Journal: Oncogene

Article Title: Glutaminase as a metabolic target of choice to counter acquired resistance to Palbociclib by colorectal cancer cells

doi: 10.1038/s41388-025-03495-w

Figure Lengend Snippet: A Cell proliferation curves for HCT116 cells treated with Palbociclib, Telaglenastat, or their combination for 96 h. Confluence was monitored in an IncuCyte ® S3 (Sartorius) live-cell analysis system. Results are shown as mean ± SD with n = 6. B Representative IncuCyte ® S3 live-cell images in phase-contrast of HCT116 cells treated with Palbociclib, Telaglenastat, or their combination at 96 h. Cell confluence was monitored every 3 h. Scale bar = 400 μm. C Doubling time of HCT116 cells treated with Palbociclib, Telaglenastat, or their combination. Shown values are the mean ± SD of six independent experiments with six replicates each. D Cell proliferation assay. HCT116 cells were treated with increasing concentrations of Palbociclib, Telaglenastat, or their combination for 96 h, and cell quantification was assessed by Hoechst 33342 staining. Left, concentration-response curves generated by data fitting with a four-parameter equation. Center, dose-response cell proliferation matrix. Results are shown as the percentage of proliferation relative to untreated cells (mean ± SD for n = 6). Right, dose-response synergy matrix. The combination index (CI) values were obtained with CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA), revealing a strong synergy (CI < 0.3) at each dose combination tested. E Cell cycle distribution in HCT116 cells treated with Palbociclib, Telaglenastat, or their combination for 96 h and determined by flow cytometry. F Effects of Palbociclib, Telaglenastat, or their combination on signaling pathways with time. P-mTOR, mTOR, MYC, GLS1, P-AKT, and HIF 1α protein levels were determined by Western blotting of total protein fractions using β-actin as a loading control. Comparative extracellular metabolic fluxes for HCT116 cells treated with Palbociclib, Telaglenastat, or their combination for ( G ) 96 h and ( H ) 192 h. Glucose and glutamine consumption and lactate and glutamate production rates were obtained after 24 h of incubation with fresh media and normalized to cell number. I Cell proliferation curves for HCT116 cells treated with Palbociclib, Telaglenastat, or their combination, in the presence or absence of dimethyl α-KG (DM-αKG) for 96 h. Confluence was monitored in the Olympus CM30 incubation monitoring system (Evident Corporation). Results are shown as mean ± SD with n = 6. Statistically significant differences between conditions are indicated with different letters. J Cell proliferation at 96 h following treatments with Palbociclib, Telaglenastat, or their combination in the presence or absence of dimethyl α-KG (DM-αKG), N-acetyl cysteine (NAC) or glutamine (Gln). Cell quantification was assessed by Hoechst 33342 staining. Results are shown as the percentage of proliferation relative to complete medium (mean ± SD for n = 6). K Cell proliferation at 96 h following treatments with Palbociclib, the glutaminase inhibitors Telaglenastat, BPTES, DON, Compound 968, Glutaminase-IN-1 (GLS-IN-1), or their combination with Palbociclib. Cell quantification was assessed by Hoechst 33342 staining. Results are shown as the percentage of proliferation relative to complete medium (mean ± SD for n = 6). L Determination of intracellular ROS levels after treatments with Palbociclib, Telaglenastat, or their combination in the presence or absence of dimethyl α-KG (DM-αKG), N-acetyl cysteine (NAC), or glutamine (Gln). ROS levels were determined by H 2 DCFDA staining. Results are shown as the percentage of proliferation relative to control cells in complete medium (mean ± SD for n = 6). Data information: Concentrations used were 2 µM for Palbociclib, 5 µM for Telaglenastat, and 5 µM for each glutaminase inhibitor. Shown values are mean ± SD for n = 3 (except otherwise indicated). Significance was determined by one-way ANOVA and two-tailed independent sample Student’s t -tests. Statistically significant differences between treated and control cells are indicated as P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), while differences between treatments are shown as P < 0.05 (#), P < 0.01 (##), and P < 0.001 (###) for Palbociclib-treated cells and P < 0.05 (¶), P < 0.01 (¶¶), and P < 0.001 (¶¶¶) for control cells.

Article Snippet: The KGA inhibitor Glutaminase-IN-1 was purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Cell Analysis, Proliferation Assay, Staining, Concentration Assay, Generated, Software, Flow Cytometry, Protein-Protein interactions, Western Blot, Control, Incubation, Two Tailed Test

Journal: Cancer research

Article Title: Undermining glutaminolysis bolsters chemotherapy while NRF2 promotes chemoresistance in KRAS-driven pancreatic cancers

doi: 10.1158/0008-5472.CAN-19-1363

Figure Lengend Snippet: A, Survival of PANC-1 cells pre-treated with CM or -Q for 72 hours followed by 36-hour gemcitabine (2μM) treatment as indicated. Values were normalized to cell survival in CM-treated condition as control, which were given a value of 100%. B, PANC-1 cells were plated and shifted to CM or media lacking Q. Cells were additionally treated with a combination of (5mM) DMKG and (1X) NEAA mixture for 72 hours following glutamine withdrawal where indicated. Gemcitabine (2μM, 36 hours) was added for respective samples, and the percent of non-viable cells was calculated using the trypan blue dye exclusion assay. C, SU.86.86 cells were plated and treated with no glutamine media as in A. Cells were treated with 0.2μM gemcitabine for the last 24 hours as indicated, and the percent of non-viable cells was calculated. D, One day after plating, HPNE-hTERT cells were shifted to CM or medium lacking Q for 48 hours then treated with or without (2μM) gemcitabine for an additional 24 hours in the presence of pretreatment as indicated. The percent of non-viable cells was determined. E, PANC-1 cells were plated in CM overnight, after which cells were treated with 10μM CB-839 or 10μM BPTES for 72 hours where indicated. An additional treatment of 2μM capecitabine, 2μM gemcitabine, or 10μM 5-FU was given for 36 hours as indicated. The percent of non-viable cells was determined. F, PANC-1 cells were treated with RSL3 (10ηM) and/or gemcitabine (2μM) for 24 hours. The percent of non-viable cells was determined as in B. A-F, Error bars represent the SEM for four independent experiments. G, Experimental design for H-P. H-O, Mice bearing tumors of KPC (H-I), MIA PaCa-2 (J-K), SU.86.86 (L-M) and PANC-1 cells (N-O) were administered drug schedules as indicated in the schematic representation G. Tumor volumes (H, J, L, N) and body weights of mice (I, K, M, O) were shown with error bars representing SEM for each group. Threshold for p-value was <0.05. P, Survival of PANC-1 tumor-bearing mice represented as a Kaplan-Meier plot. H-P, Statistical differences between treatment groups were determined with a critical p value of <0.05. Synergistic effect of CB-839+Gemcitabine is evident by comparing tumor volumes (H,J,L,N) with single compound treated groups with statistical significance whereas insignificant body weight changes has been documented (I,K,M,O) for either of four treatment groups in all cases.

Article Snippet: CB-839 (#B0084–462472) was purchased from BOC Sciences.

Techniques: Exclusion Assay

Journal: Cancer research

Article Title: Undermining glutaminolysis bolsters chemotherapy while NRF2 promotes chemoresistance in KRAS-driven pancreatic cancers

doi: 10.1158/0008-5472.CAN-19-1363

Figure Lengend Snippet: Model illustrates reprogrammed metabolic pathways in RAS-driven PDAC cells linked to KRAS mutation and NRF2 activation, which drive glutaminolysis. Mutant KRAS-mediated NRF2 activation leads to chemoresistance by regulating antioxidant genes. Modulation of nucleotide synthesis by NRF2 also supports PDAC cell growth. Anaplerotic glutamine utilization is a key feature of KRAS-driven PDAC cells. KRAS-regulated glutamine metabolic rewiring influenced the TCA cycle, which is critical for nucleotide and DNA synthesis to support cell growth and survival (12,29). CB-839 inhibits GLS, whereas gemcitabine blocks DNA synthesis. This model potentiates CB-839 treatment along with gemcitabine as a therapeutic strategy to combat chemoresistance in KRAS-driven pancreatic cancers.

Article Snippet: CB-839 (#B0084–462472) was purchased from BOC Sciences.

Techniques: Mutagenesis, Activation Assay, DNA Synthesis