Proliferation Of Human Glioblastoma Cells Research Articles

Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition.

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.

CircPITX1 Regulates Proliferation, Angiogenesis, Migration, Invasion, and Cell Cycle of Human Glioblastoma Cells by Targeting miR-584-5p/KPNB1 Axis.

Recent researches reported that several circular RNAs (circRNAs) were associated with the glioblastoma (GBM) progression, while the regulatory role of circPITX1 remains unknown in GBM. The real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify circPITX1, miR-584-5p, and karyopherin b1 (KPNB1) expression in GBM tissues and cells. The proliferation capability of cells was analyzed by Cell Counting Kit-8 (CCK-8) and colony-forming assays. The matrigel angiogenesis assay was used to assess tube formation in GBM cells. Flow cytometry assays were conducted to evaluate the cell cycle distribution of GBM cells. The migration and invasion assays were assessed by transwell assay. The Western blot assay was employed to quantify the protein expression level in GBM tissues and cells. The targets of circPITX1 and miR-584-5p were confirmed by dual-luciferase reporter and RNA pull-down assays. A xenograft experiment in nude mice was used to assess the functional role of circPITX1 in vivo. CircPITX1 was obviously overexpressed in GBM tissues and cells when compared with negative groups. The functional experiment implied that knockdown of circPITX1 suppressed proliferation, angiogenesis, migration, invasion, and tumor growth in vivo along with induced cell cycle arrest of GBM cells. Furthermore, miR-584-5p was a target gene of circPITX1, and knockdown of miR-584-5p could abolish circPITX1 silencing-induced effects on GBM cells. KPNB1 was a target gene of miR-584-5p, and functional experiments revealed that overexpression of miR-584-5p repressed proliferation, angiogenesis, migration, invasion, and cell cycle process in GBM cells by targeting KPNB1. Mechanistically, circPITX1/miR-584-5p/KPNB1 axis regulated GBM process via mediating proliferation, angiogenesis, migration, invasion, and cell cycle process of GBM cells.

Notch intracellular domain regulates glioblastoma proliferation through the Notch1 signaling pathway.

Notch intracellular domain (NICD), also known as the activated form of Notch1 is closely associated with cell differentiation and tumor invasion. However, the role of NICD in glioblastoma (GBM) proliferation and the underlying regulatory mechanism remains unclear. The present study aimed to investigate the expression of NICD and Notch1 downstream gene HES5 in human GBM and normal brain samples and to further detect the effect of NICD on human GBM cell proliferation. For this purpose, western blotting and immunohistochemical staining were performed to analyze the expression of NICD in human GBM tissues, while western blotting and reverse-transcription quantitative PCR experiments were used to analyze the expression of Hes5 in human GBM tissues. A Flag-NICD vector was used to overexpress NICD in U87 cells and compound E and small interfering (si) Notch1 were used to downregulate NICD. Cellular proliferation curves were generated and BrdU assays performed to evaluate the proliferation of U87 cells. The results demonstrated that compared with normal brain tissues, the level of NICD protein in human GBM tissues was upregulated and the protein and mRNA levels of Hes5 were also upregulated in GBM tissues indicating that the Notch1 signaling pathway is activated in GBM. Overexpression of NICD promoted the proliferation of U87 cells in vitro while downregulation of NICD by treatment with compound E or siNotch1 suppressed the proliferation of U87 cells in vitro. In conclusion, NICD was upregulated in human GBM and NICD promoted GBM proliferation via the Notch1 signaling pathway. NICD may be a potential diagnostic marker and therapeutic target for GBM treatment.

Galangin (GLN) Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells by Targeting Skp2-Induced Epithelial-Mesenchymal Transition (EMT).

BackgroundGalangin (GLN), a pure natural flavonoid compound found in plants, has been shown to exert anti-cancer effects against multiple cancer types, including glioma. However, its underlying molecular mechanism remains unclear. Epithelial-to-mesenchymal transition (EMT) performs an important function in the genesis and development of cancer. Skp2, a pivotal component of SCFSkp2 E3 ubiquitin ligase, has been shown to function as an oncogene in GBM invasion that contributes to the EMT process. Thus, we explored whether GLN inhibited Skp2-mediated EMT and the mechanism underlying the Skp2 degradation pathway.MethodsCCK-8 assay, wound healing assay and transwell assay were used to examine cell proliferation, migration, and invasion after treatment with or without GLN. RT-PCR and Western blotting analysis were performed to evaluate mRNA and protein expression, respectively. Co-immunoprecipitation was conducted to detect ubiquitinated Skp2 levels in vitro and in vivo after GLN treatment. Bioluminescence imaging was performed to examine the intracranial tumor size of U87 xenograft mice. Microscale thermophoresis (MST) experiment was used to detect interactions between Skp2 and GLN.ResultsGLN suppressed GBM cell growth, migration, and invasion, and also downregulated the expression of Skp2 and mesenchymal markers (Zeb1, N-cadherin, snail, vimentin) in vitro. Moreover, the overexpression of Skp2 in GBM cells decreased the effect of GLN on EMT. Furthermore, we demonstrated that GLN degraded skp2 protein through the ubiquitination proteasome pathway and directly interacted with skp2 protein, as shown through the MST assay.ConclusionThis study is the first to identify Skp2 as a novel target of GLN for the treatment of GBM and report of Skp2 protein degradation in a ubiquitination proteasome pathway. Results from our study indicated the potential of GLN for the treatment of GBM through ubiquitin-mediated degradation of Skp2.

β-Elemene Inhibits the Proliferation and Migration of Human Glioblastoma Cell Lines via Suppressing Ring Finger Protein 135.

β-Elemene is commonly used as an anti-cancer agent in different types of cancers and its effects on glioblastoma have been studied through different pathways. However, its effect through ring finger protein 135 (RNF135, OMIM 611358) (RNF135), which is upregulated in glioblastomas, has not yet been explored. The current study is focused on the effects of β-elemene on human glioblastoma cell lines U251, U118, A172 and U87 through RNF13 5. A cell counting kit-8 assay and wound healing assay have been utilized to test the proliferation and migration of the cells. Western blot and quantitative real-time-polymerase chain reaction (qRT-PCR) were used to evaluate the level of expression of RNF135. A model of nude mice was used to explore progression of the tumor in vivo. It was observed that increasing treatment time or dose of β-elemene remarkably decreased viability of the cells. The cells that were treated with β-elemene had a much lower speed of moving toward the gap in comparison to untreated cell lines. β-Elemene-treated cells showed a much lower level of expression of RNF135 mRNA than control groups (p <0.05) and the levels of RNF135 protein were lower in the cells treated with β-elemene than in control groups (p <0.05). Moreover, tumor progression in subcutaneous xenograft nude mice was delayed with the injection of β-elemene. Altogether, our findings suggest that β-elemene inhibits proliferation, migration and tumorigenicity of human glioblastoma cells through suppressing RNF135.

Abstract 2652: Adjuvant therapeutic potential of hypothermia in the treatment of glioblastoma: An in vitro study

Abstract Glioblastoma is a particularly aggressive and infiltrative brain tumor with an annual incidence of three per 100,000 adults worldwide. Despite multimodal treatments, associating surgical resection with combined radiotherapy and temozolomide-based chemotherapy [1], prognosis remains poor. Median survival is only 14 to 16 months because of tumor recurring in the resection margins in over 90% of patients, due to the activation of residual glioblastoma cells. Hence, there is a need for developing novel therapies that improve survival outcome. To handle this issue, we propose therapeutic hypothermia as an adjuvant treatment, to place tumor cells at the resection margin in a dormant state. The aim of our study is to evaluate the adjuvant therapeutic potential of moderate hypothermia by investigating in vitro its ability to prevent cancer cell growth. We previously published results demonstrating that continuous moderate hypothermia at 28 °C is able to significantly inhibit both cell proliferation and migration [2]. We performed in vitro experiments on four glioblastoma cell lines with different p53 status and various growth rates: A-172, U-251 MG, U-87 MG, and T98G. To study the adjuvant potential of moderate hypothermia, we investigated its inhibitory effects on the proliferation of glioblastoma cells exposed to different conditions. First, we studied the impact of different sequences in which cells were successively placed in hypothermic conditions then rewarmed to 37 °C. On the other hand, we compared the inhibitory effects of moderate hypothermia with current chemotherapy protocols. Finally, we investigated the therapeutic potential of combining chemotherapy with hypothermia. Here, we showed that moderate hypothermia significantly inhibited glioblastoma cell proliferation even after rewarming to 37 °C, with a significant influence of hypothermic preconditioning duration. In addition, moderate hypothermia reduced cell proliferation far more than chemotherapy alone, with inhibitory effects up to eight times higher for chemoresistant cell lines. Finally, combining hypothermia with chemotherapy further reduced proliferation, with more than 95% inhibition even after returning glioblastoma cells to 37 °C. Our results showed that glioblastoma cell proliferation was not fully restored upon returning to 37 °C, suggesting that the effects of moderate hypothermia on cell growth are not limited to direct conditioning, which is important for clinical applications. We also demonstrated that responses to moderate hypothermia were similar for all four glioblastoma cell lines whereas they presented different levels of sensitivity to chemotherapy, depending on their p53 status. Moreover, combining both therapeutic approaches significantly improved glioblastoma cell sensitivity to treatment for all cell lines, regardless of their p53 status. Finally, these results showed that moderate hypothermia has the potential to reduce inter-patient variability of treatment efficiency, and thus appears to be a relevant adjuvant therapy for use when treating glioblastoma patients. [1] R. Stupp et al., "Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival⋯", Lancet Oncol., 2009. [2] C. Fulbert et al., "Moderate hypothermia inhibits both proliferation and migration of human glioblastoma cells", J. Neurooncol., 2019. Citation Format: Clémentine Fulbert, Christophe Gaude, Stéphan Chabardès, David Ratel. Adjuvant therapeutic potential of hypothermia in the treatment of glioblastoma: An in vitro study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2652.

Abstract 2972: Urolithin A decreases cell proliferation of three genetically diverse glioblastoma multiforme cell lines

Abstract Glioblastoma multiforme is one of the most common and malignant tumors of the central nervous system, remaining incurable with a median survival time of only 15 months. Over the past 50 years there has been little change in this overall survival rate, due to the many challenges in treating glioblastoma, including molecular complexity, tumor location, and resistance towards standard therapies. Therefore, an improved method for treating glioblastoma is urgently needed. Urolithin A is a natural biometabolite from ellagitannins and ellagic acid, which can be found in pomegranates, berries, and nuts. Urolithin A exhibits anti-cancer activities in several cancer cell lines, including prostate, colon, and bladder cancer cells. However, the anti-cancer activities of urolithin A have yet to be characterized in glioblastoma multiforme. In this project, we investigated whether urolithin A could reduce human glioblastoma cell proliferation by conducting clonogenic assays and cell cycle analysis in three genetically diverse human glioblastoma cell lines: U87MG, T98G, and LN229. Clonogenic assay results showed that urolithin A significantly decreased cell proliferation in a dose-dependent manner in all three glioblastoma cell lines. Depending on the different cell lines, the cell proliferation was inhibited at concentrations of 10 to 60 µM urolithin A. In addition, cell cycle analysis via flow cytometry found that urolithin A was capable of arresting cell cycle progression in either the G2/M or S phase of the cell cycle in U87 and LN229 or T98G cells, respectively. In conclusion, we have found that urolithin A can reduce cell proliferation and alter cell cycle progression of three genetically diverse glioblastoma cell lines. Importantly, urolithin A can pass the blood-brain barrier and has previously shown to be non-toxic in humans, a prerequisite for a drug to treat glioblastoma. Therefore, urolithin A has the potential to be used as a novel drug which can be combined with current standard methods to improve the efficacy of glioblastoma treatment. In the future, we plan to investigate specific signaling pathways involved in apoptosis, cellular proliferation, and cell cycle arrest. Once we elucidate the specific mechanisms we will investigate the efficacy of urolithin A in vivo, using an animal model. If successful, urolithin A will have the potential to be used alongside radiation and chemotherapy to further reduce glioblastoma cell proliferation. Citation Format: Alexandra Lynn Hearne, Dakota Brockway, Shiyong Wu, Yunsheng Li. Urolithin A decreases cell proliferation of three genetically diverse glioblastoma multiforme cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2972.

5alpha-dihydroprogesterone promotes proliferation and migration of human glioblastoma cells

Glioblastomas (GBMs) are the most common and deadliest intracranial tumors. Steroid hormones, such as progesterone (P4), at physiological concentrations, promote proliferation, and migration of human GBM cells in vivo and in vitro. Neuronal and glial cells, but also GBMs, metabolize P4 and synthesize different active metabolites such as 5α-dihydroprogesterone (5α-DHP). However, their contribution to GBM malignancy remains unknown. Here, we determined the 5α-DHP effects on the number of cells, proliferation, and migration of the U87 and U251 human GBM-derived cell lines. Of the tested concentrations (1 nM-1 µM), 5α-DHP 10 nM significantly increased the number of U87 and U251 cells from day 2 of treatment, and proliferation (at day 3) in a similar manner as P4 (10 nM). The treatment with the progesterone receptor (PR) antagonist RU486 (mifepristone), blocked the effects of 5α-DHP on the number of cells and proliferation. Besides, in U251 and LN229 GBM cells, 5α-DHP promoted cell migration (from 12 to 24 h). We also determined that GBM cells expressed the 3α-hydroxysteroid oxidoreductases (3α-HSOR), which reversibly reduce 5α-DHP to allopregnanolone (3α-THP). These data indicate that 5α-DHP induces proliferation and migration of human GBM through the activation of PR.

Avasimibe exerts anticancer effects on human glioblastoma cells via inducing cell apoptosis and cell cycle arrest.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, but there is no effective drug available for GBM. Avasimibe is a potent inhibitor of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1), which was used to treat atherosclerosis. Experimental evidence and bioinformatics have shown that avasimibe has anticancer activity. In this study we investigated the anticancer effects of avasimibe on human glioblastoma cells and the underlying mechanisms. Our results showed that avasimibe dose-dependently inhibited the proliferation of U251 and U87 human glioblastoma cells with IC50 values of 20.29 and 28.27 μM, respectively, at 48 h. Avasimibe (7.5, 15, 30 μM) decreased the DNA synthesis, and inhibited the colony formation of the tumor cells. Treatment of avasimibe also dose-dependently increased the apoptotic rate of tumor cells, decreased the mitochondrial membrane potential, induced the activity of caspase-3/7, and increased the protein expression of cleaved caspase-9, cleaved PARP and Bax in U251 and U87 cells. RNA-sequencing analyses revealed that avasimibe suppressed the expression of CDK2, cyclin E1, CDK4, cyclin D, CDK1, cyclin B1, Aurora A, and PLK1, while induced the expression of p53, p21, p27, and GADD45A, which was validated by Western blot analysis. These results demonstrated that avasimibe induced mitochondria-dependent apoptosis in glioblastoma cells, which was associated with arresting the cell cycle at G0/G1 phase and G2/M phase by regulating the p53/p21 pathway, p53/GADD45A and Aurora A/PLK1 signaling pathways. In U87 xenograft nude mice model, administration of avasimibe (15, 30 mg·kg-1·d-1, ip, for 18 days) dose-dependently inhibit the tumor growth. Taken together, our results demonstrated that avasimibe might be a promising chemotherapy drug in the treatment of GBM.

Gamma-Linolenic acid alters migration, proliferation and apoptosis in human and rat glioblastoma cells

Glioblastoma multiforme (GBM) is the most malignant astrocytoma, the main treatments consist of surgical resection followed by radiotherapy and chemotherapy. Patients, after diagnosed, have a survival rate of one year. GBM cells have an invasive, proliferative and migratory characteristic, also they do not respond properly for usual cancer treatment (radiotherapy, chemotherapy). Fatty acids have been studied as an adjuvant cancer treatment in breast, colorectal and GBM. The fatty acid can alter tumoural cell metabolism causing a modification of eicosanoids production. This study has observed some cellular aspects modified by fatty acid treatment in vitro, using GBM cells (human and rat). Modifications in cell behaviour were analyzed like cell proliferation, apoptosis, migration and invasion cell capacity after treatment with fatty acid (gamma-linolenic acid). The treatment suggested in this study showed an increased number of apoptotic cells and a decreased number of proliferative and migratory cells. These data recognize that gamma-linolenic acid could be used as an alternative treatment for glioblastoma.

MiR-449b-5p inhibits human glioblastoma cell proliferation by inactivating WNT2B/Wnt/β-catenin signaling pathway.

As the most common primary brain cancer in adults, glioblastoma shows an extremely poor prognosis. Glioblastoma-associated deaths account for approximately 3%-4% of all malignancy-associated deaths. Numerous microRNAs (miRNAs) play important roles in the occurrence and progression of solid tumors. Herein, identifying functional miRNAs and the central molecular mechanisms would provide novel proofs for the development of targeted cancer therapies. In this study, we described the role of miR-449b-5p in restraining ontogenesis and progression of glioblastoma. Human glioblastoma tissues were provided by our hospital. Human U251 glioblastoma cells were infected with lentivirus induced miR-449b-5p mimics or miR-449b-5p siRNA. Real-time qPCR was carried out to determine miRNA expression. Tumor spheres formation, MTT assay, and BrdU cell proliferation assay were used to evaluate the growth ability of U251 cells. Western blot assay was performed to measure protein expression. ChIP was used to detect the capacity of β-catenin to recruit its downstream genes. Dual-Luciferase assay was conducted to detect the ability of miR-449b-5p to regulate the 3'UTR (untranslated regions) of WNT2B. TOP/FOP ratio was used to evaluate the activity of Wnt/β-catenin signaling pathway. Down-regulation of miR-449b-5p expression was found in both human glioblastoma tissues and cell lines, which was negatively associated with the clinical stages. Up-regulation of miR-449b-5p inhibited tumor spheres formation, cell viability and proliferation ability of glioblastoma cells. The expression levels of WNT2B and nuclear β-catenin were negatively associated with miR-449b-5p levels in glioblastoma cells. MiR-449b-5p inhibited Wnt/β-catenin signaling by targeting WNT2B. MiR-449b-5p acts as a tumor suppressor and retards the oncogenesis of glioblastoma, which is achieved via inactivation of Wnt/β-catenin signaling by directly targeting WNT2B.

Major Contribution of Caspase-9 to Honokiol-Induced Apoptotic Insults to Human Drug-Resistant Glioblastoma Cells.

Temozolomide (TMZ)-induced chemoresistance to human glioblastomas is a critical challenge now. Our previous studies showed that honokiol, a major bioactive constituent of Magnolia officinalis (Houpo), can kill human glioblastoma cells and suppresses glioblastoma growth. This study was further aimed to evaluate the effects of honokiol on human drug-resistant glioblastoma cells and the possible mechanisms. The results by data mining in the cancer genome atlas (TCGA) database and immunohistochemistry displayed that expression of caspase-9 mRNA and protein in human glioblastomas was induced. Human TMZ-resistant U87-MG-R9 glioblastoma cells were selected and prepared from human drug-sensitive U87-MG cells. Compared to human drug-sensitive U87-MG cells, TMZ did not affect viability of U87-MG-R9 glioblastoma cells. Interestingly, treatment with honokiol suppressed proliferation and survival of human drug-resistant glioblastoma cells in concentration- and time-dependent manners. Compared to caspase-8 activation, honokiol chiefly increased activity of caspase-9 in U87-MG-R9 cells. Successively, levels of cleaved caspase-3 and activities of caspase-3 and caspase-6 in human TMZ-tolerant glioblastoma cells were augmented following honokiol administration. In parallel, honokiol triggered DNA fragmentation of U87-MG-R9 cells. Accordingly, treatment of human TMZ-resistant glioblastoma cells with honokiol induced cell apoptosis but did not affect cell necrosis. Fascinatingly, suppressing caspase-9 activity using its specific inhibitors repressed honokiol-induced caspase-6 activation, DNA fragmentation, and cell apoptosis. Taken together, this study has shown the major roles of caspase-9 in transducing honokiol-induced mitochondria-dependent apoptosis in human drug-resistant glioblastoma cells. Thus, honokiol may be clinically applied as a drug candidate for treatment of glioblastoma patients with chemoresistance.

SOX4-mediated repression of specific tRNAs inhibits proliferation of human glioblastoma cells

Transfer RNAs (tRNAs) are products of RNA polymerase III (Pol III) and essential for mRNA translation and ultimately cell growth and proliferation. Whether and how individual tRNA genes are specifically regulated is not clear. Here, we report that SOX4, a well-known Pol II-dependent transcription factor that is critical for neurogenesis and reprogramming of somatic cells, also directly controls, unexpectedly, the expression of a subset of tRNA genes and therefore protein synthesis and proliferation of human glioblastoma cells. Genome-wide location analysis through chromatin immunoprecipitation-sequencing uncovers specific targeting of SOX4 to a subset of tRNA genes, including those for tRNAiMet Mechanistically, sequence-specific SOX4-binding impedes the recruitment of TATA box binding protein and Pol III to tRNA genes and thereby represses their expression. CRISPR/Cas9-mediated down-regulation of tRNAiMet greatly inhibits growth and proliferation of human glioblastoma cells. Conversely, ectopic tRNAiMet partially rescues SOX4-mediated repression of cell proliferation. Together, these results uncover a regulatory mode of individual tRNA genes to control cell behavior. Such regulation may coordinate codon usage and translation efficiency to meet the demands of diverse tissues and cell types, including cancer cells.

GALE Promotes the Proliferation and Migration of Glioblastoma Cells and Is Regulated by miR-let-7i-5p.

PurposeGlioma is the most common and lethal type of brain tumor. While GALE (UDP-galactose-4-epimerase) has been shown to be overexpressed in some kinds of cancers, its expression in gliomas has not been reported. MicroRNAs (miRNAs) function as tumor suppressors in many cancers, and online datasets can be used to predict whether GALE is regulated by miR-let-7i-5p. In this investigation, we explored the effect and regulatory mechanisms of GALE on glioblastoma growth via miR-let-7i-5p.MethodsWe used a Cox proportional hazards model and publicly available datasets to examine the relationship between GALE and the survival rates of glioma patients. Bioinformatics predicted the targeting of GALE by miR-let-7i-5p. The proliferation, migration, cell cycle and apoptosis of human glioblastoma cells were assessed by relevant assays. We also demonstrated the effect of GALE on glioblastoma multiforme [GBM] tumor growth using an in vivo orthotopic xenograft model.ResultsGALE was upregulated in human gliomas, especially in high-grade gliomas (e.g., GBM). An obvious decline in GALE expression was observed in human glioblastoma cell lines (U87 and U251) following treatment with a small interfering RNA (siRNA) targeting GALE or miR-let-7i-5p mimics. Knockdown of GALE or overexpression of miR-let-7i-5p (with miR-let-7i-5p mimics) inhibited U87 and U251 cell growth. miR-let-7i-5p significantly restrained the migration ability of human glioblastoma cells in vascular mimic (VM), wound healing and transwell assays, and GALE promoted glioblastoma growth in vivo.ConclusionOur findings confirm that GALE plays an important role in promoting the development of human glioma and that GALE can be regulated by miR-let-7i-5p to inhibit human glioblastoma growth.Implications for cancer survivorsOur data show that cancer survivors have low GALE expression, which indicates that GALE may be a diagnostic biomarker and a promising therapeutic target in glioblastoma.

Moderate hypothermia inhibits both proliferation and migration of human glioblastoma cells.

Glioblastoma is the most aggressive malignant brain tumor. Despite multimodal treatments, median survival is only 15months for glioblastoma patients, with tumor recurring in the resection margins after surgical removal. Hypothermia is emerging as an interesting and safe treatment for several conditions. In the context of glioblastoma, we propose that moderate hypothermia could inhibit both cell proliferation and migration, and thus help prevent secondary tumor growth. In vitro experiments on A172, U251, U87 and T98G human glioblastoma cell lines explored the effects of severe (23°C), moderate (28°C), and mild (33°C) hypothermia. We further investigated the effects of moderate hypothermia on cell proliferation, migration, morphology, and cell cycle distribution. Similar results were obtained with all four cell lines, indicating a consistent and broad effect of moderate hypothermia. Hypothermia inhibited both cell proliferation and non-oriented migration in a dose-dependent manner, with a significant reduction at 33°C and almost total arrest at 28°C. Cell proliferation arrest was long-lasting and oriented cell migration was also reduced at 28°C. Moreover, moderate hypothermia significantly altered cell cycle distribution, with cells accumulating in the G2/M phase, leading to cell cycle arrest. Lastly, hypothermia at 28°C also affected cell morphology by deteriorating cell membranes and altering cell shape. The presented results demonstrate that moderate hypothermia could be a promising adjuvant therapy for glioblastoma treatment as it strongly inhibits both cell proliferation and migration. If in vivo preclinical results corroborate our findings, therapeutic hypothermia applied at the resection margins could probably delay tumor recurrence, combined with current treatments.

Tibolone Effects on Human Glioblastoma Cell Lines

Ovarian steroid hormones are involved in modulating the growth of glioblastomas (the most common, aggressive, and lethal brain tumor) through the interaction with their intracellular receptors. Activation of sex hormone receptors is involved in glioblastomas progression. Tibolone (TIB) is a selective tissue estrogenic activity regulator widely prescribed to treat menopausal symptoms and to prevent bone lost. The effects of TIB on the growth of glioblastoma are unknown. To evaluate the effects of TIB on cell number, migration, and invasion of two derived human glioblastoma cell lines (U251MG and U87), as well as the role of this steroid in estrogen and progesterone receptors activity and content. U251-MG and U87 human glioblastoma cell lines were grown with different doses of TIB. The number of cells was determined and migration and invasion tests were carried out. Protein expression was performed by Western blot. We observed that TIB (10nM) increased the number of cells by inducing proliferation with no effects on cell migration or invasion. The increase in cell proliferation induced by TIB was blocked by estrogen (ERs) or progesterone receptor (PRs) antagonists, ICI 182, 780 and RU 486, suggesting that these receptors mediate proliferating actions of TIB; TIB also modified the content of ERs and PRs by increasing ER-α, ER-β, and PR-B, while decreased PR-A. Our results suggest that TIB increases cell number and proliferation of human glioblastoma cells through the regulation of ERs and PRs actions and content.

Jinlong capsule inhibits migration and invasion in human glioblastoma cells via the modulation of mTOR/S6 signaling pathway.

Aim: To investigate the anticancer effects of Jinlong capsule (JLC) against human glioblastoma cells and the possible underlying mechanism.Methods: Cell Counting Kit-8 and colony formation assay were adopted for the analysis of cell viability. Cell invasion and migration were evaluated by transwell and wound healing assays. Then, the expression level of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), S6 and phosphorylated S6 (p-S6) were determined by western blotting.Results: The results showed that JLC significantly inhibited human glioblastoma cell proliferation, invasion and migration in a dose-dependent manner. The expressions of p-mTOR and p-S6 were dramatically suppressed by JLC. Furtherly, inhibition of mTOR reduced the cell migration and invasion, while the mTOR agonist (MHY1485) could partially reverse the anti-migration and anti-invasion activity of JLC.Conclusion: The above results suggested that JLC would be a potential candidate for the treatment of glioblastoma.

CD164 regulates proliferation, progression, and invasion of human glioblastoma cells.

Grade IV astrocytoma, also known as glioblastoma multiforme (GBM), is the most common and aggressive intracranial glial tumor. GBM is associated with very poor survival and effective treatments have remained elusive so far. Mounting evidence indicates that CD164 contributes to stemness and tumorigenesis in normal cells and is overexpressed in various tumor types, including glioblastoma. Using tissue microarray immunohistochemistry, we show that there is a significant correlation between CD164 expression and glioma type and grade. Depletion of CD164 expression in human glioblastoma cells with siRNA reduced proliferation, migration, and invasiveness. In parallel, immunoblotting showed that downregulation of CD164 expression decreased Akt activation and modified the expression of autophagy markers by upregulating Beclin-1 and LC3B and downregulating p62. These effects were mimicked by inhibition of Akt with MK2206, which suggests that CD164 induces autophagy via Akt/Beclin-1 signaling. We propose that CD164 may serve as a GBM molecular marker and a potential target in therapeutic strategies aimed to improve outcomes for this devastating brain tumor.

Prostaglandin E2 increases migration and proliferation of human glioblastoma cells by activating transient receptor potential melastatin 7 channels.

Recent studies showed that both prostaglandin E2 (PGE2) and transient receptor potential melastatin 7 (TRPM7) play important roles in migration and proliferation of human glioblastoma cells. In this study, we tested the association between PGE2 and TRPM7. We found that PGE2 increased TRPM7 currents in HEK293 and human glioblastoma A172 cells. The PGE2 EP3 receptor antagonist L‐798106 abrogated the PGE2 stimulatory effect, while EP3 agonist 17‐phenyl trinor prostaglandin E2 (17‐pt‐PGE2) mimicked the effect of PEG2 on TRPM7. The TRPM7 phosphotransferase activity‐deficient mutation, K1646R had no effect on PGE2 induced increase of TRPM7 currents. Inhibition of protein kinase A (PKA) activity by Rp‐cAMP increased TRPM7 currents. TRPM7 PKA phosphorylation site mutation S1269A abolished the PGE2 effect on TRPM7 currents. PGE2 increased both mRNA and membrane protein expression of TRPM7 in A172 cells. Knockdown of TRPM7 by shRNA abrogated the PGE2 stimulated migration and proliferation of A172 cells. Blockage of TRPM7 with 2‐aminoethoxydiphenyl borate (2‐APB) or NS8593 had a similar effect as TRPM7‐shRNA. In conclusion, our results demonstrate that PGE2 activates TRPM7 via EP3/PKA signalling pathway, and that PGE2 enhances migration and proliferation of human glioblastoma cells by up‐regulation of the TRPM7 channel.

Knockdown of arsenic resistance protein 2 inhibits human glioblastoma cell proliferation through the MAPK/ERK pathway.

It is generally known that glioblastoma is the most common primary malignant brain tumor and that it is highly aggressive and deadly. Although surgical and pharmacological therapies have made long-term progress, glioblastoma remains extremely lethal and has an uncommonly low survival rate. Therefore, further elucidation of the molecular mechanisms of glioblastoma initiation and its pathological processes are urgent. Arsenic resistance protein 2 (Ars2) is a highly conserved gene, and it has been found to play an important role in microRNA biosynthesis and cell proliferation in recent years. Furthermore, absence of Ars2 results in developmental death in Drosophila, zebrafish and mice. However, there are few studies on the role of Ars2 in regulating tumor development, and the mechanism of its action is mostly unknown. In the present study, we revealed that Ars2 is involved in glioblastoma proliferation and we identified a potential mechanistic role for it in cell cycle control. Our data demonstrated that Ars2 knockdown significantly repressed the proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo. Further investigation clarified that Ars2 deficiency inhibited the activation of the MAPK/ERK pathway, leading to cell cycle arrest in the G1 phase, resulting in suppression of cell proliferation. These findings support the conclusion that Ars2 is a key regulator of glioblastoma progression.