Delivery of Functional Anti-miR-9 by Mesenchymal Stem Cell–derived Exosomes to Glioblastoma Multiforme Cells Conferred Chemosensitivity

Introduction: As one of the most aggressive and lethal tumors, glioblastoma multiforme (GBM) is commonly treated by surgical resection combined with radiotherapy and chemotherapy. Temozolomide (TMZ) is the preferred chemotherapy medicine against GBM. However, recurrent GBM patients exhibit TMZ resistance. As reported in previous studies, NEAT1 is over-expressed in glioma cells; thus, we explored the relationship between NEAT1 and TMZ resistance in GBM. Materials and Methods: The expression levels of NEAT1 and O -6-methylguanine-DNA methyltransferase (MGMT) in GBM tissues and cells were determined by quantitative real-time PCR. Si-RNA and the over-expression vector were transfected into GBM cells to modulate the level of related molecules. Western blot analysis was used to investigate the protein expression of MGMT. The cell viability and IC-50 were analyzed by CCK-8, and apoptosis was detected by flow cytometry assay. Result: The expression level of NEAT1 in the TMZ-sensitive GBM tissues and cells was lower than that in the TMZ-resistant GBM tissues and cells. Furthermore, due to the down-regulation of NEAT1 in TMZ-resistant GBM cells transfected with Si-RNA, the viability and IC-50 value of the GBM cell lines were decreased, and the knockdown of NEAT1 significantly enhanced TMZ-induced cell apoptosis in GBM cells. We also determined that the mRNA and protein level of MGMT were up-regulated in TMZ-resistant GBM cells. Interference with MGMT expression led to a decrease in viability and IC-50 value in the GBM cells, and the knockdown of NEAT1 suppressed the transcription and translation levels of MGMT. However, the over-expression of MGMT enhanced TMZ resistance in NEAT1-silenced U87 and U251 cells. Conclusion: NEAT1 participates in the TMZ resistance of GBM cells by regulating MGMT.

Discovery of potent and selective cytotoxic activity of new quinazoline-ureas against TMZ-resistant glioblastoma multiforme (GBM)

INTRODUCTION Glioblastoma multiforme (GBM) is an aggressive brain tumor with limited treatment options and poor prognosis. Resistance to temozolomide (TMZ), the standard chemotherapy for GBM, further complicates treatment. This study investigates the effects of MPT0G521, a dual inhibitor of LSD1 and HDAC, on both parental and TMZ-resistant GBM cells to determine its potential as a therapeutic agent. MATERIALS AND METHODS TMZ and MPT0G521 were introduced in our study. Human GBM cell lines A172 and patient-derived primary GBM cells Pt#3, along with their TMZ-resistant counterparts, were cultured under specified conditions. Assays conducted include cell proliferation (MTT), colony formation, cell cycle analysis via flow cytometry, and western blot analysis for protein expression. Statistical significance was evaluated using ANOVA. RESULTS MPT0G521 exhibited significant anti-proliferative activity and toxicity against both parental and TMZ-resistant GBM cells in a dose- and time-dependent manner. Treatment with MPT0G521 led to reduced cell viability and colony formation. Cell cycle analysis revealed G2M phase arrest and increased subG1 phase, indicating cell cycle disruption and apoptosis. Western blot analysis showed upregulation of histone H3 methylation and acetylation, confirming the dual inhibitory action of MPT0G521 on LSD1 and HDAC. Additionally, MPT0G521 modulated markers of cell cycle and apoptosis, including increased levels of cleaved caspase-3 and PARP. CONCLUSION MPT0G521 demonstrates potential as an effective therapeutic agent against GBM by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis in both parental and TMZ-resistant cells. These findings support further investigation of MPT0G521 as a promising treatment for GBM, particularly in cases resistant to standard chemotherapy.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin β1/FAK signaling pathway

Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma

Contact-based pericellular interactions play important roles in cancer progression via juxtacrine signaling pathways. The present study revealed that hypoxia-inducible factor-1α (HIF-1α), induced even in non-hypoxic conditions by cell-to-cell contact, was a critical cue responsible for the malignant characteristics of glioblastoma multiforme (GBM) cells through Notch1 signaling. Densely cultured GBM cells showed enhanced viability and resistance to temozolomide (TMZ) compared to GBM cells at a low density. Ablating Notch1 signaling by a γ-secretase inhibitor or siRNA transfection resensitized resistant GBM cells to TMZ treatment and decreased their viability under dense culture conditions. The expression of HIF-1α was significantly elevated in highly dense GBM cells even under non-hypoxic conditions. Atypical HIF-1α expression was associated with the Notch1 signaling pathway in both GBM and glioblastoma stem cells (GSC). Proteasomal degradation of HIF-1α was prevented by binding with Notch1 intracellular domain (NICD), which translocated to the nuclei of GBM cells. Silencing Notch1 signaling using a doxycycline-inducible Notch1 RNA-interfering system or treatment with chetomin, a HIF pathway inhibitor, retarded tumor development with a significant anti-cancer effect in a murine U251-xenograft model. Using GBM patient tissue microarray analysis, a significant increase in HIF-1α expression was identified in the group with Notch1 expression compared to the group without Notch1 expression among those with positive HIF-1α expression. Collectively, these findings highlight the critical role of cell-to-cell contact-dependent signaling in GBM progression. They provide a rationale for targeting HIF-1α signaling even in a non-hypoxic microenvironment.

Background: The alkylating agent temozolomide (TMZ) is widely used in glioblastoma multiforme (GBM) therapy. Unfortunately, TMZ-resistance frequently occurs in recurrent GBM and is the major cause of treatment failure. The anti-malarial drug quinacrine (QC) harbors antitumor and chemosensitivity properties, but its interactions with TMZ in GBM remain unclear. This study aimed to investigate whether QC would sensitize TMZ in TMZ-sensitive and TMZ-resistant GBM cells as well as the underlying mechanisms. Materials and Methods: The cytotoxicity of QC and TMZ in TMZ-sensitive and TMZ-resistant GBM cells was evaluated using in vitro cell viability assay and colony formation assay. Cellular apoptosis and protein expression levels were determined using TUNEL assay and immunoblotting, respectively. Results: QC substantially enhanced TMZ cytotoxicity in both TMZ-sensitive and TMZ-resistant cells. Such cytotoxic effect was accompanied by changes in the expression levels of LC3II, p62 and cleaved caspase 3, and increased cellular apoptosis. The results suggested that QC could sensitize GBM cells to TMZ at least partially through apoptosis induction, in which autophagy inhibition might be involved. Conclusion: The antimalarial drug QC may hold promise as a potentiation of TMZ treatment in GBM, especially in cases of TMZ-resistance.

Glioblastoma multiforme (GBM) is a grade IV, highly proliferative, and malignant form of brain tumor with a 5-year survival rate at ~ 5%. Current treatment strategies for GBM include surgery, radiation, and chemotherapy. Major challenges in GBM management include difficulties in surgical resection due to brain's vital functions and GBM metastasis, development of resistance to temozolomide (TMZ), and protection of tumor by blood brain barrier (BBB). Therefore, we aimed to discover a novel therapeutic for GBM by targeting its metabolic reprogramming. We screened metabolic inhibitors by their effects on GBM cell viability by MTT assay. We discovered an FDA-approved drug stiripentol (STP) in our screening of metabolic inhibitors in GBM cells. STP is used for Dravet syndrome (a rare epilepsy). We further tested efficacy of STP using proliferation assay, clonogenic assay, in vitro migration assay, cell cycle assay, apoptosis assay, and in U87 3D spheroids. We also tested the toxicity of STP, and combinations used in the study on normal human dermal fibroblasts. STP was effective in decreasing GBM cell viability, proliferation, clonogenic ability, and migration. Moreover, cell cycle changes were involved but robust apoptosis was absent in STP's anticancer effects. STP was effective in 3D spheroid models, and in TMZ-resistant cells. STP showed additive or synergistic effect with TMZ in different anticancer assays on GBM cells and was considerably less toxic in normal cells. Our results indicate that STP can be an effective GBM therapeutic that enhances the effects of TMZ on GBM cells. Importantly, STP reduced viability of TMZ-resistant cells. Our results warrant further studies in the mechanistic basis of STP's effects on GBM cells and the preclinical potential of STP in animal models.

Purpose: Several microRNAs (miRNAs) that are aberrantly expressed in glioblastoma multiforme (GBM) play a significant role in GBM formation and progression. The expression profile and functions of miR-559 in GBM remain unclear. Here, we quantified the expression and investigated the involvement of miR-559 in the oncogenicity of GBM cells in vitro and in vivo.Material and methods: Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was carried out to determine miR-559 expression in GBM tissues and cell lines. A series of functional assays was performed to evaluate the effects of miR-559 overexpression on GBM cell proliferation, apoptosis, migration, and invasion in vitro and on GBM tumor growth in vivo. The regulatory mechanisms of miR-559 action in GBM cells were then explored.Results: The expression of miR‑559 was lower in GBM tissues and cell lines and significantly correlated with the Karnofsky performance score and tumor size among patients with GBM. Exogenous miR‑559 expression inhibited GBM cell proliferation, migration, and invasion and promoted apoptosis. MiR-559 overexpression decreased tumor growth in vivo. Mechanistic experiments confirmed metadherin (MTDH) as a direct target gene of miR-559 in GBM. Silencing of MTDH induced effects similar to those of miR-559 upregulation in GBM cells, whereas MTDH expression restoration attenuated the antitumor effects of miR‑559 in GBM cells. Protein kinase B (AKT) in the phosphatase and tensin homolog (PTEN)–AKT signaling pathway was found to be deactivated in GBM cells after upregulation of miR-559 both in vitro and in vivo.Conclusion: MiR-559 acts as a tumor suppressor in GBM cells in vitro and in vivo, at least in part through the downregulation of MTDH and inhibition of AKT in the PTEN–AKT pathway. Therefore, targeting the miR-559–MTDH axis may be a promising therapeutic strategy for patients with GBM.

MicroRNA‑432 (miR‑432) has been studied in multiple tumors, but the expression status, biological functions and the mechanism of action of miR‑432 in glioblastoma multiforme (GBM) are yet to be elucidated. In the present study, miR‑432 expression in GBM was determined and its clinical significance was evaluated among patients with GBM. The effects on the malignancy of GBM invitro and invivo were examined in detail and the interactions between miR‑432 and insulin‑like growth factor 1 receptor (IGF‑1R) mRNA were then explored. miR‑432 expression in GBM tissue samples and cell lines was measured by reverse transcription‑quantitative (RT‑q)PCR. GBM cell proliferation, apoptosis, migration and invasion invitro and tumor growth invivo were evaluated by a Cell Counting Kit‑8 assay, flow‑cytometric analysis, Transwell migration and invasion assays, and a tumor xenograft experiment, respectively. Bioinformatic analysis followed by a luciferase reporter assay, RT‑qPCR and western blotting was applied to demonstrate that IGF‑1R is a direct target gene of miR‑432 in GBM cells. It was found that miR‑432 is downregulated in GBM tumors and cell lines. miR‑432 under expression obviously correlated with the Karnofsky Performance Status score and shorter overall survival among patients with GBM. Exogenous miR‑432 expression significantly reduced proliferation and induced apoptosis of GBM cells. In addition, miR‑432 overexpression impaired the migratory and invasive abilities of GBM cells invitro and decreased their tumor growth invivo. Furthermore, IGF‑1R was validated as a direct target gene of miR‑432 in GBM cells. IGF‑1R knockdown imitated the tumor‑suppressive actions of miR‑432 overexpression in GBM cells. Rescue experiments proved IGF‑1R downregulation to be essential for the effects of miR‑432 on GBM cells. The results of the present study revealed a tumor‑suppressive role of the miR‑432‑IGF‑1R axis in GBM cells and this axis may have implications for GBM therapy.

The development of resistance to chemotherapy in the case of aggressive glioblastoma multiforme (GBM) presents a significant treatment challenge. Dysregulation of the Notch signalling pathway promotes tumour proliferation in GBM cells. This study was that targeting the Notch signalling pathway could be a potential therapeutic approach for GBM. Initially, temozolomide-(TMZ)-resistant GBM cells were generated, and the effect of Notch1 on the expression of multiple resistance proteins within these cells was investigated. Subsequently, the expression of Notch-1 in GBM cells was reduced using siRNA. Results revealed a significant reduction in TMZ sensitivity in TMZ-resistant GBM cells, accompanied by a substantial increase in the expression of major vault protein-(MVP), O6-methylguanine-DNA-methyltransferase-(MGMT), and ATP-binding-cassette transporter-G2-(ABCG2). Furthermore, TMZ-resistant U87-R and U251-R cells exhibited higher proliferation rates compared to their parental control cells (U87 and U251). Additionally, we observed that downregulating Notch-1 signalling inhibited the proliferation of TMZ-resistant U87-R and U251-R cells. This downregulation led to the inactivation of MGMT, ABCG2, and MVP. Importantly, it increased chemosensitivity to TMZ, particularly by downregulating MVP expression. Consequently, Notch1 could serve as a potential therapeutic target for GBM cells and may be effective in preventing TMZ resistance by targeting MVP, as well as MGMT and ABCG2 in GBM cells.

Overexpression of PLK1 promotes autophagy-mediated resistance to TMZ in glioblastoma cells by activating NPM1.

PurposeAn increasing number of studies have documented that dysregulation of microRNAs (miRNAs) is common in glioblastoma multiforme (GBM). miR-652 is aberrantly expressed in various human cancers and plays important roles in numerous cancer-related processes. However, the expression profiles and potential roles of miR-652 in GBM remain largely unknown.Patients and methodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine miR-652 expression in GBM tissues and cell lines. The effects of miR-652 upregulation on GBM cell proliferation, clone formation, apoptosis, migration and invasion were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clone formation, flow cytometry and Transwell® migration and invasion assays, respectively. In vivo xenotransplantation was utilized to determine the effect of miR-652 on GBM tumor growth in vivo. Of note, the molecular mechanisms underlying the tumor-suppressing activity of miR-652 upregulation in GBM cells were also investigated using a series of experiments, including bioinformatics analysis, luciferase reporter assay, RT-qPCR and Western blot analysis.ResultsmiR-652 expression was considerably downregulated in GBM tissues and cell lines. Low miR-652 expression was strongly correlated with Karnofsky performance score and tumor size. Overall survival duration was shorter in GBM patients with low miR-652 expression than in those with high miR-652 expression. miR-652 resumption considerably suppressed the proliferation, clone formation, migration, and invasion and promoted the apoptosis of GBM cells in vitro. In addition, forkhead-box k1 (FOXK1) was demonstrated as the direct target gene of miR-652 in GBM cells. FOXK1 downregulation led to a tumor-suppressing activity similar to that of miR-652 upregulation. Restoration of FOXK1 expression partially neutralized the influence of miR-652 overexpression on GBM cells. Furthermore, ectopic miR-652 expression deactivated the AKT/mTOR pathway in GBM cells via FOXK1 regulation. Moreover, miR-652 impaired GBM tumor growth in vivo, probably caused by miR-652-mediated suppression of FOXK1/AKT/mTOR signaling.ConclusionmiR-652 inhibits FOXK1 and deactivates the AKT/mTOR pathway, thereby resulting in the suppression of malignant phenotypes of GBM cells in vitro and in vivo.

BACKGROUND AND PURPOSE: Some patients with glioblastoma multiforme (GBM) do not respond to temozolomide (TMZ), a target for the multidrug resistance transporter MDR1/ABCB1. The genotype of the MDR1 exon12 C1236T single nucleotide polymorphism is reported as a predictive factor of the efficacy of TMZ in GBM. The expression of MDR1 is inhibited via JNK activation. Elsewhere we reported that the overexpression of DKK3 exerted anti-tumor effects in association with JNK activation. However, the regulation of MDR1 by DKK3 remains to be elucidated. We tested our hypothesis that the DKK3-induced activation of JNK down-regulates MDR1, thereby attenuating the chemoresistance of GBM to TMZ. METHODS: We compared the results of treating GBM cells, i.e. U87MG- and primary TGB105 cells with TMZ alone and with TMZ plus Ad-DKK3. RESULTS: The viability of U87MG- and TGB105 cells was not significantly affected by TMZ or Ad-DKK3 alone. However, in combination, they augmented cell death. In a GBM xenograft mice model, treatment with TMZ or Ad-DKK3 exerted anti-tumor effects that were enhanced by their combined administration without affecting the animals' body weight. The increased expression of MDR1 in GBM cells was reduced by Ad-DKK3. The decrease in MDR1 expression elicited by Ad-DKK3 was inversely correlated with the expression of p-JNK, c-JUN, and p-c-JUN but not of JNK, which was eliminated by the JNK inhibitor SP600125. Our findings suggest that the Ad-DKK3-induced activation of JNK downregulates MDR1, thereby attenuating the chemoresistance of GBM cells to TMZ and enhancing its anti-tumor effects. CONCLUSION: Ad-DKK3 may not only exert anti-tumor effects but also serve as an adjuvant therapy for GBM. Chemotherapy targeting JNK may be a promising treatment option.

STAT3 inhibition induced temozolomide-resistant glioblastoma apoptosis via triggering mitochondrial STAT3 translocation and respiratory chain dysfunction