Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 5. Overexpression of LAT2 protein in gemcitabine-resistant CCA cells. (a) Volcano plot of differentially expressed genes of KKU-213B (parental) vs. KKU-213BGemR. Green dots indicate downregulated genes, orange dots indicate upregulated genes, and brown dots indicate stable genes. (b) The fold change of SLC7A8 expression in KKU-213B compared to KKU-213BGemR. (c + d) LAT2 expression and intensity in parental CCA cells and gemcitabine-resistant CCA cells and (e–h) LAT2, glutaminase (GLS) and glutamine synthetase (GS) expression and intensity after treatment with gemcitabine, curcumin and the combination of curcumin and gemcitabine. Original blots are presented in Supplementary Table S2. Data are expressed as mean ± SD of three replicate experiments; ns = not significant, *p < 0.01, **p < 0.01 and ***p < 0.001 compared between groups; Log2FC: Log2 fold-change.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Over Expression, Expressing

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 6. LAT2 promotes chemotherapeutic sensitivity against gemcitabine-resistant cell. Western blot analyses of (a–d) LAT2, glutaminase (GLS) and glutamine synthetase (GS) in KKU-213BGemR knockdown SLC7A8. (e) Cell morphology and confluence of cells before and after transfection at 48h (10X magnification). (f) Cell proliferation using MTT assay after transfection with siSLC7A8 at 48 h and 72 h (OD 540), treatment with (g) gemcitabine 0–2000 μM at 48 h and 72 h and (h) the combination of curcumin and gemcitabine at 24–72 h. Original blots are presented in Supplementary Table S3. Western blot data are expressed as mean ± SD of biological tripicates and cell proliferation are expressed as mean ± SD of biological and technical replicate experiments; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and #p < 0.0001 compared to control and between groups. G: gemcitabine, C: curcumin.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Western Blot, Knockdown, Transfection, MTT Assay, Control

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 7. Anti-tumor effects of curcumin, gemcitabine and their combination in gemcitabine- resistant xenograft mouse model. (a) Tumor growth rates and images of tumor tissue (N = 5 in each group), (b) LAT2 immunohistochemical staining in the untreated group, curcumin- and combination-treated groups (the gemcitabine-treated group was not included due to fluid accumulation in the tumor mass) and (c) the positive- staining area (%) of LAT2. (d–g) Protein intensities of LAT2, GLS and GS. Original blots are presented in Supplementary Table S4. All data are expressed as means ± SD of three biological independent experiments; ns = not significant, ** p < 0.01, ***p < 0.001 and #p < 0.0001 compared between groups (N = 3 in each group). CUR: Curcumin, GEM: Gemcitabine.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 8. This summary schematic demonstrates that curcumin enhances gemcitabine’s ability to (I) suppress the SLC7A8 (LAT2)-regulated glutamine pathway, (II) inhibit GLS, and (III) inhibit GS. This leads to reduced ability of glutamine (Gln) to participate in synthesis of nucleotides, and of glutamate (Glu) being unable to enter the tricarboxylic acid (TCA) cycle and synthesize glutathione and amino acids19. These mechanisms result in inhibited cell proliferation, induced cell cycle arrest, and apoptosis, ultimately inhibiting cancer progression in gemcitabine-resistant cholangiocarcinoma.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques:

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 5. Overexpression of LAT2 protein in gemcitabine-resistant CCA cells. (a) Volcano plot of differentially expressed genes of KKU-213B (parental) vs. KKU-213BGemR. Green dots indicate downregulated genes, orange dots indicate upregulated genes, and brown dots indicate stable genes. (b) The fold change of SLC7A8 expression in KKU-213B compared to KKU-213BGemR. (c + d) LAT2 expression and intensity in parental CCA cells and gemcitabine-resistant CCA cells and (e–h) LAT2, glutaminase (GLS) and glutamine synthetase (GS) expression and intensity after treatment with gemcitabine, curcumin and the combination of curcumin and gemcitabine. Original blots are presented in Supplementary Table S2. Data are expressed as mean ± SD of three replicate experiments; ns = not significant, *p < 0.01, **p < 0.01 and ***p < 0.001 compared between groups; Log2FC: Log2 fold-change.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Over Expression, Expressing

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 6. LAT2 promotes chemotherapeutic sensitivity against gemcitabine-resistant cell. Western blot analyses of (a–d) LAT2, glutaminase (GLS) and glutamine synthetase (GS) in KKU-213BGemR knockdown SLC7A8. (e) Cell morphology and confluence of cells before and after transfection at 48h (10X magnification). (f) Cell proliferation using MTT assay after transfection with siSLC7A8 at 48 h and 72 h (OD 540), treatment with (g) gemcitabine 0–2000 μM at 48 h and 72 h and (h) the combination of curcumin and gemcitabine at 24–72 h. Original blots are presented in Supplementary Table S3. Western blot data are expressed as mean ± SD of biological tripicates and cell proliferation are expressed as mean ± SD of biological and technical replicate experiments; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and #p < 0.0001 compared to control and between groups. G: gemcitabine, C: curcumin.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Western Blot, Knockdown, Transfection, MTT Assay, Control

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 7. Anti-tumor effects of curcumin, gemcitabine and their combination in gemcitabine- resistant xenograft mouse model. (a) Tumor growth rates and images of tumor tissue (N = 5 in each group), (b) LAT2 immunohistochemical staining in the untreated group, curcumin- and combination-treated groups (the gemcitabine-treated group was not included due to fluid accumulation in the tumor mass) and (c) the positive- staining area (%) of LAT2. (d–g) Protein intensities of LAT2, GLS and GS. Original blots are presented in Supplementary Table S4. All data are expressed as means ± SD of three biological independent experiments; ns = not significant, ** p < 0.01, ***p < 0.001 and #p < 0.0001 compared between groups (N = 3 in each group). CUR: Curcumin, GEM: Gemcitabine.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 8. This summary schematic demonstrates that curcumin enhances gemcitabine’s ability to (I) suppress the SLC7A8 (LAT2)-regulated glutamine pathway, (II) inhibit GLS, and (III) inhibit GS. This leads to reduced ability of glutamine (Gln) to participate in synthesis of nucleotides, and of glutamate (Glu) being unable to enter the tricarboxylic acid (TCA) cycle and synthesize glutathione and amino acids19. These mechanisms result in inhibited cell proliferation, induced cell cycle arrest, and apoptosis, ultimately inhibiting cancer progression in gemcitabine-resistant cholangiocarcinoma.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques:

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 5. Overexpression of LAT2 protein in gemcitabine-resistant CCA cells. (a) Volcano plot of differentially expressed genes of KKU-213B (parental) vs. KKU-213BGemR. Green dots indicate downregulated genes, orange dots indicate upregulated genes, and brown dots indicate stable genes. (b) The fold change of SLC7A8 expression in KKU-213B compared to KKU-213BGemR. (c + d) LAT2 expression and intensity in parental CCA cells and gemcitabine-resistant CCA cells and (e–h) LAT2, glutaminase (GLS) and glutamine synthetase (GS) expression and intensity after treatment with gemcitabine, curcumin and the combination of curcumin and gemcitabine. Original blots are presented in Supplementary Table S2. Data are expressed as mean ± SD of three replicate experiments; ns = not significant, *p < 0.01, **p < 0.01 and ***p < 0.001 compared between groups; Log2FC: Log2 fold-change.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Over Expression, Expressing

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 6. LAT2 promotes chemotherapeutic sensitivity against gemcitabine-resistant cell. Western blot analyses of (a–d) LAT2, glutaminase (GLS) and glutamine synthetase (GS) in KKU-213BGemR knockdown SLC7A8. (e) Cell morphology and confluence of cells before and after transfection at 48h (10X magnification). (f) Cell proliferation using MTT assay after transfection with siSLC7A8 at 48 h and 72 h (OD 540), treatment with (g) gemcitabine 0–2000 μM at 48 h and 72 h and (h) the combination of curcumin and gemcitabine at 24–72 h. Original blots are presented in Supplementary Table S3. Western blot data are expressed as mean ± SD of biological tripicates and cell proliferation are expressed as mean ± SD of biological and technical replicate experiments; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and #p < 0.0001 compared to control and between groups. G: gemcitabine, C: curcumin.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Western Blot, Knockdown, Transfection, MTT Assay, Control

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 7. Anti-tumor effects of curcumin, gemcitabine and their combination in gemcitabine- resistant xenograft mouse model. (a) Tumor growth rates and images of tumor tissue (N = 5 in each group), (b) LAT2 immunohistochemical staining in the untreated group, curcumin- and combination-treated groups (the gemcitabine-treated group was not included due to fluid accumulation in the tumor mass) and (c) the positive- staining area (%) of LAT2. (d–g) Protein intensities of LAT2, GLS and GS. Original blots are presented in Supplementary Table S4. All data are expressed as means ± SD of three biological independent experiments; ns = not significant, ** p < 0.01, ***p < 0.001 and #p < 0.0001 compared between groups (N = 3 in each group). CUR: Curcumin, GEM: Gemcitabine.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 8. This summary schematic demonstrates that curcumin enhances gemcitabine’s ability to (I) suppress the SLC7A8 (LAT2)-regulated glutamine pathway, (II) inhibit GLS, and (III) inhibit GS. This leads to reduced ability of glutamine (Gln) to participate in synthesis of nucleotides, and of glutamate (Glu) being unable to enter the tricarboxylic acid (TCA) cycle and synthesize glutathione and amino acids19. These mechanisms result in inhibited cell proliferation, induced cell cycle arrest, and apoptosis, ultimately inhibiting cancer progression in gemcitabine-resistant cholangiocarcinoma.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques:

Journal: Oncology Letters

Article Title: Monascin accelerates anoikis in circulating tumor cells and prevents breast cancer metastasis

doi: 10.3892/ol.2020.12029

Figure Lengend Snippet: MS inhibits 4T1 breast cancer cell aggregation under the suspension condition. (A) Phase-contrast images of 4T1 cells on the polyHEMA-coated plate treated with or without 20 µM MS for 48 h. Quantitative counts of clustered 4T1 cells reveal a significant decrease in MS treatment. (B) Western blot analysis of E-cadherin and β-catenin expression to determine the effect of MS treatment for 48 h on the floating cells. *P<0.05 vs. control. MS, monascin; Ctrl, control.

Article Snippet: After blocking with BlockPROTM Protein-Free Blocking Buffer (Visual Protein; cat. no. BF01) for 1 h at room temperature, membranes were incubated with primary antibodies against PARP (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9542), caspase-3 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9665), E-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 3195), β-catenin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9562) and β-actin (1:2,000; Cell Signaling Technology, Inc.; cat. no. 4970) primary antibodies overnight at 4°C.

Techniques: Suspension, Western Blot, Expressing, Control