etifoxine  (Adooq Bioscience LLC)

Journal: bioRxiv

Article Title: Mitochondrial sites of contact with the nucleus aid in chemotherapy evasion of glioblastoma cells

doi: 10.1101/2024.08.27.608373

Figure Lengend Snippet: (A) Schematic of the experimental design for glioblastoma cells treatment with TMZ and TSPO ligands. (B) Cell viability was evaluated via crystal violet in u87MG, u87MG + TSPO, u87MG - TSPO, T98G, A172MG and ADF treated with Etifoxine (30μM) and Lovastatin (10μM) in combination with TMZ (100μM) for 24 hours. (C) Isosurfaces derived from confocal images of immunocytochemical analyses of TSPO (green) and ATPβ (red) at 24h-time point of TMZ treatment (100μM) combined with Lovastatin (10μM) and Etifoxine (30μM) (bar=100μ). (D) Quantification of the mito-nuclear contacts. (E) Immunoblotting of TSPO, MnSOD, Actin and Lamin B2 in nuclear and cytosolic fractions of TMZ-treated U87MG (24h-timepoint, 100μM) alone and combined with Lovastatin(10μM) or Etifoxine (30μM). (F) Reports densitometry analysis of TSPO and MnSOD levels. All data are represented as mean±sem. *p≤0.05; **p≤0.01; ***p≤0.001

Article Snippet: The compounds Temozolomide (Sigma, T2577), PK11195 (Enzo Life Technologies, BML-CM 118), Etifoxine (Sigma, SML 0272), Lovastatin (Enzo, BML-G226), Cerivastatin (Sigma, SML0005) and MF-438 (Sigma, 569406) have been enrolled in the analysis at the concentration and time indicated in the specific experiments.

Techniques: Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Mitochondrial sites of contact with the nucleus aid in chemotherapy evasion of glioblastoma cells

doi: 10.1101/2024.08.27.608373

Figure Lengend Snippet: (A) Cell viability was evaluated via crystal violet in U87MG cells treated with Etifoxine (30μM) and in combination with TMZ (100μM) for 48 and 72 hours. (B) Representative images of U87MG spheroids stained with Dapi, PI and antibody for TSPO at resting conditions, following TMZ alone and combined with Etifoxine (TMZ+Etifoxine). Histograms reporting quantification of spheroids diameter in the conditions of analysis ( C ) and relative changes in fluorescent intensity ( D ). (E) Percentage of MGMT promoter methylation (MGMTp meth%) in t98G and ADF cell lines. (F) Immunoblotting of MGMT and TSPO in ADF total lysates treated with TMZ (100μM) alone or combined with Lovastatin (10μM) and Etifoxine (30μM) for 24 hours. The graph in panel ( G ) shows the relative densitometry of MGMT and TSPO normalized to GAPDH. All data are represented as mean±sem. *p≤0.05; **p≤0.01; ***p≤0.001

Article Snippet: The compounds Temozolomide (Sigma, T2577), PK11195 (Enzo Life Technologies, BML-CM 118), Etifoxine (Sigma, SML 0272), Lovastatin (Enzo, BML-G226), Cerivastatin (Sigma, SML0005) and MF-438 (Sigma, 569406) have been enrolled in the analysis at the concentration and time indicated in the specific experiments.

Techniques: Staining, Methylation, Western Blot

Journal: bioRxiv

Article Title: Mitochondrial sites of contact with the nucleus aid in chemotherapy evasion of glioblastoma cells

doi: 10.1101/2024.08.27.608373

Figure Lengend Snippet: (A) Filipin-based staining of cholesterol (bar=20µm) in U87MG cells treated with TMZ (100μM) alone and combined with Etifoxine (30μM, 24h) and Lovastatin (10μM, 24h). The cholesterol inhibitor U1866A (4μM, 24h) was used as positive control. (B) qRT-PCR analysis of CYP11 in U87MG cells treated with TMZ (100μM) alone and combined with Etifoxine (30μM) and Lovastatin (10μM) for 24hours. (C) Immunoblotting of LXRα and SREBP1 in nuclear fractions of TMZ-treated U87MG (24h-timepoint, 100μM) alone or combined with Lovastatin (10μM) and Etifoxine (30μM). Quantification (normalized on Lamin B2) is reported in (D) . (E) Axial post-contrast MRI scan of glioblastoma before surgery and at recurrence during chemotherapy. (F) Assessment of TSPO expression in a primary and recurrent case of glioblastoma (bar=50µm haematoxylin-stained sections and immunoperoxidase) quantified in (G) . (H) Pearson correlation between the relative activity of TEAD2 transcription factor (normalized enrichment score measured by expression of downstream target genes) and TSPO gene expression for TCGA GBM (n=160) and LGG (n=514) tumours. (I) Immunoblotting of YAP/TAZ, Actin in nuclear fractions of TMZ-treated U87MG (24h-timepoint, 100μM) alone or combined with Lovastatin (10μM) and Etifoxine (30μM). Quantification (normalized on Lamin B2) is reported in ( J ). All data are represented as mean±sem. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001

Article Snippet: The compounds Temozolomide (Sigma, T2577), PK11195 (Enzo Life Technologies, BML-CM 118), Etifoxine (Sigma, SML 0272), Lovastatin (Enzo, BML-G226), Cerivastatin (Sigma, SML0005) and MF-438 (Sigma, 569406) have been enrolled in the analysis at the concentration and time indicated in the specific experiments.

Techniques: Staining, Positive Control, Quantitative RT-PCR, Western Blot, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: IL-1β ELISAs were used to measure cytokine expression in supernatants of primary mouse macrophages (n=7) and microglia (n=8) that were either untreated, treated with etifoxine (5uM) (eti(5uM)), inflammasome activated (inflam), or pre-treated with etifoxine (5uM) under inflammasome-activating conditions (inflam + eti(5uM)). (A) IL-1β secretion in primary mouse macrophages was decreased when treated with etifoxine under inflammasome-activating conditions (160.2pg/mL±13.46) compared to the inflammasome-only control (1426pg/mL±100.30). (B) IL-1β secretion in primary mouse microglia was decreased when treated with etifoxine under inflammasome-activating conditions (0pg/mL) compared to the inflammasome-only control (1874pg/mL±146.32). Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. *** p <0.001.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Expressing

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: IL-1β ELISAs were used to measure cytokine expression in supernatants of primary mouse macrophages (n=7) and microglia (n=8) that were either untreated, treated with etifoxine (5uM) (eti(5uM)), inflammasome activated (inflam), or pre-treated with etifoxine (5uM) under inflammasome-activating conditions (inflam + eti(5uM)). (A) IL-1β secretion in primary mouse macrophages was decreased when treated with etifoxine under inflammasome-activating conditions (160.2pg/mL±13.46) compared to the inflammasome-only control (1426pg/mL±100.30). (B) IL-1β secretion in primary mouse microglia was decreased when treated with etifoxine under inflammasome-activating conditions (0pg/mL) compared to the inflammasome-only control (1874pg/mL±146.32). Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. *** p <0.001.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Expressing

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: IL-1β secretion measured by ELISA in primary mouse macrophages (n=2) that were untreated, inflammasome activated (inflam) (2538pg/mL±117.62), and pre-treated with etifoxine (1uM, 5uM) (eti) prior to inflammasome activation (2305pg/mL±143.65, 57.21pg/mL±7.01), with LPS (1708pg/mL±59.055, 637pg/mL±19.92), or with nigericin (2450pg/mL±91.96, 3052pg/mL±148). Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. ** p <0.01, *** p <0.001.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: A TNF ELISA was conducted on primary human macrophages (n=3) that were untreated, treated with LPS, inflammasome activated (inflam), and pre-treated with etifoxine (5,25,50uM) (eti) under inflammasome-activating conditions. IL-1β secretion significantly decreased in a dose-dependent manner when pre-treated with etifoxine (25uM,50uM) under inflammasome-activating conditions (913.95+/-42.71, 123+/-9.16) compared to the inflammasome-only control. Results are displayed as mean with standard error. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. * p <0.05, ** p <0.01

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: RT-qPCR was performed using RNA isolated from primary mouse macrophages and microglia (n=4) that were untreated, treated with etifoxine (5uM) (eti), inflammasome activated (inflam), or pre-treated with etifoxine (5uM) under inflammasome-activating conditions. (A) Fold change of il-1β mRNA in macrophages was significantly decreased when pre-treated with etifoxine under inflammasome-activating conditions (73.52±5.42) compared to the inflammasome-only control (252.72±12.81). (B) Fold change of il-1β mRNA in microglia was significantly decreased when pre-treated with etifoxine under inflammasome-activating conditions (22.21±5.84) compared to the inflammasome-only control (1147±92.54). (C) Fold change of nlrp3 mRNA in macrophages was significantly decreased when pre-treated with etifoxine under inflammasome-activating conditions (10.13±1.69) compared to the inflammasome-only control (122.28±26.05). (D) Fold change of nlrp3 mRNA in microglia was significantly decreased when pre-treated with etifoxine under inflammasome-activating conditions (4.86±1.45) compared to the inflammasome-only control (53.22±13.62). All fold changes were calculated by using the 2 -ΔΔCT method. Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. * p <0.05, ** p <0.01.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: RT-qPCR was performed on primary human macrophages (n=4) that were untreated, inflammasome-activated (inflam), and pre-treated with etifoxine (5uM, 50uM) (eti) under inflammasome-activating conditions. Western blotting for NLRP3 was also performed on primary human macrophages (n=) that were untreated, inflammasome-activated (inflam), and pre-treated with etifoxine (50uM) (eti) under inflammasome-activating conditions. Protein loading was normalized to relative β-actin. Fold change of il-1β mRNA when pre-treated with etifoxine (5uM, 50uM) under inflammasome-activating conditions (178.08±13.26, 9.26±2.21) compared to the inflammasome-only control (143.17±15.12). (B) Fold change of nlrp3 mRNA when pre-treated with etifoxine (5uM, 50uM) under inflammasome-activating conditions (2.9±0.43, 0.43±0.06) compared to the inflammasome-only control (1.65±0.13). (C) Fold change of tnf⍺-ip3 mRNA when pre-treated with etifoxine (5uM, 50uM) under inflammasome-activating conditions (25.18±2.46, 2.40±0.19) compared to the inflammasome-only control (30.37±3.06). (D) Pre-treatment with etifoxine (50uM) significantly decreased NLRP3 protein expression under inflammasome-activating conditions when compared to the inflammasome-only control. All fold changes were calculated by using the 2 -ΔΔCT method. Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: A Qiagen data analysis web-based program was used to determine the fold regulation by using the 2 -ΔΔCT method. Initially the ΔC T is calculated between the genes of interest and the reference genes, which is then followed by the ΔΔC T and fold change calculations between inflammasome-only & no treatment (A), inflammasome + etifoxine & no treatment (B), and inflammasome + etifoxine & inflammasome only (C). (n=3/group). Points are plotted as log 10 values. The scatterplot compares the normalized gene expression between the test group and the control group, the black line represents unchanged (<2-fold) gene expression and the blue lines represent the >2-fold change threshold. Upregulated genes are colored in green, unchanged genes are colored in black, and downregulated genes are colored in red. Individual points in the upper left and lower right quadrants are up- and downregulated by >2-fold in the inflammasome-only group compared to the control group. The compared groups had a biological n=3. Genes with the greatest increase in fold regulation have been labelled.

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Expressing

Journal: bioRxiv

Article Title: Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

doi: 10.1101/2023.09.19.558428

Figure Lengend Snippet: ELISAs were conducted in monocytes derived from healthy controls (n=6/group) and SPMS patients (n=6/group). The control and SPMS monocytes were either untreated or treated with LPS. (A) ELISA comparing IL-1β secretion in monocytes from healthy controls and SPMS patients. IL-1β secretion was significantly increased in SPMS patients (2174pg/mL±204.4) when compared to healthy controls (1451pg/mL±142.3). (B) ELISA comparing IL-6 secretion in monocytes from healthy controls and SPMS patients. There was no significant difference in IL-6 secretion in SPMS patients (15441pg/mL±1164) compared to healthy controls (18266pg/mL±1095). (C) ELISA comparing TNF secretion in monocytes from healthy controls and SPMS patients. TNF secretion was significantly increased in SPMS patients (2055pg/mL±408.8) when compared to healthy controls (781.5pg/mL±254.1). (D) An IL-1β ELISA was conducted on PBMCs derived from SPMS patients (n=3). PBMCs were untreated, inflammasome activated (inflam), and pre-treated with etifoxine (50uM) (eti) under inflammasome activating conditions. IL-1 secretion was significantly decreased when pre-treated with etifoxine (50uM) under inflammasome activating conditions (725.6pg/mL±77.5) when compared to inflammasome-only control (2428pg/mL±786.7). Results are displayed as mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used to determine group differences. * p <0.05. ** p <0.01

Article Snippet: 24 hours after first clinical signs (limp/flaccid tail), the mice were injected i.p. with vehicle (90% cyclodextrin, 10% ethanol), XBD-173 (10mg/kg twice daily), or etifoxine (50mg/kg once daily, saline once daily) (Sigma Aldrich) for 7 consecutive days.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay