical phenotypes in glioma.

Glioma is the commonest form of primary brain tumor in adults with varying malignancy grades and histological subtypes. Long non-coding RNAs (lncRNAs) are a novel class of non-protein-coding transcripts that have been shown to play important roles in cancer development. To discover novel tumor-related lncRNAs and determine their associations with glioma subtypes, we first applied a lncRNA classification pipeline to identify 1970 lncRNAs that were represented on Affymetrix HG-U133 Plus 2.0 array. We then analyzed the lncRNA expression patterns in a set of previously published glioma gene expression profiles of 268 clinical specimens, and identified sets of lncRNAs that were unique to different histological subtypes (astrocytic versus oligodendroglial tumors) and malignancy grades. These lncRNAs signatures were then subject to validation in another non-overlapping, independent data set that contained 157 glioma samples. This is the first reported study that correlates lncRNA expression profiles with malignancy grade and histological differentiation in human gliomas. Our findings indicate the potential roles of lncRNAs in the biogenesis, development and differentiation of gliomas, and provide an important platform for future studies.

Long-term outcome and prognostic factors after spontaneous cerebellar hemorrhage.

Cerebellar hemorrhage is the least common type of intracranial hemorrhage (ICH) encountered in clinical practice, and clinical data concerning the long-term outcomes are limited. This study aimed to investigate the long-term outcomes following spontaneous cerebellar hemorrhage in a cohort of Chinese patients. This single-center observational study was carried out between 1996 and 2010 and included 72 consecutive Chinese patients with a first spontaneous cerebellar hemorrhage. Of 440 patients with primary ICH, 72 (16.4%) had primary cerebellar hemorrhage. The mean age was 67.5 ± 12.3 years and patients were predominantly male (54%). The 30-day mortality was 16.7% with Glasgow coma scale ≤ 8 as the only predictor. At 6 months, 56.7% of patients who survived the first 30 days had a persistently poor functional status with modified Rankin scale score >2. After a mean follow-up of 4.7 years, 3.3% of patients had recurrent ICH, a recurrence rate of 7.3 per 1,000 patient-years. Ischemic stroke occurred in 12% of patients, an incidence of 25.5 per 1,000 patient-years. This study provided data on the long-term outcome of post-cerebellar hemorrhage in Chinese patients.

Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities.

Meningiomas are the most common primary intracranial tumors. There are no effective medical therapies for meningioma patients, and new treatments have been encumbered by limited understanding of meningioma biology. Here, we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, proteomic and single-cell approaches to show meningiomas are composed of three DNA methylation groups with distinct clinical outcomes, biological drivers and therapeutic vulnerabilities. Merlin-intact meningiomas (34%) have the best outcomes and are distinguished by NF2/Merlin regulation of susceptibility to cytotoxic therapy. Immune-enriched meningiomas (38%) have intermediate outcomes and are distinguished by immune infiltration, HLA expression and lymphatic vessels. Hypermitotic meningiomas (28%) have the worst outcomes and are distinguished by convergent genetic and epigenetic mechanisms driving the cell cycle and resistance to cytotoxic therapy. To translate these findings into clinical practice, we show cytostatic cell cycle inhibitors attenuate meningioma growth in cell culture, organoids, xenografts and patients.

Acupuncture as Part of Iatrogenic Facial Nerve Palsy Rehabilitation-First Report.

BACKGROUND: Facial paresis is one of the complications after treatment for vestibular schwannoma (VS). Acupuncture has been used for Bell palsy but not in iatrogenic facial paresis. The objective of this study is to measure the efficacy of using acupuncture for iatrogenic facial nerve palsy and patients' satisfaction. METHODS: This is a single-center retrospective study with patients from 2007-2019 received treatment for newly diagnosed or recurrent VS. Some patients who suffered facial paresis after surgery had self-initiated acupuncture. All patients who had facial paresis were included. Their facial nerve status before and immediately after surgery, postoperative 6 months and 12 months, were recorded. Those who received acupuncture also answered 6- and 12-month patient satisfaction surveys over the phone. Adverse effects were also assessed. RESULTS: There were 123 patients in this period. Of these, 29 patients had iatrogenic facial paresis and 23 of them received acupuncture. There was significant improvement of facial paresis for the acupuncture group compared with the nonacupuncture group at 6 and 12 months. More than 80% of patients who received acupuncture were satisfied. They had motor improvement and experienced less pain and tightness. No adverse effects were reported. CONCLUSIONS: Acupuncture for postresection VS facial paresis seemed to speed up its recovery. Both patients' recovery and satisfaction were good after acupuncture, and it seemed to be a safe procedure in trained hands.

Self-assembling peptide nanofiber scaffold promotes the reconstruction of acutely injured brain.

Traumatic brain injury (TBI) or brain surgery may cause extensive loss of cerebral parenchyma. However, no strategy for reconstruction has been clinically effective. Our previous study had shown that self-assembling peptide nanofiber scaffold (SAPNS) can bridge the injured spinal cord, elicit axon regeneration, and eventually promote locomotor functional recovery. In the present study we investigated the effect of SAPNS for the reconstruction of acutely injured brain. The lesion cavity of the injured cortex was filled with SAPNS or saline immediately after surgically induced TBI, and the rats were killed 2 days, 2 weeks, or 6 weeks after the surgery for histology, immunohistochemistry, and TUNEL studies. Saline treatment in the control animals resulted in a large cavity in the injured brain, whereas no cavity of any significant size was found in the SAPNS-treated animals. Around the lesion site in control animals were many macrophages (ED1 positive) but few TUNEL-positive cells, indicating that the TBI caused secondary tissue loss mainly by means of necrosis, not apoptosis. In the SAPNS-treated animals the graft of SAPNS integrated well with the host tissue with no obvious gaps. Moreover, there were fewer astrocytes (GFAP positive) and macrophages (ED1 positive) around the lesion site in the SAPNS-treated animals than were found in the controls. Thus, SAPNS may help to reconstruct the acutely injured brain and reduce the glial reaction and inflammation in the surrounding brain tissue. FROM THE CLINICAL EDITOR: Self-assembling peptide nanofiber scaffold (SAPNS) was reported earlier to bridge the injured spinal cord, elicit axon regeneration, and promote locomotor recovery. In this study the effect of SAPNS for the reconstruction of acutely injured brain was investigated. In SAPNS-treated animals the graft integrated well with the host tissue with no obvious gaps. SAPNS may help to reconstruct the acutely injured brain and reduced the glial reaction and inflammation in the surrounding brain tissue.

Repair of Anterior Skull Base Defect by Dual-Layer/Split-Frontal Pericranial Flap.

INTRODUCTION: Normal skull base structures are destroyed either by the skull base pathology itself or during surgery that results in cerebrospinal fluid (CSF) leak. Skull base repair is usually performed transnasally by using a nasal septal flap. But when NSF is not available and failed repeated transnasal repair, a cranionasal repair with frontal pericranial flap (PF) can be attempted to achieve the highest successful chance. We performed a dual layer/split PF repair of a skull base defect using cranionasal approach and here we describe the technique. CASE DESCRIPTION: A 74-year-old man suffered from CSF leak and ventriculitis after multiple transnasal surgeries for pituitary macroadenoma despite multiple repairs with intranasal vascularized flaps. We performed repair by cranionasal approach and using frontal PF. The frontal PF was divided into left and right halves. The left half went intradural, through the chiasmatic window to reach the sella and cover the tubercular and sellar defect from above. The right half went through the nasion into the nose to cover the defect from below. So the defect was covered by two vascularized flap from a single frontal PF. The repair was successful with no CSF leak and infection successfully treated by antibiotics. CONCLUSION: We introduced the cranionasal dual layer/split PF technique to repair anterior skull base and successfully stopped the CSF leak in a patient after multiple EEA surgeries. This technique should only be considered in specific difficult situation where usual repair by EEA is impossible.

Improving Survival with Tranexamic Acid in Cerebral Contusions or Traumatic Subarachnoid Hemorrhage: Univariate and Multivariate Analysis of Independent Factors Associated with Lower Mortality.

BACKGROUND: Fall with head injury is a pervasive challenge, especially in the aging population. Contributing factors for mortality include the development of cerebral contusions and delayed traumatic intracerebral hematoma. Currently, there is no established specific treatment for these conditions. OBJECT: This study aimed to investigate the impact of independent factors on the mortality rate of traumatic brain injury with contusions or traumatic subarachnoid hemorrhage. METHODS: Data were collected from consecutive patients admitted for cerebral contusions or traumatic subarachnoid hemorrhage at an academic trauma center from 2010 to 2016. The primary outcome was the 30-day mortality rate. Independent factors for analysis included patient factors and treatment modalities. Univariate and multivariate analyses were conducted to identify independent factors related to mortality. Secondary outcomes included thromboembolic complication rates associated with the use of tranexamic acid. RESULTS: In total, 651 consecutive patients were identified. For the patient factors, low Glasgow Coma Scale on admission, history of renal impairment, and use of warfarin were identified as independent factors associated with higher mortality from univariate and multivariate analyses. For the treatment modalities, univariate analysis identified tranexamic acid as an independent factor associated with lower mortality (P = 0.021). Thromboembolic events were comparable in patients with or without tranexamic acid. CONCLUSION: Tranexamic acid was identified by univariate analysis as an independent factor associated with lower mortality in cerebral contusions or traumatic subarachnoid hemorrhage. Further prospective studies are needed to validate this finding.

Iatrogenic Sigmoid Sinus Occlusion Rescued by Graft Repair and Endovascular Thrombolysis.

BACKGROUND: Iatrogenic cerebral venous sinus injury and occlusion may occur during resection of parasagittal meningioma and lateral skull base surgery. The former involves the superior sagittal sinus, and direct surgical repair is associated with good results. Outcome of direct repair of transverse-sigmoid sinus injury is less clear. We present a patient with iatrogenic sigmoid sinus injury in whom direct repair was complicated by subsequent thrombosis that was successfully salvaged by combined endovascular mechanical and chemical thrombolysis. CASE DESCRIPTION: A 60-year-old man with left tentorial atypical meningioma had disease recurrence after 3 excisions. Angiography revealed that the straight sinus and torcular and bilateral transverse sinuses were occluded. He underwent a fourth craniotomy with inadvertent occlusion of the transverse-sigmoid sinus junction. Direct surgical repair was done but was complicated by thrombosis. Mechanical endovenous thrombectomy was done followed by continuous urokinase infusion for 1 week. Digital subtraction angiography performed 7 days after endovascular treatment showed improved venous drainage through the left transverse-sigmoid sinus junction. The patient was ambulatory and fully independent, with no new neurologic deficit. CONCLUSIONS: This case emphasizes the need to preserve every vein, especially when major venous sinuses have been obliterated. Detailed study of high-quality preoperative digital subtraction angiography is extremely important. Venous injury should be repaired immediately whenever possible. Postrepair venous sinus thrombosis may be effectively salvaged by endovascular thrombectomy for rapid recannulation, with or without combined use of continuous in situ thrombolytic therapy.

CRNDE Expression Positively Correlates with EGFR Activation and Modulates Glioma Cell Growth.

BACKGROUND: The long non-coding RNA CRNDE has emerged as an important regulator in carcinogenesis and cancer progression. While CRNDE has previously been found to be the most highly upregulated lncRNA in glioma, detailed information on its roles in regulating cancer cell growth remains limited. OBJECTIVE: In the present study, we aimed at exploring the functional roles and underlying mechanisms of CRNDE in glioma. METHODS: We applied microarray data analysis to determine the prognostic significance of CRNDE in glioma patients and its correlation with epidermal growth factor receptor (EGFR) activation. EGFR inhibition was used to confirm the role of EGFR in regulating CRNDE expression. Functional studies were performed upon CRNDE silencing to explore its role in gliomagenesis. RESULTS: We confirm that CRNDE acts as an oncogene that is highly up-regulated in glioma, and high CRNDE expression correlates with poor prognosis in glioma patients. We further demonstrate that the expression of CRNDE correlates with EGFR activation. EGF and EGFR tyrosine kinase inhibitor (TKI) enhance and block the up-regulation of CRNDE expression, respectively, suggesting that EGFR signaling may positively regulate CRNDE expression. Functional assays show that CRNDE depletion inhibits glioma cell growth both in vitro and in vivo, and is associated with induced cellular apoptosis with decreased Bcl2/Bax ratio. CONCLUSIONS: Our findings suggest that the aberrant expression of CRNDE may be mediated by activated EGFR signaling and play significant roles in gliomagenesis.

Primary Central Nervous System Natural Killer Cell Lymphoma in a Chinese Woman with Atypical (11)C-Choline Positron Emission Tomography and Magnetic Resonance Spectrometry Findings.

BACKGROUND: Primary central nervous system (CNS) natural killer (NK)-cell lymphoma is rare with only 7 cases reported previously. Magnetic resonance spectroscopy (MRS) and [(18)F]fluorodeoxyglucose (FDG) positron emission tomography (PET) are frequently used for disease diagnosis and monitoring. Choline (CHO) PET is gaining popularity for identifying CNS lesions because of its high disease to background radioactivity ratio compared with FDG. Normally, CNS lymphoma shows high choline uptake on CHO-PET and a high choline peak on MRS. We present an unusual case of primary CNS NK-cell lymphoma with high choline uptake but absence of a high choline peak on MRS. CASE DESCRIPTION: A 39-year-old woman presented with subacute onset of cognitive deterioration. Magnetic resonance imaging of the brain showed a gadolinium-enhancing lesion in the left temporal lobe. MRS showed suppressed N-acetyl-aspartate and the absence of a high choline peak. CHO-PET confirmed that it was the only hypermetabolic lesion in the body with moderate uptake of choline. The differential diagnoses included encephalitis and neoplasm. She was initially treated for the former but did not respond to steroids, intravenous immunoglobulin, and plasmapheresis. A surgical biopsy later confirmed NK-cell lymphoma. She was then treated as primary CNS NK-cell lymphoma with intravenous and intrathecal chemotherapy. CONCLUSIONS: We presented a unique case of primary CNS NK-cell lymphoma with atypical imaging findings characterized by moderately increased uptake of choline without a corresponding high choline peak on MRS. Although CHO-PET was suggestive of malignancy, surgical biopsy was required to confirm the diagnosis.

Use of aspirin in Chinese after recovery from primary intracranial haemorrhage.

Intracranial haemorrhage (ICH) accounts for ~35% of all strokes in Chinese. Anti-platelet agent is often avoided after an index event due to the possibility of recurrent ICH. This single-centered observational study included 440 consecutive Chinese patients with a first spontaneous ICH surviving the first month performed during 1996-2010. The subjects were identified, and their clinical characteristics, anti-platelet therapy after ICH, and outcomes including recurrent ICH, ischaemic stroke, and acute coronary syndrome were checked from hospital records. Of these 440 patients, 56 patients (12.7%) were prescribed aspirin (312 patient-aspirin years). After a follow-up of 62.2 ± 1.8 months, 47 patients had recurrent ICH (10.7%, 20.6 per 1,000 patient years). Patients prescribed aspirin did not have a higher risk of recurrent ICH compared with those not prescribed aspirin (22.7 per 1,000 patient-aspirin years vs. 22.4 per 1,000 patient years, p=0.70). Multivariate analysis identified age > 60 years (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.07-3.85, p=0.03) and hypertension (HR: 2.0, 95% CI: 1.06-3.75, p=0.03) as independent predictors for recurrent ICH. In a subgroup analysis including 127 patients with standard indications for aspirin of whom 56 were prescribed aspirin, the incidence of combined vascular events including recurrent ICH, ischaemic stroke, and acute coronary syndrome was statistically lower in patients prescribed aspirin than those not prescribed aspirin (52.4 per 1,000 patient-aspirin years, vs. 112.8 per 1,000 patient-years, p=0.04). In conclusion, we observed in a cohort of Chinese post-ICH patients that aspirin use was not associated with an increased risk for a recurrent ICH.

Invasion of human glioma cells is regulated by multiple chloride channels including ClC-3.

BACKGROUND: Glioblastoma is a type of highly malignant primary brain tumour. By means of ion excretion and the associated obligatory water loss, glioma cells can change shapes and undergo extensive migration and invasion. This study investigated the effects of inhibition of ion excretion in glioma cells. MATERIALS AND METHODS: The expression of chloride channels (ClCs) and metalloproteinase-2 (MMP-2) was studied in two human glioma cell lines (STTG1 and U251-MG). The effects of ClC inhibition with chlorotoxin (a ClC-3 inhibitor), 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB) (a non-specific ClC inhibitor), and ClC-3 siRNA knockdown were studied. RESULTS: Both STTG1 and U251-MG cells expressed ClC family members ClC-2, -3, -4, -5, -6 and -7, as well as MMP-2. Glioma cell invasion was markedly but not completely inhibited by ClC-3 and MMP-2 siRNA knockdown, and by chlorotoxin treatment. Addition of chlorotoxin to siRNA-treated glioma cells only slightly increased the suppression of invasion. In contrast, invasion was completely blocked by the non-specific ClC blocker NPPB. CONCLUSION: ClCs are crucial in glioma cell migration and invasion. Blockade of a single ClC, however, is not sufficient to achieve complete inhibition of glioma cell invasion, suggesting that any future therapy should be targeted at pharmacological blockade of multiple ClCs.