Journal: Frontiers in Chemistry

Article Title: Synthesis, radiolabeling, and evaluation of 68Ga-labeled aminoquinoxaline derivative as a potent PFKFB3-targeted PET tracer

doi: 10.3389/fchem.2023.1158503

Figure Lengend Snippet: Structures of the representative PFKFB3 inhibitors (compounds 1–4) and compound 5.

Article Snippet: All regents used in this section were purchased from Sigma, and the Human Recombinant PFKFB3 Protein (His and GST Tag) was purchased from SinoBiological.

Techniques:

Journal: Frontiers in Chemistry

Article Title: Synthesis, radiolabeling, and evaluation of 68Ga-labeled aminoquinoxaline derivative as a potent PFKFB3-targeted PET tracer

doi: 10.3389/fchem.2023.1158503

Figure Lengend Snippet: Crystal complex of compound 4 with PFKFB3 (PDB code 6IBX) (A) and overlay between compounds 4 and 5 with PFKFB3 (B) .

Article Snippet: All regents used in this section were purchased from Sigma, and the Human Recombinant PFKFB3 Protein (His and GST Tag) was purchased from SinoBiological.

Techniques:

Journal: Frontiers in Chemistry

Article Title: Synthesis, radiolabeling, and evaluation of 68Ga-labeled aminoquinoxaline derivative as a potent PFKFB3-targeted PET tracer

doi: 10.3389/fchem.2023.1158503

Figure Lengend Snippet: Expression of PFKFB3 in the indicated human tumor tissues was analyzed using immunofluorescence staining. (A) NUGC3; (B) HCC827; (C) MKN45, (D) H1975. Scale bar, 100 µm.

Article Snippet: All regents used in this section were purchased from Sigma, and the Human Recombinant PFKFB3 Protein (His and GST Tag) was purchased from SinoBiological.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Chemistry

Article Title: Synthesis, radiolabeling, and evaluation of 68Ga-labeled aminoquinoxaline derivative as a potent PFKFB3-targeted PET tracer

doi: 10.3389/fchem.2023.1158503

Figure Lengend Snippet: The IC 50 of compounds 4 and 5 in biochemical activity and cellular inhibition assays.

Article Snippet: All regents used in this section were purchased from Sigma, and the Human Recombinant PFKFB3 Protein (His and GST Tag) was purchased from SinoBiological.

Techniques: Activity Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells

doi: 10.3390/ijms20215415

Figure Lengend Snippet: Oxa up-regulates the expression of PFKFB3. ( a ) Following treatment with Oxa for 6 h, culture media were collected and subjected to lactate assay. ( b ) Real-time PCR was performed to detect the mRNA expression of PFKFB3 following treatment with Oxa for 6 h. ( c – f ) SW480 cells were treated with Oxa ( e : 50 μM) upon to 2 h ( c : 2 h). Cell lysates were subjected to immunoblotting with the indicated antibodies, and the relatively ratios of PFKFB3 and p-Ulk1 to Actin were shown in the histogram graphs. * p < 0.05 vs. control, and ** p < 0.01 vs. control. Data represent three independent experiments.

Article Snippet: The siRNA specific for human MAP LC3β (sc-43390), PFKFB3 (sc-44011), and Ulk1 (sc-44182) were purchased from Santa Cruz Biotechnology along with the control siRNA (sc-37007).

Techniques: Expressing, Lactate Assay, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells

doi: 10.3390/ijms20215415

Figure Lengend Snippet: PFKFB3 inhibition lessens Oxa-induced autophagy. ( a , b ) Cell lysates were subjected to immunoblotting following treatment of SW480 cells with Oxa, or together with PFK-15 (6 μM; unless otherwise indicated) in the presence or absence of CQ for 2 h. ( c , d ) SW480 cells were transfected with the PFKFB3 siRNA or control siRNA (mock) for 48 h. Cell lysates were collected and subjected to immunoblotting following treatment with Oxa in the presence or absence of CQ for 2 h. All data were acquired from at least three independent experiments.

Article Snippet: The siRNA specific for human MAP LC3β (sc-43390), PFKFB3 (sc-44011), and Ulk1 (sc-44182) were purchased from Santa Cruz Biotechnology along with the control siRNA (sc-37007).

Techniques: Inhibition, Western Blot, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells

doi: 10.3390/ijms20215415

Figure Lengend Snippet: PFKFB3 inhibition enhances the cytotoxicity of Oxa. ( a , b ) Colony growth assay was performed with Oxa (1.5 μM) in the presence or absence of PFK-15. ( c , d ) Cell viability was analyzed by MTS assay following the treatment with Oxa in the presence or absence of PFK-15 for 24 h ( c ); cell lysates from cells treated same as ( c ) were subjected to immunoblotting with indicated antibodies ( d ). ( e – h ) SW480 cells were transfected with the PFKFB3 siRNA or control siRNA for 48 h. Colony growth assay was performed with Oxa (1.5 μM) ( e ), and relative clones number to control was shown in ( f ). Cell viability was analyzed by MTS assay following the treatment with Oxa for 24 h ( g ). Cell lysates were subjected to immunoblotting with indicated antibodies following treatment with Oxa for 24 h ( h ). For histogram graph data, * p < 0.05 vs. control, and ** p < 0.01 vs. control. Similar experiments were repeated three times.

Article Snippet: The siRNA specific for human MAP LC3β (sc-43390), PFKFB3 (sc-44011), and Ulk1 (sc-44182) were purchased from Santa Cruz Biotechnology along with the control siRNA (sc-37007).

Techniques: Inhibition, Growth Assay, MTS Assay, Western Blot, Transfection, Control, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells

doi: 10.3390/ijms20215415

Figure Lengend Snippet: Primer sequences for Real-time PCR analysis.

Article Snippet: The siRNA specific for human MAP LC3β (sc-43390), PFKFB3 (sc-44011), and Ulk1 (sc-44182) were purchased from Santa Cruz Biotechnology along with the control siRNA (sc-37007).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 1 Alisertib inhibits the proliferation, migration, and invasion of ATC cells in vitro and vivo. A, C CCK-8 assays showed that Alisertib (5 μmol and 10 μmol) treatment significantly suppressed cell proliferation in 8305 C (A) and CAL-62 (C) cells in a dose-dependent pattern. B, D After treatment with the indicated concentration of Alisertib for 2 days, the migration/invasion assay was performed in 8305 C (B) and CAL-62 (D) cells. Representative images are shown. The above data are presented as the mean ± SD of three independent experiments. E Five representative tumors from NSG mice injected with CAL-62 are shown, n = 5 mice per group. The subcutaneous xenografts were dissected and are shown on day 13. F, G Tumor volumes were measured with growth curves (F) and tumor weights (G) in the control group (DMSO) and Alisertib group (30 mg/kg). H Representative IHC images and p-Aurora-A and Ki-67 staining in xenograft tumor specimens of control and Alisertib groups are shown. p-Aurora-A and Ki-67 expression were significantly decreased after treatment with Alisertib. Scale bars, 50 μm. The data are presented as the mean ± SD. All *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Migration, In Vitro, CCK-8 Assay, Concentration Assay, Invasion Assay, Injection, Control, Staining, Expressing

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 2 Aurora-A promotes the proliferation, migration and invasion of TC cells, and its expression is correlated to the poor prognosis of TC. A Representative IHC images of low expression and high expression of Aurora-A were shown. Scare bar is 50 µm. B Varying expression levels of Aurora-A in 20 normal samples, 126 DTC samples and 29 ATC samples. **P < 0.01, ***P < 0.001, compared with control normal samples. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with DTC samples. C Kaplan–Meier survival analysis showed that high Aurora-A expression correlated with short survival time in ATC patients. D Aurora-A expression in TPC-1 cells was detected by western blotting in the Aurora-A knockdown group. E CCK-8 assays showed that Aurora-A downregulation significantly enhanced cell proliferation in TPC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. F, G Representative images of transwell assay of migration (F) and invasion (G) of TPC-1 cells with or without Aurora-A knockdown. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. H Aurora-A expression in KTC-1 cells was detected by western blotting in Aurora-A overexpressed group. I CCK-8 assays showed that Aurora-A upregulation significantly enhanced cell proliferation in KTC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. J, K Representative images of transwell assay of migration (J) and invasion (K) of KTC-1 cells with or without Aurora-A overexpression. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. Above data are presented as the mean ± SD of three independent experiments. L Representative bioluminescent images and statistical analysis of the two groups on days 35 after tail vein injection (n = 5 per group) M Representative H&E staining of metastatic tumors in the lungs and livers. N Representative immunohistochemical staining for Ki-67 and p-Aurora-A. ***P < 0.001.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Migration, Expressing, Control, Western Blot, Knockdown, CCK-8 Assay, Transwell Assay, Over Expression, Injection, Staining, Immunohistochemical staining

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 3 Multiomics and bioinformatics analyses indicated Aurora-A regulates the glycolysis in TC cells. A The Venn plot of proteome and RNA-seq revealed 5309 genes had significant alterations at the mRNA and protein levels after Aurora-A knockdown. B KEGG analysis of 5309 genes showed significant alterations in both proteome and RNA-seq. Glycolysis pathway can be remarkably enriched. C Analysis of TCGA database revealed Aurora-A was positively correlated with the glycolysis pathway. D Volcano plot highlighting phosphorylation changes of proteins. 318 proteins with 423 sites were identified with greater than 1.5-fold change in Aurora-A knockdown relative to control TPC-1 cells and plotted in red (upregulation) or blue (downregulation), respectively. E KEGG analysis of the phosphorylation-changed proteins after Aurora-A knockdown in TPC-1 cells. The glycolysis pathway was significantly enriched.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: RNA Sequencing, Knockdown, Phospho-proteomics, Control

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 4 Aurora-A-mediated PFKFB3 phosphorylation regulates the glycolysis of TC cells. A The interaction between Aurora-A and PFKFB3 was verified by an IP assay in TPC-1 cells. B Interaction between Aurora-A and PFKFB3 is examined by GST pull-down. C Phosphorylation level on PFKFB3 was determined by Anti-PFKFB3 (phospho S461) antibody after in vitro kinase assay. D The expression of p-PFKFB3/PFKFB3 in KTC- 1 cells with Aurora-A overexpression and Alisertib-treated 8305 C and CAL-62 cells was analyzed by western blotting. E Comparable level of glycolysis, glycolytic reserve and capacity of 8305 C, TPC-1, and KTC-1 cells measured by Seahorse XF Extracellular Flux analysis. ECAR = extracellular acidification rate. F The ADP/ATP ratio was measured in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3, in TPC-1 cells with Aurora-A knockdown and in 8305 C cells with Alisertib treatment, respectively. G The expression of p-Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, AKT in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3. H, I The proliferation, migration and invasion ability in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3 was analyzed by CCK-8 assay (H) and transwell of migration and invasion assay (I), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with Aurora-A upregulation group. J Western blotting analyzed the expression of p- Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, and AKT in 8305 C cells with Alisertib treatment or combined with exogenous ATP (2 mM). K CCK-8 assays showed that Alisertib (10 μmol) significantly inhibited cell proliferation in 8305 C, which can be remarkably restored by exogenous ATP (2 mM) supplementation. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. L Transwell of migration and invasion assay detected the migration and invasion ability of 8305 C cells pretreated with Alisertib for 2 days or combined with exogenous ATP (2 mM). *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. The data are presented as the mean ± SD of three independent experiments.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Expressing, Over Expression, Western Blot, Knockdown, Migration, CCK-8 Assay, Invasion Assay, Control

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 5 Targeting Aurora-A improves the efficacy of Sorafenib treatment in TC cells in vitro. A, D Western blotting detected p-ERK, ERK, p- AKT, and AKT expression in 8305 C (A) and TPC-1 (D) cells, respectively, exposed to Alisertib (5 μmol), Sorafenib (1 μmol) and their combination for 48 h. B, E The CCK-8 assay was performed in 8305 C (B) and TPC-1 (E) cells exposed to Alisertib (5 μmol), Sorafenib (1 μmol) and both drugs for 96 h. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. C, F Transwell of migration and invasion assay detected the migration and invasion ability of 8305 C (C) and TPC-1 (F) cells pretreated with Alisertib (5 μmol), Sorafenib (1μmol) or both drugs for 2 days. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. The data are presented as the mean ± SD of three independent experiments.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: In Vitro, Western Blot, Expressing, CCK-8 Assay, Control, Migration, Invasion Assay

Journal: Cell death & disease

Article Title: Targeting Aurora-A inhibits tumor progression and sensitizes thyroid carcinoma to Sorafenib by decreasing PFKFB3-mediated glycolysis.

doi: 10.1038/s41419-023-05709-z

Figure Lengend Snippet: Fig. 6 The combination of Alisertib and Sorafenib is a promising therapeutic approach for advanced TC in vivo. A Representative images of subcutaneous xenografts in NSG mice injected 8305 C cells treated with Alisertib (30 mg/kg), Sorafenib (30 mg/kg) or both drugs. n = 5 mice per group. The subcutaneous xenografts were dissected on day 13. B, C Tumor volumes were measured with tumor weights (B) and growth curves (C) in each group. D The expression levels of Ki67 and p-Aurora-A in xenografts of each group were assessed by immunohistochemical staining. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the combination group. The data are presented as the mean ± SD.

Article Snippet: His-tagged PFKFB3 (Abcam, ab268355) was mixed with GST or GST-tagged Aurora-A (MCE, HY-P71199) were in binding buffer at 4 °C overnight, followed by the addition of glutathione-sepharose beads.

Techniques: In Vivo, Injection, Expressing, Immunohistochemical staining, Staining, Control

Journal: bioRxiv

Article Title: Mechanistic studies of PFKFB2 reveals a novel inhibitor of its kinase activity

doi: 10.1101/2024.12.25.630325

Figure Lengend Snippet: Development of a PFKFB kinase activity assay and screening of potential modulators. ( A ) Schematic of the coupled enzyme activity assay protocol. Purple indicates the reaction mix containing purified PFKFB isoforms. Red indicates enzymes added for the coupled assay. The ultimate product is glycerol-3-phosphate and the assay measures the disappearance of NADH at 340nm. ( B ) Plate-reader screening of candidate compounds (all at 10 μM in DMSO; wells A3 and E10 ( red boxes ) were DMSO controls). ( C ) IC50 determination of candidate inhibitor B2 (structure on inset ) for PFKFB2 ( top ) and PFKFB3 ( lower ) kinase activities. ( D ) IC50 determination of known PFKFB3 kinase inhibitor PFK158 (structure on inset ) for PFKFB2 ( top ) and PFKFB3 ( lower ) kinase activities.

Article Snippet: PFKFB2 and PFKFB3 constructs (Origene) were subcloned into GST expression vector.

Techniques: Kinase Assay, Enzyme Activity Assay, Purification