Journal: bioRxiv

Article Title: A novel pan class-I glucose transporter inhibitor DRB18 exhibits synergistic effects in vitro and in vivo with paclitaxel against human non-small cell lung cancer

doi: 10.1101/2024.12.15.628558

Figure Lengend Snippet: 2 μM of DRB18 combined with or without other drugs to treat A549 cells for 48 hours. The inhibition of cell proliferation was compared to single compound treatment to identify anti-proliferation synergism. 4A. DRB18 combined with 10 µM V9302 showed more inhibition against A549 cells. 4B . DRB18 combined with 100 nM or 300 nM of CB839 showed more cytotoxicity against A549. 4C . DRB18 combined with 5 µM or 10 µM of sunitinib (Sutent) showed more inhibition against A549 cells. 4D . DRB18 combined with 10 µM V9302 showed more inhibition against Panc1 cells. 4E . DRB18 combined with 100 nM or 300 nM of CB839 showed more inhibition against Panc1 cells. 4F . DRB18 combined with 5 µM or 10 µM of sunitinib showed more inhibition against Panc1 cells. 4G . 5 µM of DRB18 combined with 0.75 µM of brigatinib showed more cytotoxicity against A549 cells. 4H . 5 µM of DRB18 combined with 15 µM of trametinib showed more inhibition against A549 cells.

Article Snippet: V9302 (HY-112683) and CB839 (HY-12248) were purchased from MedChemExpress.

Techniques: Inhibition

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Dose–response curves of the NSCLC cell lines panel treated with increasing concentrations of CB-839. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. The average of three independent experiments is reported. ( B ) Dose–response curves of the NSCLC LU99 and H358 LKB1 isogenic systems treated with increasing concentrations of CB-839. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. The average of three independent experiments is reported. ( C ) GLS1 RNAseq gene expression data retrieved from the CCLE , in ten NSCLC cell lines. ( D ) Western Blot analysis of GLS1 protein levels in the ten NSCLC cell lines used. Ran was used as loading control. The figure is representative of at least three independent experiments. ( E ) Fold change in abundance (normalized peak area) of extracellular glucose uptake and lactate release in NSCLC CB-839 treated vs. untreated cells (500 nM CB-839, 6 h treatment). Mean ± SD of triplicate culture/conditions.

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: MTS Assay, RNA sequencing, Gene Expression, Western Blot, Control

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Schematic representation of the 13-C glutamine labelling experiment. ( B ) M + 0 and M + 5 glutamine and glutamate abundance in two sensitive and two resistant H1299-derived clones with or without CB-839 (500 nM, 24 h treatment). ( C ) Isotope labelling of the TCA cycle in the presence of 13C 5 -glutamine with or without CB-839 (500 nM, 24 h treatment) in two sensitive (S1, KRASwt/LKB1wt, S2, KRASmut/LKB1wt) and two resistant (R1, KRASwt/LKB1del, R2, KRASmut/LKB1del) H1299-derived clones (normalized peak area). ( D ) Schematic representation of intracellular pyruvate metabolism. ( E ) Intracellular lactate levels (normalized peak area) with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( F ) Intracellular alanine levels (normalized peak area) with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( G ) Normalized fold change of extracellular alanine levels vs. unconditioned RPMI-1640 with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. ( H ) Schematic representation of fatty acid biosynthesis. ( I ) Intracellular levels of fatty acids with or without CB-839 (500 nM, 24 h treatment) in two sensitive and two resistant H1299-derived clones. Mean ± SD of triplicate culture/conditions. Statistical significance * p < 0.05 untreated vs CB-839 treated cells, # p < 0.05 untreated sensitive vs untreated resistant cells, Wilcoxon Mann–Whitney test (M + 0, M + 2, M + 3, M + 4, M + 5).

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: Derivative Assay, Clone Assay, MANN-WHITNEY

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Changes in extracellular alanine levels in the panel of ten NSCLC cell lines, CB-839-treated vs. untreated, cells (500 nM, 24 h treatment). ( B ) Unlabeled and 13C 3 -labelled alanine incorporation (M + 0, M + 3) in two sensitive (LU99, A549) and two resistant (H358, H520) cells, either untreated or CB-839 treated (500 nM, 24 h treatment). ( C ) The 13C 3 -alanine-derived carbon labelling (M + 2, M + 3, normalized peak area) of TCA cycle intermediates and citrate, malate, glutamate and glutathione derived metabolites in CB-839 treated sensitive and resistant cells. ( D ) GPT2 mRNA expression as RNA-seq, retrieved from the CCLE in the sensitive and resistant cells. ( E ) Western blot analyses of GPT2 expression in the NSCLC cell line panel. Ran was used as loading control. ( F ) Pearson correlation between alanine uptake from each cell line’s medium and GPT2 mRNA expression, retrieved from the CCLE. The average of three independent experiments is reported. Statistical significance # p < 0.05 refer to significant differences (one-way ANOVA and Tukey Kramer test) of M + 2 and M + 3 in the resistant vs. sensitive cells.

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: Derivative Assay, Expressing, RNA Sequencing, Western Blot, Control

Journal: Cells

Article Title: Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation

doi: 10.3390/cells9081766

Figure Lengend Snippet: ( A ) Schematic representation of metabolic strategy to overcome CB-839 resistance, targeting GPT2 by l -cycloserine (cyclo). ( B ) Dose–response curves of H1975 (left panel) and H520 (right panel) cells treated with increasing concentrations of CB-839, alone (CB-839) or with l -cycloserine (CB + cyclo) at the reported concentrations. The response to the drugs was assessed with the MTS assay 72 h from the treatment start. ( C ) Schematic representation of metabolic strategy to induce CB-839 resistance in the sensitive cell lines by pyruvate supplementation, with inhibition of GLS1, alone or together with GPT2. ( D ) Dose–response curves of LU99 (left panel) and A549 (right panel) cells treated with increasing concentrations of CB-839, grown and treated in standard (CB-839) or pyruvate-enriched medium (CB-839 Pyr). The response to the drug was assessed with the MTS assay 72 h from treatment start. ( E ) Histograms of LU99 and A549 cells grown in standard or pyruvate-enriched medium (Pyr) and treated with CB-839 (CB, 12 nM for LU99 and 111 nM for A549) and l -cycloserine (cyclo, 75 µM for LU99 and 100 µM for A549), either alone or in combination. The response to the drug was assessed 72 h from the start of treatment with the MTS assay. ( F ) Histograms of H520 and H1975 cells grown in standard or pyruvate-enriched medium (Pyr) and treated with CB-839 (CB, 1000 nM) and l -cycloserine (cyclo), 250 µM for H520 and 500 µM for H1975), either alone or in combination. The response to the drug was assessed with the MTS assay 72 h from treatment start. The average of three independent experiments is reported. For the sake of clarity, the statistical significance one-way ANOVA and Bonferroni post-test for multiple comparisons (panel B , D , E , F ) are reported as , respectively).

Article Snippet: CB-839 (TargetMol, Wellesley Hills, MA, USA) DMSO stock solutions (10 mM) were dissolved in medium just before use. l -cycloserine (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in sterile water and diluted in medium just before use.

Techniques: MTS Assay, Inhibition