Drug Resistance In Glioma Cells Research Articles

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/MGMT axis

To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells. The inhibitory effect of 10, 20, and 40 μg/mL curcumol were observed in human glioma cell lines A172 and U251. UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol (40 μg/mL), temozolomide (TMZ; 10 μg/mL), or both, and the changes in cell viability, clone formation capacity and apoptosis were assessed using MTT assay, cell clone formation experiment, and flow cytometry; UTX activity in the cells was determined using a UTX detection kit, and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR. The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry. In a nude mouse model bearing glioma xenografts, the effects of curcumol (20 mg/kg), TMZ (20 mg/kg) and their combination on tumor growth and expressions of UTX, H3K27me3 and MGMT were evaluated. Curcumol significantly inhibited the proliferation (P<0.05) and promoted apoptosis of cultured glioma cells (P<0.01). Curcumol, but not TMZ, produced significant inhibitory effect on tumor growth in the tumor-bearing mice (P<0.01). Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells. UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis (P<0.01). Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region (P<0.05) and decreased MGMT protein expression, which was reversed by UTX overexpression. In both the in vivo and in vitro experiments, curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells, reduced the expression of its downstream target gene MGMT, and enhanced TMZ sensitivity of the glioma cells. Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.

Potential mechanisms underlying the promoting effects of 3D collagen scaffold culture on stemness and drug resistance of glioma cells

Background3D collagen scaffold culture is a good tool to study glioma metastasis and recurrence in vitro. MethodsThe effect of 3D collagen culture on the colony formation, the sphere formation, and drug sensitivity of glioma cells was observed by soft-agar colony formation assays, sphere formation assays, and CCK-8 assays, respectively. 3D-glioma-drug genes were identified by previous results and online databases. Gene enrichment and PPI analyses were performed by R software and Metacsape. Hub 3D-glioma-drug genes were screened by STRING and Cytoscape. TCGA and CGGA databases and R software were used to analyze the distribution of hub genes in glioma and their effects on the prognosis. Western Blot was used to verify the effect of 3D collagen culture on the expression of hub genes. miRNAs targeting hub genes were predicted by ENCORI. Results3D collagen scaffold culture promoted colony formation, sphere formation, and drug resistance of glioma cells. There were 77 3D-glioma-drug genes screened, and the pathways enriched in the protein interaction network mainly included responses to stressors, DNA damage and repair, and drug metabolism. Hub 3D-glioma-drug genes were AKT1, ATM, CASP3, CCND1, EGFR, PARP1, and TP53. These genes and predicted miRNAs were expressed differentially in glioma samples and partially affected the prognosis of patients with glioma. These findings suggested these hub genes and miRNAs may play a key role in the effects generated by the 3D culture model and become new markers for glioma diagnosis and treatment.

STUB1-SMYD2 Axis Regulates Drug Resistance in Glioma cells.

SET and MYND domain-containing protein 2 (SMYD2) is an important epigenetic regulator that methylates histone and non-histone proteins. The study aimed to investigate the oncogenic role of SMYD2 in gliomas and explore its degradation mechanism induced by cisplatin. Tumor tissue microarray of 441 patients with glioma was collected for SMYD2 immunohistochemical staining. Kaplan-Meier survival curves were constructed using the overall survival values. mRNA-sequencing analysis was performed for understanding the downstream mechanisms mediated by SMYD2. The half-inhibitory concentrations (IC50) of temozolomide and cisplatin in AZ505-treated and control cells were calculated. The potential E3 ubiquitin ligase of SMYD2 was predicted in UbiBrowser and confirmed by a knockdown test. The effect of SMYD2 and its E3 ligase on apoptosis and migration of glioma cells was determined via cell-function assays. High SMYD2 expression correlated with a high WHO stage (P = 0.004) and a low survival probability (P = 0.012). The inhibition of SMYD2 suppressed the process of epithelial to mesenchymal transition (EMT) by downregulating the expression of Collagen 1A1 (COL1A1). AZ505 treatment significantly increased the drug sensitivity of glioma cells. SMYD2 expression was markedly reduced by cisplatin treatment via STIP1 Homology And U-Box Containing Protein 1 (STUB1)-mediated degradation. The knockdown of STUB1 could partly reverse the cell function impairment induced by cisplatin. Our findings suggested that SMYD2 could be a potential drug target for the treatment of gliomas, and STUB1-mediated degradation of SMYD2 plays an important role in reversing chemotherapy resistance in patients with gliomas.

Long-stranded non-coding RNA HCG11 regulates glioma cell proliferation, apoptosis and drug resistance via the sponge MicroRNA-144COX-2 axis.

In the current study, we analyzed long non-coding RNA HCG11 regulates the proliferation, apoptosis and drug resistance of glioma cells by spongy microRNA-144COX-2 axis. For this purpose, glioma cells at the logarithmic growth stage were divided into blank, up-regulated and down-regulated groups. The blank group did not undergo any treatment. Bacteria were inserted into the complete culture medium of the up-regulated group and down-regulated group for co-culture for 24 h. The down-regulated group was transfected with Mir-HCG11 inhibitor. The expressions of Mir-HCG11, Mir-144-3p and COX-2 in each group were observed, and the proliferation and apoptosis of glioma cells were analyzed, and their drug resistance was analyzed. Results showed that compared with the blank group, the expression of Mir-HCG11 and Mir-144 was increased and the expression of COX-2 was decreased in the up-regulated group (P < 0.05). Compared with the up-regulated group, the down-regulated group increased the expression of Mir-HCG11 and Mir-144 and decreased the expression of COX-2 (P < 0.05). Compared with the blank group, the proliferation rate of glioma cells in the up-regulated group (24h, 48h, 72h) was increased (P < 0.05); Compared with the up-regulated group, the proliferation rate of glioma cells in the down-regulated group (24h, 48h, 72h) was decreased (P < 0.05); Compared with the blank group, apoptosis rate of glioma cells in the up-regulated group (24h, 48h, 72h) was decreased (P < 0.05); Compared with the up-regulated group, the apoptosis rate of glioma cells in the down-regulated group (24h, 48h, 72h) was increased (P < 0.05); The IC50 values of Imatinib, VP-16 and TMZ in blank group and up-regulated group was compared (P > 0.05). Compared with the blank group and up-regulated group, the IC50 values of Imatinib, VP-16 and TMZ in the down-regulated group were decreased (P < 0.05). In general, down-regulation of long non-coding RNA-HCG11 can regulate the microRNA-144COX-2 axis in glioma, thus reducing the proliferation rate of glioma cells and improving the apoptosis rate of glioma cells. In addition, down-regulation of long non-coding RNA-HCG11 is also involved in the drug resistance mechanism of Imatinib, VP-16 and TMZ chemotherapy drugs.

Mechanisms Underlying the Promoting Effects of 3D Collagen Scaffold Culture on Stemness and Drug Resistance of Glioma Cells

Mechanisms Underlying the Promoting Effects of 3D Collagen Scaffold Culture on Stemness and Drug Resistance of Glioma Cells

Drug Resistance in Glioma Cells Induced by a Mesenchymal-Amoeboid Migratory Switch.

Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/β inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal–amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.

CDKL5 promotes proliferation, migration, and chemotherapeutic drug resistance of glioma cells via activation of the PI3K/AKT signaling pathway.

Gliomas, the most prevalent cancer in the central nervous system, are characterized by high morbidity and mortality, emphasizing the need to understand their etiology. Here, we report that cyclin‐dependent kinase‐like 5 (CDKL5) is highly expressed in gliomas, and CDKL5 overexpression promotes invasion, proliferation, migration and drug (β‐lapachone) resistance of glioma cells. In vitro, CDKL5 overexpression enhanced invasion, growth and migration of glioma cells, and stimulated the phosphoinositide 3‐kinase (PI3K)/AKT axis. Furthermore, CDKL5 overexpression in vivo promoted glioma proliferation, whereas CDKL5 knockdown had opposing effects. The effect of CDKL5 on drug resistance was eliminated if the PI3K/AKT axis was suppressed, and cisplatin combined with the PI3K/AKT suppressor XL147 remarkably prohibited proliferation in xenografts overexpressing CDKL5. Collectively, our findings suggest that CDKL5 acts through the PI3K/AKT axis in glioma cells, and indicate a possible role for CDKL5 in glioma therapy.

NCK1-AS1 Increases Drug Resistance of Glioma Cells to Temozolomide by Modulating miR-137/TRIM24.

Background: Glioma is a common malignant tumor. The purpose of this study was to investigate the effect and molecular mechanism of long noncoding RNA (lncRNA) NCK1-AS1 on the drug resistance of temozolomide (TMZ) in glioma cells. Methods: The fresh and recurrent glioma tissues and peritumoral brain edema (PTBE) were collected from the same patient. U251 and A172 cells were treated with TMZ to screen TMZ-resistant cells. The expression levels of NCK1-AS1, miR-137, or TRIM24 were detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, in situ hybridization (ISH), or RNA pull-down assay. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide (MTT) assay. In addition, the relationship between NCK1-AS1 and miR-137 or TRIM24 and miR-137 was confirmed by dual luciferase activity assay. Results: NCK1-AS1 expression was increased in regular and recurrent glioma tissues and TMZ-resistant cells. Cell viability was increased in TMZ-resistant cells, and the IC50 of TMZ also increased in TMZ resistant cells. However, knockdown of NCK1-AS1 inhibited these increases. Moreover, suppression of NCK1-AS1 increased miR-137 expression, whereas overexpression of miR-137 decreased TRIM24 expression. Then, expression of miR-137 alleviated the NCK1-AS1 overexpression-induced increased expression of TRIM24. In addition, the decreases of cell viability and IC50 induced by NCK1-AS1 knockdown were reversed after adding TRIM24 in U251/TMZ and A172/TMZ cells. Conclusion: NCK1-AS1 could increase drug resistance of glioma cells to TMZ by modulating miR-137/TRIM24 pathway.

Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma

BackgroundFAM92A1–289(abbreviated FAM289) is recognized as one of the newly-discovered putative oncogenes. However, its role and molecular mechanisms in promoting cancer progression has not yet been elucidated. This study was performed to reveal its oncogenic functions and molecular mechanisms in human glioblastoma multiforme (GBM) cell models with knockdown or overexpression of FAM289 in vitro and in vivo.MethodsTo elucidate the molecular mechanisms underlying FAM289-mediated tumor progression, the protein-protein interaction between FAM289 and Galectin-1 was verified by co-immunoprecipitation, followed by an analysis of the expression and activity of Galectin-1-associated signaling molecules. Knockdown and overexpression of FAM289 in glioma cells were applied for investigating the effects of FAM289 on cell growth, migration and invasion. The determination of FAM289 expression was performed in specimens from various stages of human gliomas.ResultsFAM289-galectin-1 interaction and concomitant activation of the extracellular signal-regulated kinase (ERK) pathway participated in FAM289-mediated tumor-promoting function. Since the expression of DNA methyl transferase 1 (DNMT1) and DNA methyl transferase 3B (DNMT3B) was regulated by FAM289 in U251 and U87-MG glioma cells, Galectin-1 interaction with FAM289 may promote FAM289 protein into the cell nucleus and activate the ERK pathway, thereby upregulating DNMTs expression. Drug resistance tests indicated that FAM289-mediated TMZ resistance was through stem-like property acquisition by activating the ERK pathway. The correlation between FAM289, Galectin-1 expression and the clinical stage of gliomas was also verified in tissue samples from glioblastoma patients.ConclusionsOur results suggest that high expression of FAM289 in GBM tissues correlated with poor prognosis. FAM289 contributes to tumor progression in malignant glioma by interacting with Galectin-1 thereby promoting FAM289 protein translocation into the cell nucleus. FAM289 in the nucleus activated the ERK pathway, up regulated DNMTs expression and induced stem-like property gene expression which affects drug resistance of glioma cells to TMZ. This study provided functional evidence for FAM289 to be developed as a therapeutic target for cancer treatment.

Abstract 1736: Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene

Abstract Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of FGFR and ERK pathways when treated with TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 26 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398 and PD173074. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Lynette M. Phillips, Elizabeth A. Ballard, Mary E. Forbes, ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1736.

MKK7 transcription positively or negatively regulated by SP1 and KLF5 depends on HDAC4 activity in glioma.

JNK activity has been implicated in the malignant proliferation, invasion and drug-resistance of glioma cells (GCs), but the molecular mechanisms underlying JNK activation are currently unknown. Here, we reported that MKK7, not MKK4, directly activates JNK in GCs and exerts oncogenic effects on tumor formation. Notably, MKK7 expression in glioma tissues was closely correlated with the grade of the glioma and JNK/c-Jun activation. Mechanistically, MKK7 transcription critically depends on the complexes formed by HDAC4 and the transcriptional factors SP1 and Krüppel-like factor-5 (KLF5), wherein HDAC4 directly deacetylates both SP1 and KLF5 and synergistically upregulates MKK7 transcription through two SP1 sites located on its promoter. In contrast, the increases in acetylated-SP1 and acetylated-KLF5 after HDAC4 inhibition switched to transcriptionally suppress MKK7. Selective inhibition of HDAC4 by LMK235, siRNAs or blockage of SP1 and KLF5 by the ectopic dominant-negative SP1 greatly reduced the malignant capacity of GCs. Furthermore, suppression of both MKK7 expression and JNK/c-Jun activities was involved in the tumor-growth inhibitory effects induced by LMK235 in U87-xenograft mice. Interestingly, HDAC4 is highly expressed in glioma tissues, and the rate of HDAC4 nuclear import is closely correlated with glioma grade, as well as with MKK7 expression. Collectively, these findings demonstrated that highly expressed MKK7 contributes to JNK/c-Jun signaling-mediated glioma formation. MKK7 transcription, regulated by SP1 and KLF5, critically depends on HDAC4 activity, and inhibition of HDAC4 presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c-Jun signaling in GCs.

Downregulation of miR‑186 promotes the proliferation and drug resistance of glioblastoma cells by targeting Twist1.

Temozolomide (TMZ) is widely used as a chemotherapeutic agent in the treatment of glioma; however, the development of drug resistance remains a major obstacle in the effective treatment of glioblastoma. Increasing evidence has indicated that microRNAs (miRs) are involved in the drug resistance of glioma; however, the role of miR‑186‑5p in the TMZ resistance of glioblastoma remains unknown. In the present study, the role of miR‑186‑5p in the resistance of glioblastoma to TMZ was investigated. mRNA and protein expression levels were detected via reverse transcription‑quantitative PCR and western blot analysis, respectively. It was determined that miR‑186‑5p was significantly downregulated in glioblastoma tissues and cell lines. Additionally, the expression of miR‑186‑5p was decreased, whereas that of Twist1 was upregulated during the development of drug resistance in glioma cells. The introduction of miR‑186 into glioblastoma cells via transfection decreased the proliferation and TMZ resistance of glioblastoma cells, as determined via 5‑ethynyl‑2'‑deoxyuridine and Cell Counting Kit‑8 assays, whereas the inhibition of miR‑186‑5p induced opposing effects. Furthermore, luciferase reporter and expression rescue assays revealed that miR‑186‑5p bound to the 3'‑untranslated region of Twist‑related protein 1 (Twist1). In conclusion, the present study demonstrated that downregulation of miR‑186‑5p may contribute to the proliferation and drug resistance of glioblastoma cells via the regulation of Twist1 expression. These results suggested that miR‑186‑5p may be a novel therapeutic target in the treatment of glioblastoma.

CIRP regulates BEV-induced cell migration in gliomas.

PurposeA better understanding of the underlying molecular mechanisms in treatment failure of bevacizumab (BEV) for malignant glioma would contribute to overcome therapeutic resistance.MethodsHere, we used a quantitative proteomic method to identify molecular signatures of glioblastoma cell after BEV treatment by two-dimensional liquid chromatography-tandem mass spectrometry analysis and 6-plex iTRAQ quantification. Next, the function of cold-inducible RNA-binding protein (CIRP), one of the most significantly affected proteins by drug treatment, was evaluated in drug resistance of glioma cells by invasion assays and animal xenograft assays. Target molecules bound by CIRP were determined using RNA-binding protein immunoprecipitation and microarray analysis. Then, these mRNAs were identified by quantitative real-time PCR.ResultsEighty-seven proteins were identified with significant fold changes. The biological functional analysis indicated that most of the proteins were involved in the process of cellular signal transduction, cell adhesion, and protein transport. The expression of CIRP greatly decreased after BEV treatment, and ectopic expression of CIRP abolished cell migration in BEV-treated glioma cells. In addition, CIRP could bind mRNA of CXCL12 and inhibit BEV-induced increase of CXCL12 in glioma cells.ConclusionThese data suggested that CIRP may take part in BEV-induced migration of gliomas by binding of migration-relative RNAs.

YB-1 modulates the drug resistance of glioma cells by activation of MDM2/p53 pathway.

BackgroundY-box-binding protein-1 (YB-1) is aberrantly expressed in a variety of cancers. However, the biological functional role of YB-1 in glioma is not yet clear.MethodsThe expression of MDM2 and YB-1 was analyzed by real time PCR. Overexpression and knockdown of YB-1 in glioma cells were created by transfection of pcDNA-YB-1 and siRNA against YB-1, respectively. Cell viability was performed by CCK8 assay.ResultsOur findings showed that glioma tissues had higher expressions of YB-1 than that in cancer-free tissues in 54 glioma patients, which were also positively correlated with Murine MDM2 expression. Overexpression of YB-1 or MDM2 renders a drug resistance feature in glioma cell exposed to temozolomide (TMZ), by directly targeting p53. Genetic or chemical inhibition of MDM2 significantly blocked YB-1-modulated response of glioma cells to TMZ. Moreover, inhibition of YB-1 or MDM2 reduced glioma cells metastasis and mortality in mice.ConclusionYB-1 facilitates the resistance of glioma cells to TMZ by direct activation of MDM2/p53 signaling and represents a promising molecular target for glioma treatment.

MiR‑501‑3p sensitizes glioma cells to cisplatin by targeting MYCN

Cisplatin, a commonly used chemotherapeutic agent for glioma patients, treatment often leads to chemoresistance. Accumulating evidence has demosntrated that microRNA (miRNA/miR) is involved in drug resistance of glioma cells. Nevertheless, the role of miR‑501‑3p in glioma cell resistance to cisplatin is unclear. In the present study, it was revealed that miR‑501‑3p expression was decreased in glioma tissues and further underexpressed in cisplatin‑resistant glioma cells compared with wild‑type (WT) glioma cells. Furthermore, cisplatin treatment inhibited the level of miR‑501‑3p in a time‑dependent way. Ectopic expression of miR‑501‑3p suppressed glioma cell growth and invasion, but increased cisplatin‑resistant glioma cell apoptosis. Furthermore, miR‑501‑3p sensitized glioma cells to cisplatin‑induced proliferation arrest and death. Mechanistically, it was demonstrated that miR‑501‑3p targeted MYCN in glioma cells. In addition, it was revealed that miR‑501‑3p inhibited MYCN expression by a luciferase reporter assay and reverse transcription‑quantitative polymerase chain reaction. Notably, restoration of MYCN reversed the effects of miR‑501‑3p in cisplatin‑resistant glioma cells. In conclusion, these results suggested that miR‑501‑3p may serve a promising marker for cisplatin resistance.

Coaxial extrusion bioprinted shell-core hydrogel microfibers mimic glioma microenvironment and enhance the drug resistance of cancer cells

Glioblastoma (GBM) is the most common primary malignant central nervous system tumor. The current treatment is mainly surgical resection combined with radiotherapy, chemotherapy and other comprehensive treatment methods. However, the treatment effect is unsatisfactory, the resistance of cancer cells to alkylating agent is the major reason for the recurrence of GBM. It is necessary to develop an ideal in vitro model to investigate the drug resistance of glioma cells. In this study, shell-glioma stem cell GSC23/core-glioma cell line U118 (G/U) hydrogel microfibers with high cell viability were constructed by coaxial extrusion bioprinting. It was found that core-U118 cells gradually proliferated to form fiber-like cell aggregates and the interactions between cell-cell and cell-extracellular matrix (ECM) were increased. Furthermore, compared with shell/core-U118 (U) hydrogel microfibers, the expressions of matrix metalloproteinase-2 (MMP2), MMP9, vascular endothelial growth factor receptor-2 (VEGFR2) and O6-methylguanine-DNA methyltransferase (MGMT) which are related to tumor invasion and drug resistance were significantly enhanced in G/U hydrogel microfibers. Moreover, U118 cells derived from G/U microfibers had greater drug resistance in vitro and the level of MGMT promoter methylation in G/U cultured U118 cells was significantly lower than that of U cultured cells. In summary, coaxial extrusion bioprinted G/U hydrogel microfiber is a preferable platform for mimicking glioma microenvironment, as well as for drug development and screening.

39 Lysosome disruptor’s siramesine and the kinase inhibitor lapatinib induce synergistic cell death through ferroptosis in glioma cells

Glioblastoma is inherently resistant to radiation and drug treatments. This is mediated by the most common forms of cell death are often actively inhibited. Identifying and exploiting alternative cell death pathways are essential to overcoming or bypassing drug resistance. Ferroptosis, a newly described, morphologically and biochemically distinct, cell death mechanism is characterized by iron-dependent cellular accumulation of reactive oxygen species. The combination of siramesine, a lysosome disruptor, and lapatinib, a dual tyrosine kinase inhibitor (TKI), synergistically induced death in glioma cancer cells. This cell death had characteristics of ferroptosis: it was blocked by the ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine. In addition, the amount of ROS and lipid peroxidation were increased in glioma cells. Iron transport protein remained unchanged but reactive iron levels increased. One target for kinase inhibitors is protein bisulfate isomerase (PDI). Knockdown of PDI in combination with siramesine increased cell death that was blocked by ferrostatin-1. Taken together, drug combinations that alter reactive iron and ROS levels might induce ferroptosis and overcome drug resistance in glioma cells.

Growth Suppression of Glioma Cells Using HDAC6 Inhibitor, Tubacin.

In cancer research, autophagy has been revealed as one of the major ways to maintain the metabolism of cancer cells, including glioma cells, through protein degradation. Meanwhile, autophagy is also regarded as a kind of mechanism to protect glioma cells from a harmful stimulus, such as chemical and radiation treatment. So, the inhibition of autophagy may be very helpful in curing glioma. This study aimed to determine the effect of autophagic inhibition on glioma cells using tubacin, a specific inhibitor of histone deacetylase 6(HDAC6). According to the results, tubacin inhibited the growth of both U251 and LN229 cells, which was accompanied by lower HDAC6 activity and accumulated autophagosome. The inhibition of HDCA6 also led to accumulation of autophagosome and death of glioma cells. Moreover, the combined treatment of tubacin and temozolomide, an alkylating agent used to treat glioblastoma, induced more severe glioma cell death. Thus, it can be concluded that inhibition of HDAC6 suppressed growth and drug resistance of glioma cells in-vitro through autophagic suppression and blocking of fusion of autophagosome and lysosome.

Upregulation of CASC2 sensitized glioma to temozolomide cytotoxicity through autophagy inhibition by sponging miR-193a-5p and regulating mTOR expression

ObjectiveNumerous studies suggested autophagy was involved in temozolomide (TMZ) resistance in glioma. Long non-coding RNA (lncRNA) CASC2 was shown to be downregulated in glioma tissues and cell lines, and was related to the TMZ resistance. However, whether CASC2 affects TMZ resistance through regulating autophagy is unknown. The aim of this study was to assess the role and mechanism of CASC2 in TMZ-induced drug resistance in glioma cells. MethodsGlioma and the adjacent non-cancerous tissues from 32 patients were collected. The expressions of CASC2 and miR-193a-5p were determined by PCR, and their correlation was analyzed. The correlation between CASC2 expression and the clinical characteristics of patients was also studied. Glioma cells were treated with TMZ to acquire the TMZ-resistant cell lines in which the expressions of CASC2, miR-193a-5p, and mTOR were measured. The regulatory roles of CASC2, miR-193a-5p, and mTOR were defined through the loss of function and luciferase reporter assays. Autophagy was inhibited by autophagy inhibitor 3-MA, CASC2 and mTOR overexpression, or miR-193a-5p inhibitor, and the effect of which on cell viability, apoptosis, and migration of TMZ-resistant glioma cells was evaluated. ResultsCASC2 downregulation and miR-193a-5p upregulation was found to be associated with advanced clinical stage and TMZ response in patients with glioma. CASC2 negatively regulates miR-193a-5p expression by direct interaction in glioma cells. Overexpression of CASC2 or inhibition of miR-193a-5p reduced TMZ-induced autophagy via mTOR upregulation, which makes the glioma cells become sensitive to TMZ cytotoxicity. ConclusionCASC2 is downregulated in gliomas, resulting in increased miR-193a-5p level and a decrease in mTOR expression, which further induces protective autophagy, leading to TMZ resistance. Inhibition of autophagy helps to increase the efficacy of TMZ.