Dysregulated inter-mitochondrial crosstalk in glioblastoma cells revealed by in situ cryo-electron tomography.

Glioblastomas (GBMs) are the most lethal primary brain tumors with limited survival, even under aggressive treatments. The current therapeutics for GBMs are flawed due to the failure to accurately discriminate between normal proliferating cells and distinctive tumor cells. Mitochondria are essential to GBMs and serve as potential therapeutical targets. Here, we utilize cryo-electron tomography to quantitatively investigate nanoscale details of randomly sampled mitochondria in their native cellular context of GBM cells. Our results show that compared with cancer-free brain cells, GBM cells own more inter-mitochondrial junctions of several types for communications. Furthermore, our tomograms unveil microtubule-dependent mitochondrial nanotunnel-like bridges in the GBM cells as another inter-mitochondrial structure. These quantified inter-mitochondrial features, together with other mitochondria-organelle and intra-mitochondrial ones, are sufficient to distinguish GBM cells from cancer-free brain cells under scrutiny with predictive modeling. Our findings decipher high-resolution inter-mitochondrial structural signatures and provide clues for diagnosis and therapeutic interventions for GBM and other mitochondria-related diseases.

Identification of CDKL3 as a critical regulator in development of glioma through regulating RRM2 and the JNK signaling pathway.

Glioma is one of the most commonly diagnosed intracranial malignancies. The molecular mechanism underlying the development of glioma is still largely unknown. In this study, we present the first report concerning the function and mechanism of cyclin-dependent kinase-like 3 (CDKL3) in the development and prognosis of glioma. It is shown that CDKL3 was upregulated in glioma tissues and could independently predict poor prognosis of patients. Silencing CDKL3 in glioma cells could inhibit cell proliferation and migration and induce cell apoptosis and cell cycle arrest, whereas the overexpression of CDKL3 promoted cell proliferation. The in vivo experiments also indicated that knockdown of CDKL3 significantly suppressed tumor growth of glioma. Gene expression profiling of CDKL3 knockdown U87 cells identified RRM2 as a potential target of CDKL3, which was proved to have direct interaction with CDKL3. Given similar effects on glioma development with CDKL3, knockdown of RRM2 could rescue the effects of CDKL3 overexpression on glioma cells. Moreover, knockdown of CDKL3 or RRM2 suppressed the activity of JNK signaling, whereas CDKL3 overexpression produced the opposite effect. In conclusion, our results identified CDKL3 as a promotor for glioma, probably through the regulation of RRM2 and activation of the JNK signalling pathway, highlighting the significance of CDKL3 as a promising therapeutic target of glioma.

The efficacy of laser interstitial thermal therapy for brain metastases with in-field recurrence following SRS: systemic review and meta-analysis.

OBJECTIVE: To study the efficacy of LITT for BM patients experiencing in-field recurrence following SRS. METHODS: A literature search was conducted to identify studies investigating local control (LC) rate and overall survival (OS) of LITT for BMs with IFR following SRS. RESULTS: Analysis included 14 studies (470 patients with 542 lesions). The 6-month (LC-6) and 12-month (LC-12) local control rates were 78.5% (95% CI: 70.6-84.8%) and 69.0% (95% CI: 60.0-76.7%) separately. Pooled median OS was 17.15 months (95% CI: 13.27-24.8). The overall OS-6 and OS-12 rates were 76.0% (95% CI: 71.4-80.0%) and 63.4% (95% CI: 52.9-72.7%) separately. LITT provided more favorable local control efficacy in RN than BM recurrence (LC-6: 87.4% vs. 67.9%, p = 0.009; LC-12: 76.3% vs. 59.9%, p = 0.041). CONCLUSIONS: LITT is an effective treatment for BM patients experiencing IFR following SRS. For different pathological entities, LITT showed more satisfactory local control efficacy on RN than BM recurrence.

An eight-mRNA signature outperforms the lncRNA-based signature in predicting prognosis of patients with glioblastoma.

BACKGROUND: The prognosis of the glioblastoma (GBM) is dismal. This study aims to select an optimal RNA signature for prognostic prediction of GBM patients. METHODS: For the training set, the long non-coding RNA (lncRNA) and mRNA expression profiles of 151 patients were downloaded from the TCGA. Differentially expressed mRNAs (DEGs) and lncRNAs (DE-lncRNAs) were identified between good prognosis and bad prognosis patients. Optimal prognostic mRNAs and lncRNAs were selected respectively, by using univariate Cox proportional-hazards (PH) regression model and LASSO Cox-PH model. Subsequently, four prognostic scoring models were built based on expression levels or expression status of the selected prognostic lncRNAs or mRNAs, separately. Each prognostic model was applied to the training set and an independent validation set. Function analysis was used to uncover the biological roles of these prognostic DEGs between different risk groups classified by the mRNA-based signature. RESULTS: We obtained 261 DEGs and 33 DE-lncRNAs between good prognosis and bad prognosis patients. A panel of eight mRNAs and a combination of ten lncRNAs were determined as predictive RNAs by LASSO Cox-PH model. Among the four prognostic scoring models using the eight-mRNA signature or the ten-lncRNA signature, the one based on the expression levels of the eight mRNAs showed the greatest predictive power. The DEGs between different risk groups using the eight prognostic mRNAs were functionally involved in calcium signaling pathway, neuroactive ligand-receptor interaction pathway, and Wnt signaling pathway. CONCLUSION: The eight-mRNA signature has greater prognostic value than the ten-lncRNA-based signature for GBM patients based on bioinformatics analysis.

Hippo signaling promotes JNK-dependent cell migration.

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam-Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

Prognostic factors and clinical outcomes in adult primary gliosarcoma patients: a Surveillance, Epidemiology, and End Results (SEER) analysis from 2004 to 2015.

Objective: Gliosarcoma (GSC), a rare malignant brain tumor, is considered as a variant of isocitrate dehydrogenase 1 wild type (IDH1-WT) glioblastoma (GBM). This study aimed to retrospectively analyze the clinical characteristics of GSC and compare whether there are some differences of treatment strategies and outcomes between GSC and GBM patients through Surveillance, Epidemiology, and End Results (SEER) database.Patients and methods: The clinical data of adults diagnosed with primary GSC between 2004 and 2015 were queried from SEER database. The Kaplan-Meier curve and the Cox model were performed to analyze the relationships between clinical parameters and patients' prognosis. Similar analyses were conducted for all primary GBM patients of SEER.Results: In total, 527 GSC and 20,541 GBM patients with complete and valid clinical information were finally enrolled for further analysis. Compared with GBM, GSC owned a proclivity to temporal lobe rather than frontal lobe (p < 0.001), a less conservative extension of resection (EOR) (p < 0.001), and a higher sensitivity to radiotherapy (p < 0.001). As shown by univariate analysis, surgery, radiotherapy and chemotherapy could prolong the overall survival (OS) time of GSC, but EOR did not confer an advantage to the outcomes of patients, no matter whether combined radio/chemotherapy was given. In multivariate analysis, age more than 60 and lack of radio/chemotherapy were identified as independent risk factors for OS of GSC patients.Conclusions: Our study found that although EOR seemed to be important to GBM, the extent of surgery did not show a clear relationship with the improved prognosis of GSC. Additionally, radiotherapy and chemotherapy could prolong patients' survival time significantly, which suggests a more positive role of them in treating GSC and needs further investigations.

Circular RNA hsa_circ_0008344 regulates glioblastoma cell proliferation, migration, invasion, and apoptosis.

BACKGROUND: Recent studies have found circular RNAs (circRNAs) involved in the biological process of cancers. However, little is known about their functional roles in glioblastoma. METHODS: Human circRNA microarray analysis was performed to screen the expression profile of circRNAs in IDH1 wild-type glioblastoma tissue. The expression of hsa_circ_0008344 in glioblastoma and normal brain samples was quantified by qRT-PCR. Functional experiments were performed to investigate the biological functions of hsa_circ_0008344, including MTT assay, colony formation assay, transwell assay, and cell apoptosis assay. RESULTS: CircRNA microarray revealed a total of 417 abnormally expressed circRNAs (>1.5-fold, P < .05) in glioblastoma tissue compared with the adjacent normal brain. Hsa_circ_0008344, among the top differentially expressed circRNAs, was significantly upregulated in IDH1 wild-type glioblastoma. Further in vitro studies showed that knockdown of hsa_circ_0008344 suppressed glioblastoma cell proliferation, colony formation, migration, and invasion, but increased cell apoptotic rate. CONCLUSIONS: Hsa_circ_0008344 is upregulated in glioblastoma and may contribute to the progression of this malignancy.

High expression of TIG3 predicts poor survival in patients with primary glioblastoma.

TIG3 (tazarotene-induced gene 3) has been reported to suppress the progression of several malignancies, where this gene is universally downregulated. However, the expression of TIG3 in primary glioblastoma and its relevance to patient's prognosis have not been elaborated. Thus, this study was aimed to evaluate TIG3 expression level in primary glioblastoma and investigate the prognostic value of TIG3 for patients. The Cancer Genome Atlas database was first utilized to analyze the expression and prognostic potential of TIG3 in 528 glioblastoma cases. Compared with control group, glioblastoma showed significantly elevated TIG3 expression (p < 0.001). Log-rank analysis revealed that higher expression of TIG3 was associated with shorter overall survival (358vs 383 days, p = 0.039). Furthermore, TIG3 protein expression detected by immunohistochemistry confirmed positive correlation of TIG3 expression and glioma grade and upregulation of TIG3 in our cohort of 101 primary glioblastoma patients compared to 16 normal brains. Finally, Kaplan-Meier analysis and Cox regression analysis identified high TIG3 expression as an independent risk factor for overall survival of primary glioblastoma patients (overall survival, 10 vs 13 months, p = 0.033; hazard ratio = 1.542, p = 0.046). Together, this study indicated that increased expression of TIG3 in primary glioblastoma is a novel biomarker for predicting poor outcome of patients. We then hypothesize that TIG3 may function in a different pattern in glioblastoma.

Genome-wide analysis of YB-1-RNA interactions reveals a novel role of YB-1 in miRNA processing in glioblastoma multiforme.

Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.

Degradation of diclofenac by UV-activated persulfate process: Kinetic studies, degradation pathways and toxicity assessments.

Diclofenac (DCF) is the frequently detected non-steroidal pharmaceuticals in the aquatic environment. In this study, the degradation of DCF was evaluated by UV-254nm activated persulfate (UV/PS). The degradation of DCF followed the pseudo first-order kinetics pattern. The degradation rate constant (kobs) was accelerated by UV/PS compared to UV alone and PS alone. Increasing the initial PS dosage or solution pH significantly enhanced the degradation efficiency. Presence of various natural water constituents had different effects on DCF degradation, with an enhancement or inhibition in the presence of inorganic anions (HCO3- or Cl-) and a significant inhibition in the presence of NOM. In addition, preliminary degradation mechanisms and major products were elucidated using LC-MS/MS. Hydroxylation, decarbonylation, ring-opening and cyclation reaction involving the attack of SO4•- or other substances, were the main degradation mechanism. TOC analyzer and Microtox bioassay were employed to evaluate the mineralization and cytotoxicity of solutions treated by UV/PS at different times, respectively. Limited elimination of TOC (32%) was observed during the mineralization of DCF. More toxic degradation products and their related intermediate species were formed, and the UV/PS process was suitable for removing the toxicity. Of note, longer degradation time may be considered for the final toxicity removal.

Expression and prognostic significance of TCTN1 in human glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and lethal intracranial malignancy in adults, with dismal prognosis despite multimodal therapies. Tectonic family member 1 (TCTN1) is a protein involved in a diverse range of developmental processes, yet its functions in GBM remain unclear. This study aims to investigate expression profile, prognostic value and effects of TCTN1 gene in GBM. METHODS: Protein levels of TCTN1 were assessed by immunohistochemical staining using a tissue microarray constructed by a Chinese cohort of GBM patients (n=110), and its mRNA expression was also detected in a subset of this cohort. Kaplan-Meier analysis and Cox regression were performed to estimate the prognostic significance of TCTN1. Similar analyses were also conducted in another two independent cohorts: The Cancer Genome Atlas (TCGA) cohort (n=528) and the Repository for Molecular Brain Neoplasia Data (REMBRANDT) cohort (n=228). For the TCGA cohort, the relationships between TCTN1 expression, clinical outcome, molecular subtypes and genetic alterations were also analysed. Furthermore, proliferation of TCTN1 overexpressed or silenced GBM cells was determined by CCK-8 assays. RESULTS: As discovered in three independent cohorts, both mRNA and protein levels of TCTN1 expression were markedly elevated in human GBMs, and higher TCTN1 expression served as an independent prognostic factor predicting poorer prognosis of GBM patients. Additionally, in the TCGA cohort, TCTN1 expression was dramatically decreased in patients within the proneural subtype compared to other subtypes, and significantly influenced by the status of several genetic aberrations such as CDKN2A/B deletion, EGFR amplification, PTEN deletion and TP53 mutation. The prognostic value of TCTN1 was more pronounced in proneural and mesenchymal subtypes, and was also affected by several genetic alterations particularly PTEN deletion. Furthermore, overexpression of TCTN1 significantly promoted proliferation of GBM cells, while its depletion evidently hampered cell growth. CONCLUSIONS: TCTN1 is elevated in human GBMs and predicts poor clinical outcome for GBM patients, which is associated with molecular subtypes and genetic features of GBMs. Additionally, TCTN1 expression impacts GBM cell proliferation. Our results suggest for the first time that TCTN1 may serve as a novel prognostic factor and a potential therapeutic target for GBM.

Liver-Derived Ketogenesis via Overexpressing HMGCS2 Promotes the Recovery of Spinal Cord Injury.

The liver is the major ketogenic organ of the body, and ketones are reported to possess favorable neuroprotective effects. This study aims to elucidate whether ketone bodies generated from the liver play a critical role in bridging the liver and spinal cord. Mice model with a contusive spinal cord injury (SCI) surgery is established, and SCI induces significant histological changes in mice liver. mRNA-seq of liver tissue shows the temporal changes of ketone bodies-related genes, ß-hydroxybutyrate dehydrogenase (BDH1) and solute carrier family 16 (monocarboxylic acid transporters), member 6 (SLC16A6). Then, an activated ketogenesis model is created with adult C57BL/6 mice receiving the tail intravenous injection of GPAAV8-TBG-Mouse-Hmgcs2-CMV- mCherry -WPRE (HMGCS2liver ) and mice receiving equal AAV8-Null being the control group (Vectorliver ). Then, the mice undergo either a contusive SCI or sham surgery. The results show that overexpression of HMG-CoA synthase (Hmgcs2) in mice liver dramatically alleviates SCI-mediated pathological changes and promotes ketogenesis in the liver. Amazingly, liver-derived ketogenesis evidently alleviates neuron apoptosis and inflammatory microglia activation and improves the recovery of motor function of SCI mice. In conclusion, a liver-spinal cord axis can be bridged via ketone bodies, and enhancing the production of the ketone body within the liver has neuroprotective effects on traumatic SCI.

UBDP1 pseudogene and UBD network competitively bind miR­6072 to promote glioma progression.

Increasing evidence suggests that pseudogenes play crucial roles in various cancers, yet their functions and regulatory mechanisms in glioma pathogenesis remain enigmatic. In the present study, a novel pseudogene was identified, UBDP1, which is significantly upregulated in glioblastoma and positively correlated with the expression of its parent gene, UBD. Additionally, high levels of these paired genes are linked with a poor prognosis for patients. In the present study, clinical samples were collected followed by various analyses including microarray for long non­coding RNAs, reverse transcription­quantitative PCR, fluorescence in situ hybridization and western blotting. Cell lines were authenticated and cultured then subjected to various assays for proliferation, migration, and invasion to investigate the molecular mechanisms. Bioinformatic tools identified miRNA targets, and luciferase reporter assays validated these interactions. A tumor xenograft model in mice was used for in vivo studies. In vitro and in vivo studies have demonstrated that UBDP1, localized in the cytoplasm, functions as a tumor­promoting factor influencing cell proliferation, migration, invasion and tumor growth. Mechanistic investigations have indicated that UBDP1 exerts its oncogenic effects by decoying miR­6072 from UBD mRNA, thus forming a competitive endogenous RNA network, which results in the enhanced oncogenic activity of UBD. The present findings offered new insights into the role of pseudogenes in glioma progression, suggesting that targeting the UBDP1/miR­6072/UBD network may serve as a potential therapeutic strategy for glioma patients.

A targetable PRR11-DHODH axis drives ferroptosis- and temozolomide-resistance in glioblastoma.

Temozolomide (TMZ) is a widely utilized chemotherapy treatment for patients with glioblastoma (GBM), although drug resistance constitutes a major therapeutic hurdle. Emerging evidence suggests that ferroptosis-mediated therapy could offer an appropriate alternative treatment option against cancer cells that are resistant to certain drugs. However, recurrent gliomas display robust ferroptosis resistance, although the precise mechanism of resistance remains elusive. In the present work, we report that proline rich protein 11 (PRR11) depletion significantly sensitizes GBM cells to TMZ by inducing ferroptosis. Mechanistically, PRR11 directly binds to and stabilizes dihydroorotate dehydrogenase (DHODH), which leads to glioma ferroptosis-resistant in a DHODH-dependent manner in vivo and in vitro. Furthermore, PRR11 inhibits HERC4 and DHODH binding, by suppressing the recruitment of E3 ubiquitin ligase HERC4 and polyubiquitination degradation of DHODH at the K306 site, which maintains DHODH protein stability. Importantly, downregulated PRR11 increases lipid peroxidation and alters DHODH-mediated mitochondrial morphology, thereby promoting ferroptosis and increasing TMZ chemotherapy sensitivity. In conclusion, our results reveal a mechanism via which PRR11 drives ferroptosis resistance and identifies ferroptosis induction and TMZ as an attractive combined therapeutic strategy for GBM.

Exploring cerebral structural and functional abnormalities in a mouse model of post-traumatic headache induced by mild traumatic brain injury.

Mild traumatic brain injury (mTBI)-induced post-traumatic headache (PTH) is a pressing public health concern and leading cause of disability worldwide. Although PTH is often accompanied by neurological disorders, the exact underlying mechanism remains largely unknown. Identifying potential biomarkers may prompt the diagnosis and development of effective treatments for mTBI-induced PTH. In this study, a mouse model of mTBI-induced PTH was established to investigate its effects on cerebral structure and function during short-term recovery. Results indicated that mice with mTBI-induced PTH exhibited balance deficits during the early post-injury stage. Metabolic kinetics revealed that variations in neurotransmitters were most prominent in the cerebellum, temporal lobe/cortex, and hippocampal regions during the early stages of PTH. Additionally, variations in brain functional activities and connectivity were further detected in the early stage of PTH, particularly in the cerebellum and temporal cortex, suggesting that these regions play central roles in the mechanism underlying PTH. Moreover, our results suggested that GABA and glutamate may serve as potential diagnostic or prognostic biomarkers for PTH. Future studies should explore the specific neural circuits involved in the regulation of PTH by the cerebellum and temporal cortex, with these two regions potentially utilized as targets for non-invasive stimulation in future clinical treatment.

AGBL4 promotes malignant progression of glioblastoma via modulation of MMP-1 and inflammatory pathways.

Introduction: Glioblastoma multiforme (GBM), the most common primary malignant brain tumor, is notorious for its aggressive growth and dismal prognosis. This study aimed to elucidate the molecular underpinnings of GBM, particularly focusing on the role of AGBL4 and its connection to inflammatory pathways, to discover viable therapeutic targets. Methods: Single-cell sequencing was utilized to examine the expression levels of AGBL4 and functional assays were performed to assess the effects of AGBL4 modulation. Results: Our findings identified the significant upregulation of AGBL4 in GBM, which correlated with adverse clinical outcomes. Functional assays demonstrated that AGBL4 knockdown inhibited GBM cell proliferation, migration, and invasion and influenced inflammatory response pathways, while AGBL4 overexpression promoted these activities. Further investigation revealed that AGBL4 exerted its oncogenic effects through modulation of MMP-1, establishing a novel regulatory axis critical for GBM progression and inflammation. Discussion: Both AGBL4 and MMP-1 may be pivotal molecular targets, offering new avenues for targeted therapy in GBM management.

Overcoming therapeutic resistance in oncolytic herpes virotherapy by targeting IGF2BP3-induced NETosis in malignant glioma.

Oncolytic virotherapy holds promise for cancer treatment, but the factors determining its oncolytic activity remain unclear. Neutrophil extracellular traps (NETs) are associated with cancer progression, yet their formation mechanism and role in oncolytic virotherapy remain elusive. In this study, we demonstrate that, in glioma, upregulation of IGF2BP3 enhances the expression of E3 ubiquitin protein ligase MIB1, promoting FTO degradation via the ubiquitin-proteasome pathway. This results in increased m6A-mediated CSF3 release and NET formation. Oncolytic herpes simplex virus (oHSV) stimulates IGF2BP3-induced NET formation in malignant glioma. In glioma models in female mice, a BET inhibitor enhances the oncolytic activity of oHSV by impeding IGF2BP3-induced NETosis, reinforcing virus replication through BRD4 recruitment with the CDK9/RPB-1 complex to HSV gene promoters. Our findings unveil the regulation of m6A-mediated NET formation, highlight oncolytic virus-induced NETosis as a critical checkpoint hindering oncolytic potential, and propose targeting NETosis as a strategy to overcome resistance in oncolytic virotherapy.

RLIP76 is overexpressed in human glioblastomas and is required for proliferation, tumorigenesis and suppression of apoptosis.

The guanosine triphosphatase-activating protein RLIP76 is overexpressed in many malignant tumor cells, but it is unclear if RLIP76 overexpression contributes to the high proliferative potential of glioma cells. We demonstrate that RLIP76 messenger RNA and protein expression are positively correlated with glioma grade and that higher RLIP76 expression correlates with shorter patient survival. Immunohistochemical staining revealed that RLIP76 expression was positively correlated with the expression of Ki-67, a biomarker for cell proliferation. Inhibition of RLIP76 expression in U87 and U251 glioma cell lines by stable transfection of a targeted siRNA suppressed anchorage-independent growth and enhanced apoptosis in vitro. Conversely, overexpression of RLIP76 in SW1088 and U251 cell lines enhanced proliferation and reduced apoptosis. Inhibition of RLIP76 in U251 cells also significantly suppressed tumorigenicity and induced apoptosis in an endotopic xenograft mouse model. Moreover, we demonstrate that knockdown of RLIP76 increases apoptosis in different human gliomas independently of p53 status. In addition, a constitutively active Rac1 reversed both the suppression of proliferation and the promotion of apoptosis induced by the RLIP76-targeted siRNA, indicating that RLIP76 is an upstream activator of Rac1. Rac1-mediated suppression of apoptosis and promotion of proliferation were dependent on intact c-jun N-terminal kinase (JNK) signaling. Furthermore, we demonstrate that RLIP76 promotes proliferation and suppresses glioma cell apoptosis through a mechanism independent of Rho-selective GTPase-activating protein. Instead, we found that the adenosine triphosphatase function of Rlip76 modulates Rac1 activity by regulating Rac1 protein ubiquitylation and degradation. These data demonstrate that RLIP76 may suppress apoptosis and promote the proliferation of glioma cells by direct adenosine triphosphate-dependent xenobiotic transport and by activating the Rac1-JNK signaling pathway. Inhibition of RLIP76 signaling is a potential treatment for malignant glioma.

Knockdown of RLIP76 expression by RNA interference inhibits invasion, induces cell cycle arrest, and increases chemosensitivity to the anticancer drug temozolomide in glioma cells.

RLIP76, a GTPase-activating protein, is a central regulator in multiple pathways that respond to redox states and control cell growth, motility, division, and apoptosis in many malignant cancer cells. In this study, human glioblastoma cell lines U87 and U251 were stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting RLIP76. shRNA knockdown of RLIP76 induced cell cycle arrest in U87 and U251 cells and inhibited their invasiveness. Quantitative Western blot analysis revealed that cells stably underexpressing RLIP76 showed lower expression of cyclin D1 and decreased expression and activity of matrix metalloproteinase 2 compared to cells stably transfected with a control vector. Furthermore, RLIP76 expression levels were correlated with IC(50) values for the antitumor drug temozolomide (TMZ). Compared with TMZ alone (17.19 ± 1.78 and 22.18 ± 1.99 µg/mL in U87 and U251 cells, respectively) or combined shGFP and TMZ (18.04 ± 1.07 and 23.040 ± 1.77 µg/mL in U87 and U251 cells, respectively), combined shRNA and TMZ therapy resulted in a significant decrease in IC(50) value (7.61 ± 2.99 and 6.91 ± 2.59 µg/mL in U87 and U251 cells, respectively). Combined RLIP76 knockdown and TMZ treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Furthermore, RLIP76 downregulation enhanced chemosensitivity to TMZ without affecting protein expression of MDR1 and MRP1. The results indicate that inhibition of RLIP76 expression may be an effective means for overcoming RLIP76-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach for glioma treatment.