Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: Binding of CAPE and PK7242 with p53 Y220C . (A) Superimposed structures of CAPE (red) and PK7242 (green) bound at the mutation crevice of p53 Y220C molecule. The orientation of both the molecules at the docked site was found to be similar. Three-dimensional representation of PK7242 ( B ) and CAPE ( D ) at the mutation crevice of p53 Y220C molecule after docking. Molecular interaction of PK7242 ( C ) and CAPE ( E ) with p53 Y220C

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Binding Assay, Mutagenesis

Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: Analysis of molecular dynamics simulation trajectories. (A) Root mean square deviations of heavy atoms in the simulated structures with respect to the starting structure over 200 ns of simulation run. (B) Number of hydrogen bonds formed between the ligands and p53 Y220C crevice during the last 100 ns of each simulation. Interactions fraction diagram of CAPE ( C ) and PK7242 ( D ) at the mutation crevice of p53 Y220C . The fraction of simulation time during which hydrogen bonds, hydrophobic contacts, ionic contacts, and water bridges are retained with different residues at the mutation crevice of p53 Y220C is shown in these plots

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Mutagenesis

Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: Docking scores and calculated free energies of binding of CAPE and PK7242 at p53 Y220C mutation crevice (the errors indicate the standard error of the mean)

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Binding Assay, Mutagenesis

Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: PRIME energies and structural integrity of the simulated complexes. (A) PRIME energies of p53 stable-variant, p53 Y220C , p53 Y220C + CAPE complex and p53 Y220C + PK7242 complex during last 100 ns of simulation. CAPE bound to p53 Y220C had the lowest PRIME energy. The error bars indicate standard error of the mean. (B) Structure of CAPE-bound p53 Y220C (purple) superimposed with structure of PK7242-bound p53 Y220C (green). (C) Structures of CAPE-bound p53 Y220C (purple) and PK7242-bound p53 Y220C (green) superimposed with structure of the p53 stable-variant (yellow). The RMSD of the superimposed structures did not indicate any significant structural alterations in the complexes after binding with ligands and subsequent simulations

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Variant Assay, Binding Assay

Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: Activation of wild type like p53 activity in p53 mutant cell line. (A) Dose dependent cytotoxicity of CAPE in HuH-6 and HuH-7 cells. (B–E) Detection of p53 wt -dependent luciferase activity by reporter plasmids containing the p53-binding synthetic sequence (PG-13Luc) ( B , C ) of p21 WAF−1 promoter (WWP-Luc) ( D , E ). Increase in p53 wt -dependent luciferase activity was detected in CAPE treated HuH-6 cells ( B , D ) and HuH-7 ( C , E ) cells

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Activation Assay, Activity Assay, Mutagenesis, Luciferase, Binding Assay, Sequencing

Journal: Discover. Oncology

Article Title: Caffeic acid phenethyl ester (CAPE) confers wild type p53 function in p53 Y220C mutant: bioinformatics and experimental evidence

doi: 10.1007/s12672-021-00461-2

Figure Lengend Snippet: Expression of p53 wt -downstream effector proteins in CAPE treated HuH-6 and HuH-7 cells. (A) CAPE-treated HuH-6 cells showed dose-dependent decrease in mortalin and increase in p53, p21 WAF−1 and BAX proteins. (B) CAPE-treated HuH-7 cells showed dose-dependent decrease in mortalin and increase in p21 WAF−1 and BAX proteins. Of note, p53 Y220C did not show increase in expression. However, has restored p53 wt function as evidenced by increase in p21 WAF−1 and BAX, and reporter assays shown in Fig.

Article Snippet: Human isogenic hepatocarcinoma HuH-6 (representative of wild type p53) and HuH-7 (harboring Y220C p53 mutation) cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank, Tokyo, Japan).

Techniques: Expressing

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Summary of the structures downloaded from PDB and PubChem along with their identification number.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Docking scores of the ligands which bound with mutated p53/p62 complexes.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamic simulation study of natural metabolites on phosphor mutant p53-p62 complex. A) Conformational changes observed in the structure of mutant type p53 compared with wild-type p53. B) Conformational changes observed in the structure of mutant type p53 after Cuc-B interaction. C) Conformational changes observed in the structure of mutant type p53 after Wi-N interaction. D) Conformational changes observed in the structure of mutant type p53 after Wi-A interaction. E) Root mean square deviation plot of wild-type as well as mutant p53 complex with p62 before and after Cuc-B interaction simulated for 200ns. F) Root mean square deviation plot of mutant p53 complex with p62 before and after Wi-N, Withaferin-A, CAPE and ARC interaction simulated for 200ns. G) Interaction fraction diagram of wild type p53 with amino acids of p62. H) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Mutagenesis

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation analysis of natural metabolites in restoring wild type p53 activity. A) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Cuc-B intervention. B) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-N intervention. C) Interaction fraction diagram of phospho-mutant p53 with amino acids of p62 after Wi-A intervention. D) Change in MMMGBSA binding energy before and after Cuc-B interaction. E) Change in MMMGBSA binding energy before and after the Wi-N interaction. F) Change in MMMGBSA binding energy before and after the Wi-A interaction. G) Change in Vander Wal, electrostatic and hydrogen bonding energy of interacting molecules. H) Hydrogen bonding plot observed between p53 and p62 before and after Cuc-B intervention.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Molecular dynamics simulation study of Mortalin p53 abrogation by natural metabolites. A) Three-dimensional visualization of interaction between Mortalin and p53 (docked using HADDOCK server and simulated for 200ns). B) Three-dimensional visualization of Interaction between Cuc-B and p53 binding domain of Mortalin. C) Three-dimensional visualization of Interaction between Wi-N and p53 binding domain of Mortalin. D) Three-dimensional visualization of interaction between Wi-A and p53 binding domain of Mortalin. E) Three-dimensional visualization of interaction between CAPE and p53 binding domain of Mortalin. F) Three-dimensional visualization of Interaction between Cuc-B and Mortalin binding domain of p53. G) Root mean square deviation plot of natural ligands bound Mortalin complex showing stable interactions. H) Root mean square deviation plot of Cuc-B bound p53 complex showing stable interaction.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Dock scores and MMGBSA binding energy for the interaction of the ligands with p53 and Mortalin.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: Binding Assay

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: Interacting residues of p53 and Mortalin with the ligands after simulation for 200ns.

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques:

Journal: Current Research in Structural Biology

Article Title: Comparative computational and experimental analyses of some natural small molecules to restore transcriptional activation function of p53 in cancer cells harbouring wild type and p53 Ser46 mutant

doi: 10.1016/j.crstbi.2022.09.002

Figure Lengend Snippet: In vitro analysis of the comparative wild type p53 activation function of five natural compounds. A) Western blotting of control and treated U2OS cells for p53 and p21 proteins showed increase in the treated cells. Highest increase was observed in cells treated with Withaferin-A followed by Withanone, Cucurbitacin B, CAPE and Artepillin C. B) Immunocytostaining of control and treated U2OS cells showed increase in expression of p53 and p21 in the later. C) p53-dependent luciferase reporter assay in control and treated U2OS cells showed increase in wild type p53 activity on treated cells and was in accordance to the expression analysis. D) Western blotting of control and treated HSC3 cells (harboring p53 Ser46mutant ) for p21 showed its increase in the later. Blots (A and D) were probed with β-Actin as an internal loading positive control. The results from three independent experiments are shown with statistical analysis indicated as p values. p ​> ​0.05 (non-significant), p ​≤ ​0.05 (∗significant), p ​≤ ​0.01 (∗∗ very significant), p ≤ 0.001 (∗∗∗highly significant), and p ​≤ ​0.0001 (∗∗∗∗ extremely significant).

Article Snippet: Human osteosarcoma (U2OS; wild type p53) and oral squamous carcinoma (HSC3; p53 Ser46mutant ) cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan).

Techniques: In Vitro, Activation Assay, Western Blot, Control, Expressing, Luciferase, Reporter Assay, Activity Assay, Positive Control

Journal: BMC Cancer

Article Title: p53-independent structure-activity relationships of 3-ring mesogenic compounds’ activity as cytotoxic effects against human non-small cell lung cancer lines

doi: 10.1186/s12885-016-2585-6

Figure Lengend Snippet: Involvement of p53 in compound C1–induced G2/M arrest. a A549 cells were treated with C1 for 12 h and harvested for western blotting of p53 and p21 and cell cycle analyses. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 12 h after irradiation for Western blotting analyses of p53 and p21; actin was used as a loading control. Representative blots of two different experiments are shown [ upper panel ]. Representative histograms of two different experiments are shown [ lower panel ]. b A549 cells preincubated with the caffeine were cultured in the presence of 10 μM C1 for 24 h [ left panel ] or cultured for 12 h after 10 Gy-irradiation [ right panel ]. The cells were harvested, and cell cycle analysis was performed. Representative histograms of two different experiments are shown. c – d A549 cells treated with p53 siRNA were cultured in the presence of C1 at 10 μM for 12 h, and then western blotting of p53 ( c ) and cell cycle profiles ( d ) were analyzed. Representative results of two different experiments are shown. e A549 cells were treated with C1 for 12–24 h and harvested for western blotting of phospho-cdc2 (p-cdc2), cyclin B1, and phospho-histone H3 (p-H3). Representative results of two different experiments are shown

Article Snippet: LU99 (p53 wild type) and EBC-1 (p53 mutant) human lung carcinoma cells were purchased from the JCRB Cell Bank (Osaka, Japan).

Techniques: Western Blot, Positive Control, Irradiation, Control, Cell Culture, Cell Cycle Assay

Journal: BMC Cancer

Article Title: p53-independent structure-activity relationships of 3-ring mesogenic compounds’ activity as cytotoxic effects against human non-small cell lung cancer lines

doi: 10.1186/s12885-016-2585-6

Figure Lengend Snippet: Involvement of DNA damage-signaling pathway and p53 in compounds-induced cell death of A549 cells. a A549 cells cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h were harvested, and intracellular phospho-ATM expression was analyzed. Representative histograms of three different experiments are shown. The dotted line histogram indicates cells stained with Alexa Fluor® 488-conjugated secondary antibody alone. The broken line and filled gray histograms indicate phosphor-ATM expression in cells treated with vehicle and compound, respectively. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 30 min after irradiation. b A549 cells preincubated with the caffeine (2 mM) were cultured in the presence of 10 μM C1 or C2 for 72 h. The cells were harvested, and annexin V/propidium iodide staining was performed. Percentages of annexin V(+) cells are presented as the mean ± SE of three independent experiments; * and ** indicate p < 0.05 and p < 0.01. c – d A549 cells treated with p53-targeting siRNA were cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h. The cells were harvested, and then western blotting ( c ) and cell death analysis ( d ) were performed. Representative results of two different experiments are shown

Article Snippet: LU99 (p53 wild type) and EBC-1 (p53 mutant) human lung carcinoma cells were purchased from the JCRB Cell Bank (Osaka, Japan).

Techniques: Cell Culture, Expressing, Staining, Positive Control, Irradiation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11000 bound to and stabilized p53 DBD. (A) Melting curve of the stabilized p53-Y220C DBD (T-p53C-Y220C) recorded via differential scanning fluorimetry in absence (blue) or in presence of 1 mM PK11000 (magenta). (B) Inhibition of T-p53C-Y220C aggregation by PK11000. Aggregation kinetics were recorded by monitoring light scattering at 500 nm with 3 μM protein at 37 °C in standard assay buffer [25 mM KPi pH 7.2, 150 mM NaCl, 1 mM TCEP, and 5% (vol/vol) DMSO]. Unlike for noncovalent binders that inhibit aggregation where the amplitude of the reaction remains unchanged and just the rate constants being lower, the amplitude of light scattering was substantially decreased at 250, 500, and 1,000 µM of PK11000, suggesting the presence of two species: unmodified p53-Y220C and a covalent adduct of PK11000 and p53. (C) Mapping of PK11000 induced peak shifts in the 15N-1H HSQC NMR spectrum onto the structure of the p53-Y220C DBD. Large peak shifts are highlighted in red; medium shifts in orange; and small shifts in yellow.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: DSF ΔTm values of different p53 mutants (8 μM) after incubation with diverse 2-sulfonylpyrimidine compounds (250 μM) for 30 min.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11000 alkylated cysteines of p53 DBD. (A) SNAr reaction mechanism for PK11000 cysteine alkylation. (B) ESI (ES+) mass spectra of 50 µM T-p53C Y220C incubated for 4 h at 20 °C with no compound (black) or 250, 500, 1,000, and 5,000 µM PK11000 (red).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: ESI (ES+) mass spectra of different cysteine to p53 core DBD serine mutants after incubation without (black) and with PK11000 (red). (A) T-p53C C124S/C182S/C277S and (D) T-p53C C182S/C277S mutants showed no cysteine modification by PK11000, whereas (B) T-p53C C124S/C182S and (C) T-p53C C124S/C277S showed one cysteine modification by PK11000.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: 15N-1H HSQC NMR spectrum of the p53 Y220C core domain (red) with 1,000 µM (blue), 436 µM (yellow), and 218 µM (green) PK11000 at 293 K.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Alkylation of full-length p53 with 2-sulfonylpyrimdines does not compromise its DNA binding capability. Stabilized full-length p53 was incubated with 1 mM PK11000, PK11007, PK11010, and 5% DMSO for 2 h at 4 °C and then titrated into a cuvette containing 20 nM carboxyfluoresceine-labeled Gadd45a DNA. Fluorescence polarization data were recorded and fitted to the Hill equation including a linear drift term.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Binding Assay, Incubation, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Structural effect of Cys182 modification by PK11000. Superposition of the structure of the p53 cancer mutant Y220C with (gray) and without (green) PK11000 shows that Cys182 on the surface of the L2 loop is modified by the alkylating agent, with the covalent modification pointing toward the solvent. Chain A of the asymmetric unit is shown. Alkylation fixes Cys182 in a defined conformation, but there is little interaction between the modification and the rest of the protein. The figure was generated using PyMOL (www.pymol.org).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Modification, Mutagenesis, Solvent, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Effects of diverse 2-sulfonylpyrimidines on p53 stabilization. (A) Library of diverse 2-sulfonylpyrimidines and structurally related compounds. (B and C) Time-dependent stabilization (DSF ΔTm) of T-p53C-Y220C after 15-, 30-, 60-, and 120-min incubation at room temperature with stabilizing or destabilizing/nonreactive 2-sulfonylpyrimidine compounds, respectively.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Biological effects of PK11007 on diverse cancer cell lines and one human fibroblast cell line. (A) Concentration-dependent viability reduction of WI-38 (fibroblast), NUGC-4 (p53 WT), HUH-6 (p53 WT), SJSA-1 (p53 WT), SW480 (p53 R273H/P309S), NUGC-3 (p53 Y220C), HUH-7 (p53 Y220C), and MKN1 (p53 V143A) cells after treatment with PK11007 for 24 h. The mutant p53 cells HUH-7, MKN1, and NUGC-3 were more sensitive to PK11007 treatment as indicated by strong viability reduction at low compound concentrations. Cell viability was measured in quadruplicate and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM. (B) Incubation of the isogenic H1299 (p53−/−), H1299 (p53 H175), HCT116, and HCT116 p53−/− cancer cells with PK11007 yielded a comparable viability reduction after 24 h. (C) Cell viability of HUH-6, HUH-7, and MKN1 after p53 knockdown via siRNA. Cells were treated with PK11007 for 24 h (48 h for 15 µM PK11007 MKN1 sample). In HUH-6 and HUH-7, cell death was independent of p53, whereas it was partially dependent on p53 in MKN1 cells. (D) Western blots of NUGC-4, NUGC-3, MKN1, HUH-6, and HUH-7 cancer cells after 3 h (6 h for MKN1) treatment with PK11007. p53 target genes p21, PUMA, and MDM2 were up-regulated not only in MKN1 (p53-V143A), NUGC-3, and HUH-7 (both p53-Y220C), but also in HUH-6 and NUGC-4 (both p53 WT) cells. With increasing PK11007 concentration, the molecular weight of p53 gradually increased to ∼3 kDa for the mutant p53 cell lines (HUH-7, MKN1, and NUGC-3), suggesting hyperalkylation of unfolded/aggregated p53. The protein level of the antioxidative gene GSTP1 was higher in NUGC-4 (p53 WT) than in NUGC-3. The asterisks at the p53 target gene levels of HUH-6 and HUH-7 cells highlight the genes for which a different β-actin control was used. (E) Quantification of relative mRNA levels of p53 target genes via real-time PCR. Cells were treated with DMSO or 15 µM (MKN1, HUH-7) or 20 µM (NUGC-3, HUH-6) PK11007 for 6 h (4.5 h for HUH-7). p21 and PUMA mRNA levels were especially up-regulated in mutant p53 cells, whereas in p53 WT cells (HUH-6) no increased transcription was observed. Significance levels were calculated with the Student t test (***P < 0.001; **P < 0.01; *P < 0.05). (F) Western blots of protein levels of UPR key markers in MKN1, HUH-6, and HUH-7 cells. PK11007 treatment for 3 h (6 h for MKN1) increased levels of spliced XBP-1 and CHOP (especially in HUH-7). (G) Inhibition of cellular glutathione synthesis by buthionine sulfoximine strongly potentiated cell viability reduction by PK11007 in HUH-7, NUGC-3, and MKN1 mutant p53 cancer cells. (H) Determination of relative intracellular ROS levels via CellROX Deep Red fluorescence after incubating four cancer cell lines with 30 or 60 µM PK11007 for 2 h. PK11007 caused increase of ROS in all tested cell lines. However, at high doses, the increase of relative ROS levels was higher in HUH-7, NUGC-3, and MKN1 cells. Median fluorescence levels were determined in triplicate with error bars depicting the SE. Significance levels were calculated using a one-way ANOVA with the Bonferroni post hoc test for mean comparison (***P < 0.001; **P < 0.01; *P < 0.05).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Concentration Assay, Mutagenesis, Incubation, Knockdown, Western Blot, Molecular Weight, Control, Real-time Polymerase Chain Reaction, Inhibition, Fluorescence, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Cell viability time course for (A) HUH-6 (p53 WT), HUH-7 (p53 Y220C), and (B) MKN1 cancer cells at 30 or 60 µM PK11007 (10 and 30 µM for MKN1). Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Biological effects of 2-sulfonylpyrimidines on diverse cell lines. Concentration-dependent viability reduction of WI-38 (p53 WT, noncancer), NUGC-4 (p53 WT), HUH-6 (p53 WT), SJSA-1 (p53 WT), SW480 (p53 R273H/P309S), NUGC-3 (p53 Y220C), HUH-7 (p53 Y220C), and MKN1 (p53 V143A) cells after compound treatment for 24 h. The strongest effects on cell viability were observed for PK11007 and PK11012. Mutant p53 cell lines were mostly significantly more sensitive for these compounds than p53 WT and noncancer cell lines. This behavior was also observed for PK11010 and PK11029, however only at higher compound concentrations. PK11003 led to a strong viability decrease; however, it did not distinguish clearly between mutant and WT p53 cell lines. Compounds that contain a negatively charged carboxyl group, such as PK11000 and PK11009, did not lead to a significant cell viability decrease in the tested cell lines at concentrations below 120 µM. Mutant p53 cell lines were also extra sensitive to compounds that destabilized the p53 core domain in vitro, such as PK11012 and PK11015. Cell viability was measured in quadruplicate and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Concentration Assay, Mutagenesis, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Viability reduction of APR-246 in NUGC-3 (p53 WT), HUH-6 (p53 WT), NUGC-4 (p53 Y220C), and HUH-7 (p53 Y220C) cancer cells after treatment for 24 h. Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: NAC prevents PK11007-mediated ROS formation. (A) Determination of intracellular ROS levels via CellROX Deep Red fluorescence after incubating HUH-6 and HUH-7 cancer cells with 5 mM NAC, 400 µM tert-Butyl hydroperoxide (TBHP), or a combination of 30 µM PK11007 and 5 mM NAC for 1.25 h. NAC does not only decrease basal ROS levels, but also completely prevents PK11007 from increasing ROS levels. (B) Relative intracellular ROS levels via CellROX Deep Red fluorescence after incubating four cancer cell lines with 30 or 60 µM PK11007 for 2 h. PK11007 led to an increase of ROS in all tested cell lines; however, at high doses the relative ROS level was significantly higher in HUH-7 and NUGC-3 cells, whereas the ROS levels in MKN1 cells remained on the level of the p53 WT cell lines. Median fluorescence levels were determined in triplicates. Error bars depict SEM values.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11007 changes the folding state of mutant p53 (Y220C) in NUGC-3 cells. PK11007 treatment led to a significant reduction of unfolded p53 (Pab 240) and a slight increase in folded p53 (Pab 1620). Hoechst 33342 dye was used to stain the nucleus. To facilitate comparison of Pab 1620 and Pab 240 fluorescence levels, we increased exposure of all raw pictures by 2 eV.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Mutagenesis, Staining, Comparison, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Protein levels of p53 after treatment with nontarget or p53 siRNA in MKN1, HUH-6, and HUH-7 cancer cells.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: