Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Ag120 binds to isolated ASCT2 protein and inhibits the metabolism of CRC cells. (A) Modeling of Ag120 (green stick) docking superimposed on ASCT2 protein (PDB: 5LLM), with the interacting ASCT2 amino acids represented by orange sticks. Inset shows higher magnification. (B) SPR analysis of the binding affinity of Ag120 for purified ASCT2. (C–I) Intracellular glutamine (C) , glutamate (D) , α-KG (E) , ATP (F) , NAD + /NADH (G) , GSH (H) , and ROS (I) levels in HCT116 and HT29 cells treated with Ag120 or V9302 for 72 h. Mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01 by Student’s t -test.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Isolation, Binding Assay, Purification

Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Ag120 inhibits CRC cell proliferation via suppression of ERK and mTOR signaling pathways. (A) Quantification of HCT116 and HT29 cells after culture for 72 h with Ag120 or V9302 was performed using a CCK-8 kit. (B,C) Plate images (B) and quantification (C) of colony formation by HCT116 and HT29 cells cultured for 10 days with Ag120 or V9302. (D–F) Fluorescence microscopy images (D,E) and quantification (F) of EdU incorporation in HCT116 and HT29 cells cultured for 3 days with Ag120 or V9302. Scale bars, 50 µm. (G) Immunoblot analysis of ERK1, p-ERK1, mTOR, p-mTOR (S2448), P70S6K, and p-P70S6K expression in CRC cells treated with Ag120 or V9302 for 72 h. Mean ± SD of at least three independent experiments. ns, no significance; * p < 0.05, ** p < 0.01 by Student’s t -test.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Protein-Protein interactions, CCK-8 Assay, Cell Culture, Fluorescence, Microscopy, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Ag120 induces autophagy in CRC cells. (A) Transmission electron microscopy images of HCT116 cells treated with AG120 or V9302 for 72 h. Scale bars, 1 µm. (B,C) Fluorescence microscopy images of CRC cells incubated with Ag120 or V9302 for 72 h and labeled with MDC to visualize autophagic vesicles (B) or with LysoTracker to visualize lysosomes (C) . Scale bars, 50 µm. (D) Immunoblot analysis of LC3, LAMP1, beclin-1, ATG7, and ATG5 expression in CRC cells treated with Ag120 or V9302 for 72 h.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Incubation, Labeling, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Effects of Ag120 and glutamine depletion on CRC cells. (A,B) 2-HG production (A) and immunoblot analysis of IDH1mt expression (B) in CRC cells treated with Ag120 or V9302 for 72 h. (C) Quantification of viable HCT116 and HT29 cells treated with vehicle or Ag120 (45 µM) for 72 h in DMEM medium containing 2 or 0 mM L -glutamine. Cell numbers were measured using a CCK-8 kit. Contribution of Ag120 to cell viability was calculated based on the data in . (D) Colony formation by CRC cells after 10 days of treatment with 0 or 45 µM Ag120 in 0 or 2 mM L -glutamine-containing medium. (E,F) Fluorescence microscopy images of EdU incorporation in HCT116 (E) and HT29 (F) cells treated for 72 h with 0 or 45 µM ASCT2 in 0 or 2 mM L -glutamine-containing medium. Scale bars, 50 µm. Mean ± SD of three experiments. * p < 0.05, ** p < 0.01 by Student’s t -test.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Western Blot, Expressing, CCK-8 Assay, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Effects of Ag120 on glutamine metabolism and proliferation in CRC cells following ASCT2/SLC1A5 knockdown or overexpression. (A,B) Immunoblot analysis of ASCT2 in HCT116 and HT29 cells stably transfected with ASCT2 shRNA (shSLC1A5#1/shSLC1A5#2) or control shRNA (A) or stably transfected with a control or SLC1A5 DNA-lipid overexpression complex (B) . (C,D) Glutamine uptake in SLC1A5-depleted or SLC1A5-overexpressing HCT116 and HT29 cells treated for 2 h with Ag120 (15 µM). The contribution of Ag120 to glutamine uptake was calculated based on the data in . (E,F) Viability of SLC1A5-depleted or SLC1A5-overexpressing HCT116 and HT29 cells treated for 72 h with Ag120 (45 µM) was quantified using a CCK-8 kit. The contribution of Ag120 to cell viability was calculated based on the data in . (G,H) As described for (E,F) except colony formation was measured after 10 days. Mean ± SD of three experiments. * p < 0.05, ** p < 0.01 by Student’s t -test.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Knockdown, Over Expression, Western Blot, Stable Transfection, Transfection, shRNA, Control, CCK-8 Assay

Journal: Frontiers in Pharmacology

Article Title: Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells

doi: 10.3389/fphar.2022.871392

Figure Lengend Snippet: Effect of Ag120 on HCT116 xenograft growth in mice. (A) Experimental protocol. Groups of athymic BALB/c nude mice were injected subcutaneously with 10 6 HCT116 cells and treatments were initiated 7 days later. Ag120 was injected once daily and V9302 was injected every 3 days via the intraperitoneal route. On day 28, animals were sacrificed and tumors and blood samples were collected for analysis. (B) Body weight change. (C) Tumor volumes. (D,E) Weights (D) and photographs (E) of tumor xenografts excised on day 28. (F) Blood glutamine levels on day 28. (G) Fluorescence microscopy images of EdU incorporation in sections of excised tumors. Scale bars, 50 µm. (H) Representative images of ASCT2, Ki67, and LC3 immunostaining in sections of excised tumors. Scale bars, 50 µm. (I) Schematic diagram of the proposed anti-tumor mechanism of action of Ag120 via regulation of glutamine metabolism in CRC cells. Mean ± SD of five mice per group. Symbols represent individual mice. * p < 0.05, ** p < 0.01 by Student’s t -test.

Article Snippet: Ag120 and the ASCT2 inhibitor V9302 were obtained from Selleck and were resuspended in phosphate-buffered saline (PBS) for experiments.

Techniques: Injection, Fluorescence, Microscopy, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: ASCT2 (SLC1A5) protein expression was up-regulated in Almonertinib-induced energy metabolic transport channels. (A) After stimulating H1975 cells with Almonertinib at IC50/2 (4 µM) for 18 h, the cells were collected and LC-MS was used to analysis differences in the expression of related proteins. The arrow shows the glutamine transporter SLC1A5. (B,C) Western blotting (WB) was used to examine the expression of SLC1A5 protein in H1975 cells stimulated by Almonertinib at different times. (D,E) Immunofluorescence was used to detect the expression of SLC1A5 protein in H1975 cells stimulated by Almonertinib at different times.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: Effects of SLC1A5 inhibition or knockdown on cell proliferation and glutamine uptake in NSCLC cells. (A) CCK8 assay was used to detect different concentrations of V9302 on H1975 and A549 cells for 24, 48 and 72 h. (B) Inhibition SLC1A5 or knockdown of SLC1A5 can reduce the uptake of glutamine in NSCLC cells. Significance: * p < 0.05, ** p < 0.01,*** p < 0.001, ## p < 0.01.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: Inhibition, Knockdown, CCK-8 Assay

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: V9302 enhanced the inhibitory effect of Almonertinib on the proliferation of NSCLC cells. (A) The effect of different concentrations of Almonertinib and 10 µM V9302 on cells alone or in combination was used for 24 h to test the effect of H1975 or A549 cell viability by CCK8. (B) Cell morphological changes observed under an inverted microscope after 12 μM (or 6 μM) Almonertinib and 10 μM V9302 were used separately or in combination for 24 h. (C) Effects of Almonertinib and V9302 alone or in combination on the colony forming ability. A549 cells (A: blank group B: 1 µM V9302 C: 1.2 µM Almonertinib D: 1 µM V9302 and 1.2 µM Almonertinib); H1975 cells (A: blank group B: 1 µM V9302 C: 0.8 µM Almonertinib D: 1 µM V9302 and 0.8 µM Almonertinib). (D) After the intervention of Almonertinib and V9302 alone or in combination for 24 h, the apoptosis rate of A549 cells was detected by double staining. (E) Protein expression of caspase8, caspase3 and PARP. The effects of Almonertinib and V9302 alone or in combination on the expression of A549 or H1975 cells for 24 h were detected by WB. (F) DAPI fluorescence staining was used to observe the nuclear changes of H1975 cells after Almonertinib and V9302 were treated alone or in combination for 24 h. (G) The submicroscopic structure of H1975 cells was observed by electron microscopy after treat with 6 µM Almonertinib combined with 10 µM V9302 for 12 h. Significance: * p < 0.05, ** p < 0.01.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: Inverted Microscopy, Double Staining, Expressing, Fluorescence, Staining, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: V9302 enhanced the antitumor effect of Almonertinib in vivo . (A) Representative tumors from each treatment group. (B) Tumor weight of the mice. (C) Tumor volume of the mice. (D) Evaluation of toxicity in vivo in nude mice. AST, ALT, BUN and Cr of serum were measured by assay kits, respectively. (E) H&E-stained sections of the tumor, liver, lung and kidney from the mice after treatment. (F) TUNEL was used to detect apoptosis in tumor tissues. (G) Protein expression of caspase8, caspase3 and PARP were detected by WB in tumor tissues. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: In Vivo, Measured Assay, Staining, TUNEL Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: Effect of V9302 on Almonertinib-induced autophagy. (A,B) Effect of different drug groups on protein expression of autophagy marker protein LC3-II and P62 in cell lines by WB. (C) Almonertinib alone or combined with chloroquine to observe the effect on the protein level of LC3-II. (D) . Effect of different drug groups on protein expression of autophagy marker protein LC3-II and P62 in tumor tissues. (E) A549 (H1975) cells were treated with 12 μM (6 μM) Almonertinib alone or in combination with 10 μM V9302 for 12 h, then fixed and observed with an electron microscope. (F) mCherry-GFP-LC3B was used to detect the autophagy flux level of Almonertinib combined with V9302 in H1975 cells for 12 h. Significance: * p < 0.05, ** p < 0.01.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: Expressing, Marker, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet: CQ increased the proliferation inhibitory effect of Almonertinib combined with V9302 on A549 cells and induces apoptosis. (A) CCK8 assay was used to determine the effect of CQ on the proliferation inhibition of Almonertinib and V9302 co-treated A549 cells. (B,C) Double staining assay was used to detect the effect of CQ on the induction of apoptosis of A549 cells treated with Almonertinib and V9302. Significance: * p < 0.05, ** p < 0.01,*** p < 0.001.

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: CCK-8 Assay, Inhibition, Double Staining

Journal: Frontiers in Pharmacology

Article Title: Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

doi: 10.3389/fphar.2021.671328

Figure Lengend Snippet:

Article Snippet: After incubation for 24 h, Almonertinib and/or SLC1A5 inhibitor V9302 (MedChemexpress, NJ, United States) at diverse doses (Jiangsu Haoseh Pharmaceutical Group Co., Ltd.) was used to co-incubate H1975 and A549 cells for 24 h. After incubation, CCK-8 assay (Dojin Chemical, Kumamoto, Japan) was conducted to determine cell viability in accordance with specific instructions.

Techniques: Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: SLC1A5-mediated kynurenine metabolism drives AHR-FANCD2 axis to remodel chromatin and induce T cell exhaustion in lung adenocarcinoma

doi: 10.1186/s12964-026-02732-3

Figure Lengend Snippet: SLC1A5 knockout in T cells enhances the therapeutic efficacy of anti-PD1 in mice. A IDO1-overexpressing LLC cells were injected into SLC1A5 fl/fl or SLC1A5 CKO mice through the tail vein, followed by treatment with anti-PD-1 monoclonal antibody (mAb) or control IgG. B Number of metastatic nodules formed by LLC cells in the lungs of mice. C Kaplan–Meier survival analysis of the mice. D GFP-IDO1-overexpressing LLC cells were implanted into C57BL/6 mice, followed by treatment with V-9302 (an SLC1A5 inhibitor), anti-PD-1 mAb, or a combination of both. E – F Analysis of bioluminescence intensity in the mice at 7, 14, and 28 d post-treatment. G-H. FACS analysis of the number of GZMB + CD8 + ( G ) and TNFA + CD8 + ( H ) T cells in tumor tissues. I FACS analysis of the number of TEX (PD1 + TIM3 + ) and TPEX (PD1 + TIM3. − ) cells in LLC-induced tumors. J Kaplan–Meier survival analysis of the mice. Animal experiments involved six mice per group. Data are presented as dot plots and bars. Statistical significance was set at p < 0.05

Article Snippet: In the immunotherapy experiment, mice were randomly assigned to receive the following treatments: PBS, SLC1A5 small molecule inhibitor V-9302 (60 mg/kg, oral gavage daily), IgG control, PD-1 monoclonal antibody (mAb) alone (BioXcell, BE0146, 200 μg, intraperitoneal injection every other day), or the combination of V-9302 and the PD-1 mAb.

Techniques: Knock-Out, Drug discovery, Injection, Control