Effects of mTOR inhibitor, everolimus, on proliferation, autophagy and temozolomide sensitivity of glioma cells

Purpose: To study the effect of the mTOR inhibitor, everolimus, on glioma cell proliferation, autophagy, and drug sensitivity to temozolomide (TMZ).Methods: Human glioma cell lines were cultured in vitro, and the effects of different concentrations of everolimus on the proliferation of brain glial cells were determined using CCK-8 method. The effect of different concentrations of everolimus on brain glial cell levels of autophagy protein were assayed by western blot method.Results: The results of CCK-8 analysis showed that everolimus inhibited the proliferation of glial cells in a time- and concentration-dependent manner. Western blot results showed that the expression levels of autophagy proteins, LC3-II and LC3-II/I, were gradually and concentration-dependently up-regulated, while p62 protein level was gradually decreased concentration-dependently, when compared with blank control (p < 0.05). Treatment with different concentrations of TMZ alone, and in combination with everolimus for 48 h inhibited the proliferation of brain glial cells in a concentration-dependent manner, but the inhibition due to TMZ-everolimus combination was significantly higher than that of TMZ singletreatment (p < 0.05). After 48 h, the expression level of Beclin-1 increased with the ratio of LC3-II/LC-I in TMZ-everolimus group, while the expression level of p62 decreased, when compared with TMZ alone, or control (p < 0.05).Conclusion: Everolimus significantly inhibits the proliferation of glioma cells and promotes the occurrence of autophagy. Combined use of TMZ and everolimus significantly enhances the sensitivity of TMZ to glioma cells, inhibits cell proliferation, and promotes autophagy better than TMZ alone. Keywords: mTOR inhibitor, Everolimus, Glioma cells, Proliferation, Autophagy

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Curcumol inhibits malignant biological behaviors and TMZ-resistance of glioma cells via reducing long noncoding RNA FoxD2-As1 promoted EZH2 activation

10.21203/rs.3.rs-415245/v1 ◽

2021 ◽

Author(s):

Xuyang Lv ◽

Jiangchuan Sun ◽

Linfeng Hu ◽

Ying Qian ◽

Chunlei Fan ◽

...

Keyword(s):

Cell Proliferation ◽

Noncoding Rna ◽

Glioma Cell ◽

Glioma Cells ◽

Migration And Invasion ◽

Dependent Manner ◽

Antitumor Effects ◽

Self Renewal

Abstract Background: Although curcumol has been shown to possess antitumor effects in several cancers, its effects on glioma are largely unknown. Recently, lncRNAs have been reported to play an oncogenic role through epigenetic modifications. Therefore, here, we investigated whether curcumol inhibited glioma progression by reducing FOXD2-AS1-mediated enhancer of zeste homolog 2 (EZH2) activation.Methods: MTT, colony formation, flow cytometry, Transwell, and neurosphere formation assays were used to assess cell proliferation, cell cycle, apoptosis, the percentage of CD133+ cells, the migration and invasion abilities, and the self-renewal ability. qRT-PCR, western blotting, immunofluorescence, and immunohistochemical staining were used to detect mRNA and protein levels. Isobologram analysis and methylation-specific PCR were used to analyze the effects of curcumol on TMZ resistance in glioma cells. DNA pull-down and Chip assays were employed to explore the molecular mechanism underlying the functions of curcumol in glioma cells. Tumorigenicity was determined using a xenograft formation assay. Results: Curcumol inhibited the proliferation, metastasis, self-renewal ability, and TMZ resistance of glioma cells in vitro and in vivo. FOXD2-AS1 was highly expressed in glioma cell lines, and its expression was suppressed by curcumol treatment in a dose- and time-dependent manner. The forced expression of FOXD2-AS1 abrogated the effect of curcumol on glioma cell proliferation, metastasis, self-renewal ability, and TMZ resistance. Moreover, the forced expression of FOXD2-AS1 reversed the inhibitory effect of curcumol on EZH2 activation.Conclusions: We showed for the first time that curcumol is effective in inhibiting malignant biological behaviors and TMZ-resistance of glioma cells by suppressing FOXD2-AS1-mediated EZH2 activation on anti-oncogenes. Our findings offer the possibility of exploiting curcumol as a promising therapeutic agent for glioma treatment and may provide an option for the clinical application of this natural herbal medicine.

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Curcumol inhibits malignant biological behaviors and TMZ-resistance of glioma cells via reducing long noncoding RNA FoxD2-As1 promoted EZH2 activation

10.21203/rs.3.rs-415245/v2 ◽

2021 ◽

Author(s):

Xuyang Lv ◽

Jiangchuan Sun ◽

Linfeng Hu ◽

Ying Qian ◽

Chunlei Fan ◽

...

Keyword(s):

Cell Proliferation ◽

Noncoding Rna ◽

Glioma Cell ◽

Glioma Cells ◽

Migration And Invasion ◽

Dependent Manner ◽

Antitumor Effects ◽

Self Renewal

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Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2021-0056 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Mohammad Reza Shiran ◽

Elham Mahmoudian ◽

Abolghasem Ajami ◽

Seyed Mostafa Hosseini ◽

Ayjamal Khojasteh ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Animal Model ◽

Protein Expression ◽

Western Blot ◽

Xenograft Model ◽

Dependent Manner ◽

Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

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Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

10.21203/rs.3.rs-88666/v1 ◽

2020 ◽

Author(s):

Tao Yan ◽

Xin Chen ◽

Hua Zhan ◽

Penglei Yao ◽

Ning Wang ◽

...

Keyword(s):

Cell Cycle ◽

Hyaluronic Acid ◽

Tumor Microenvironment ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Glioma Cell ◽

Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

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EXTH-54. ENHANCED SENSITIVITY OF HUMAN GLIOMA CELLS FOR TEMOZOLOMIDE (TMZ) BY COMBINING THERAPY WITH HEAT SHOCK PROTEIN 90

Neuro-Oncology ◽

10.1093/neuonc/noaa215.408 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii99-ii99

Author(s):

Pratibha Sharma ◽

Lakshmi Shree K Mahadevan ◽

Aaron Argall ◽

Jihong Xu ◽

Deepa Sampath ◽

...

Keyword(s):

Cell Proliferation ◽

Heat Shock ◽

Heat Shock Protein ◽

Blood Brain Barrier ◽

Glioma Cell ◽

Glioma Cells ◽

Heat Shock Protein 90 ◽

Brain Barrier ◽

Client Proteins

Abstract BACKGROUND Heat-shock protein 90 (HSP90) is a molecular chaperone involved in the conformational maturation of many client proteins that regulate cell proliferation, survival, and apoptosis. Due to the limited solubility of natural Hsp90 inhibitors, synthetic inhibitors with a more potent impact are being developed. In this study we examined the biological activity of a potent synthetic small molecule Hsp90 inhibitor, SNX-5422 (PF-04929113)and assessed its ability of to inhibit the growth of glioma and to synergize with temozolomide. We also examined the ability of SNX-5422 to cross the blood brain barrier (BBB) and to achieve target inhibition in vivo. METHODS Using a combination of in vitro techniques, the effect of SNX-5422 on the biological impact and HSP90 client protein signaling were studied in glioma lines and patient-derived glioma stem like cells. Its efficacy as a single agent or in combination with TMZ was also assessed in vitro. To assess SNX-5422 ability to cross blood brain barrier, brain and plasma pharmacokinetics was performed in non-tumor bearing mice. RESULTS SNX-5422 exhibited potent growth inhibition in both glioma cells and GSCs with an IC50 range of 100-500nM, and inhibited pro-survival signal kinases, phospho-Akt, p-ERK1/2 and p-S6 following treatment in GSC262 and GSC811. This was accompanied by accumulation of apoptotic cells following SNX-5422 exposure. Combination studies with TMZ showed a synergestic impact on glioma cell proliferation. Pharmacokinetics studies showed a significant drug penetrance into the intact brain further supported by elevated levels of HSP70 (molecular maker for HSP90 inhibition) by IHC. CONCLUSIONS SNX-5422 is effective in downregulating Hsp90 client proteins required for glioma cell survival. In addition, SNX-5422 inhibits tumor growth by promoting apoptosis through modulation of several key signaling pathways and sensitizes glioma cells to TMZ. Given also that SNX-5422 crosses BBB, it warrants further investigation as a clinical agent for treatment of gliomas.

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Novel Roles of Chloroquine and Hydroxychloroquine in Graves’ Orbitopathy Therapy by Targeting Orbital Fibroblasts

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa161 ◽

2020 ◽

Vol 105 (6) ◽

pp. 1906-1917 ◽

Cited By ~ 1

Author(s):

Yan Guo ◽

Hai Li ◽

Xueying Chen ◽

Huasheng Yang ◽

Hongyu Guan ◽

...

Keyword(s):

Western Blot ◽

Real Time ◽

Fluorescent Protein ◽

Inhibitory Effect ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Graves Orbitopathy ◽

Orbital Fibroblasts

Abstract Context Graves’ orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis. Objectives Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs. Design/Setting/Participants OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 μM), and subsequently examined in vitro. Main Outcome Measures CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR) , and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses. Results CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 μM) induced GO-OF apoptosis without aggravating oxidative stress. Conclusions The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors.

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Toxicity of Industrially Relevant Chlorinated Organic Solvents In Vitro

International Journal of Toxicology ◽

10.1177/1091581813482006 ◽

2013 ◽

Vol 32 (2) ◽

pp. 136-145 ◽

Cited By ~ 13

Author(s):

Catherine McDermott ◽

James J.A. Heffron

Keyword(s):

Cell Proliferation ◽

Organic Solvents ◽

Caspase 3 ◽

Free Calcium ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Chlorinated Organic ◽

Free Calcium Concentration ◽

Ros Formation

The cytotoxic effects of 4 industrially important chlorinated organic solvents, dichloromethane (DCM), 1,2-dichloroethane (DCE), trichloroethylene (TCE), and tetrachloroethylene (PERC) in vitro, were investigated. Jurkat T cells were exposed to the solvents individually for 72 hours and changes in reactive oxygen species (ROS) formation, cell proliferation, intracellular free calcium concentration ([Ca2+]), and caspase-3 activity were measured. There was a concentration-dependent increase in the ROS formation and intracellular free [Ca2+] following exposure to each of the solvents. This was accompanied by a decrease in the cell proliferation. Solvent potency decreased in the following order: PERC > TCE > DCM > DCE. Caspase-3 activity was increased in a concentration-dependent manner by TCE and PERC but was not significantly altered by DCM or DCE. n-Acetyl-l-cysteine pretreatment showed that changes in the intracellular free [Ca2+] and caspase-3 activity were independent of ROS formation. However, increased ROS formation did play a causal role in the decreased cell proliferation observed.

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In Vitro Evidence for Effects of Magnesium Supplementation on Quinolone-treated Horse and Dog Chondrocytes

Veterinary Pathology ◽

10.1354/vp.38-2-143 ◽

2001 ◽

Vol 38 (2) ◽

pp. 143-148 ◽

Cited By ~ 22

Author(s):

M. Egerbacher ◽

B. Wolfesberger ◽

C. Gabler

Keyword(s):

Cell Proliferation ◽

Magnesium Deficiency ◽

Great Part ◽

Control Group ◽

Dependent Manner ◽

Free Medium ◽

Culture Dish ◽

Magnesium Supplementation ◽

Concentration Dependent Manner

Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg2+ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 μg/ml) and enrofloxacin (100 and 150 μg/ml). Mg2+ was added at a concentration of 0.0612 mg/ml (MgCl) and 0.0488 mg/ml (MgSO4) or a triple dose. In addition, cells were cultivated in Mg-free medium and accordingly treated with Mg2+ supplementation. After 5 days in culture, the number of adherent cells per milliliter was determined. The number of chondrocytes in quinolone-treated groups decreased to 12-36% that of the control group within the culture period. With Mg2+ supplementation, the number of attached cells increased to 40-70% that of control cells. The threefold dose of Mg2+ led to better results than did the single dose. Cell proliferation tested by immunohistochemical staining with Ki67 (clone MIB5) decreased from 70% in control groups to 55%, 48%, and 30% in enrofloxacintreated groups in a concentration dependent manner (50, 100, and 150 μg/ml). Addition of Mg2+ did not increase the rate of cell proliferation. These results suggest that a great part of quinolone-induced damage is due to magnesium complex formation, as Mg2+ supplementation is able to reduce the effects in vitro. However, quinolone effects on cell proliferation seem to be an independent process that is not influenced by magnesium supplementation.

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The effect of histone deacetylase inhibitor Trichostatin A on IL-17A-induced transformation of MRC5 into myofibroblasts and its mechanism

10.1101/2021.03.31.437856 ◽

2021 ◽

Author(s):

qian Zhang ◽

Zexin Guo ◽

Jing Zhang ◽

Wei Zhou ◽

Yueqing Wu ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Expression ◽

Western Blot ◽

Trichostatin A ◽

Combined Treatment ◽

Collagen I ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Mrc5 Cell ◽

Tgf Β1

Objective To elucidate potential IL-17A- and TSA-mediated regulation of fibroblasts transformation. Methods MTT assay, HDAC1 activity assay, cell immunofluorescence and Western blot were employed to detect the expression of related indicators. Results MRC5 cells expressed only a small amount of Vimentin. IL-17A treatment upregulated MRC5 cell proliferation, in a concentration-dependent manner. TSA treatment, however, suppressed MRC5 cell proliferation. IL-17A treatment also upregulated HDAC1 activity in MRC5 cells, in a concentration-dependent manner. Using immunofluorescence, we demonstrated that IL-17A-treated MRC5 cells had markedly elevated Vimentin, Collagen-I and a-SMA levels, compared to controls. However, a combined treatment of IL-17A and TSA resulted in markedly reduced levels of the Vimentin, Collagen-I and a-SMA, compared to IL-17A alone, yet the amount was higher than controls. Using western blot analysis, we also revealed that the IL-17A-treated MRC5 cells had markedly elevated levels of Vimentin, a-SMA, HDAC1, p-Smad2, and p-Smad3, and markedly reduced level of Smad7, compared to controls. In TSA intervention group, the expression effect of the above protein was opposite. Moreover, no discernible difference was observed in the levels of Smad2 and Smad3 among the treated and un-treated groups. Conclusion IL-17A stimulates proliferation of MRC5 cells and increases HDAC1 activity and protein expression. It also transforms MRC5 cells into myofibroblasts via activation of the TGF -β1/ Smads signaling network. TSA, on the other hand, strongly suppresses TGF -β1/ Smads pathway-mediated fibrosis by ceasing HDAC1 activity and protein expression.

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Downregulation of TET1 Promotes Glioma Cell Proliferation and Invasion by Targeting Wnt/β-Catenin Pathway

Analytical Cellular Pathology ◽

10.1155/2021/8980711 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Jianwen Ji ◽

Qiuxiang You ◽

Jidong Zhang ◽

Yutao Wang ◽

Jing Cheng ◽

...

Keyword(s):

Cell Proliferation ◽

Glioma Cell ◽

Molecular Mechanisms ◽

Glioma Cells ◽

Adult Brain ◽

Migration And Invasion ◽

Downstream Targets ◽

Glioma Cell Proliferation

Glioma is the most common malignant tumor in adult brain characteristic with poor prognosis and low survival rate. Despite the application of advanced surgery, chemotherapy, and radiotherapy, the patients with glioma suffer poor treatment effects due to the complex molecular mechanisms of pathological process. In this paper, we conducted the experiments to prove the critical roles TET1 played in glioma and explored the downstream targets of TET1 in order to provide a novel theoretical basis for clinical glioma therapy. RT-qPCR was adopted to detect the RNA level of TET1 and β-catenin; Western blot was taken to determine the expression of proteins. CCK8 assay was used to detect the proliferation of glioma cells. Flow cytometry was used to test cell apoptosis and distribution of cell cycle. To detect the migration and invasion of glioma cells, wound healing assay and Transwell were performed. It was found that downregulation of TET1 could promote the proliferation migration and invasion of glioma cells and the concomitant upregulation of β-catenin, and its downstream targets like cyclinD1 and c-myc were observed. The further rescue experiments were performed, wherein downregulation of β-catenin markedly decreases glioma cell proliferation in vitro and in vivo. This study confirmed the tumor suppressive function of TET1 and illustrated the underlying molecular mechanisms regulated by TET1 in glioma.

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