Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Synthesis, in silico modelling, and in vitro biological evaluation of substituted pyrazole derivatives as potential anti-skin cancer, anti-tyrosinase, and antioxidant agents

doi: 10.1080/14756366.2023.2205042

Figure Lengend Snippet: Cytotoxicity of pyrazole (celecoxib analogs), isoxazole, pyrazolone, and positive control compounds P1 – P25 (structures, Figure 3 ) against cells of human cutaneous melanoma and non-melanoma skin cancer lines relative to standard control noncancerous immortalised HaCaT cells.

Article Snippet: Human-derived GFP-expressing melanoma A375 and epidermoid carcinoma A431 cell lines were purchased from Angio-Proteomie (Boston, MA).

Techniques: Positive Control, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The invasive capacity of BRAF mutated (A375, SK-MEL-28 and RPMI-7951), NRAS mutated (SK-MEL-119) and BRAF-NRAS wild type (Hs294T) melanoma cells was determined in vitro using Boyden chamber assay. Melanoma cells (3×10 4 cells/200 µl serum-reduced medium) were placed in the upper chamber of Boyden chamber containing 0, 5, 10 and 20 µM of fisetin. The lower chamber contained 110 µl of medium supplemented with 10% FBS. After 24 hours of incubation, the invaded cells on the lower surface of the membranes were fixed with chilled methanol and stained with crystal violet. A representative picture from three independent experiments is shown. The invaded cells were counted in at least four to five randomly selected microscopic fields on the membrane and the results are summarized and expressed as the mean number of invaded cells ± SEM per microscopic field. Significant difference versus control group, * P <0.05, ** P <0.01. Bar = 100 µm.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: In Vitro, Boyden Chamber Assay, Incubation, Staining, Membrane, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: [ A ] The three-dimensional skin equivalents containing A375 cells were treated with fisetin (5–20 µM) for 7 days. After treatment with fisetin, skin samples were collected and H&E was performed. A representative picture from three independent experiments is shown. Arrows indicate invading A375 cells. Bar = 25 µm. [ B ] The three-dimensional skin equivalents containing A375 cells were treated with 20 µM of fisetin, PD98059 or CAPE for 12 days. After treatment samples were collected and H&E was performed. A representative picture from three independent experiments is shown. Arrows indicate invading A375 cells. Bar = 25 µm. [ C ] A375 cells (3×10 4 cells/200 µl serum-reduced medium) were placed in the upper chamber of Boyden chamber containing 10 and 20 µM of fisetin, PD98059 or CAPE. The lower chamber contained 110 µl of medium supplemented with 10% FBS. After 24 hours of incubation, the invaded cells on the lower surface of the membranes were fixed with chilled methanol and stained with crystal violet. The invaded cells were counted on the membrane in at least four to five randomly selected microscopic fields. The results are summarized and expressed as the mean number of invaded cells ± SEM per microscopic field. Significant difference versus control group, * P <0.05, ** P <0.01. [D] A375 cells were transfected with MEK1-GFP or GFP-N2 control vector using Xfect Transfection Reagent as per the manufacturer’s protocol. Forty-eight hours after transfection, the cells were harvested and invasion assay was performed as described in “Materials and Methods” section. Significant difference versus control group, ** P <0.01.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Incubation, Staining, Membrane, Control, Transfection, Plasmid Preparation, Invasion Assay

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24 hours) and then the cells were harvested. Total cell lysates were prepared and [ A ] Western blot analysis for protein expression and [ B ] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24 hours) and then the cells were harvested. Nuclear and cytosolic lysates were prepared and [A&C] Western blot analysis for protein expression and [B&D] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for Lamin (nuclear fraction) and β-actin (cytosolic fraction). The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24hours) and then cells were harvested. Total cell lysates were prepared and [ A ] Western blot analysis for protein expression and [ B ] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The tissue-engineered three-dimensional skin equivalents consisting A375 cells were treated with (10 and 20 µM fisetin for 7 days. After treatment, samples were collected, fixed in formalin and paraffin blocks were prepared. Five micormeter sections were cut, deparaffinized, rehydrated and were heated at 95°C for 20 min in citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated with primary antibody against E-cadherin or vimentin overnight at 4°C followed by incubation with specific Alexa Flour 488 or 594 labeled secondary antibody for 1 hour at room temperature in the dark. After washing, the sections were incubated with vectashield mounting media containing DAPI for 10 min in the dark and analyzed under microscope immediately. A representative picture from three independent experiments is shown. E-cadherin is shown in red, vimentin in green and DAPI in blue. Bar = 25 µm.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Incubation, Labeling, Microscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: IκBα/scFvMEL specifically binds to gp240 antigen-positive melanoma cells, as assessed by ELISA. IκBα/scFvMEL binds to the (A) gp240 antigen-positive cell line A375M, (B) gp240-negative cell line T-24, (C) A375M but was competed by preadded ZME-018 to the cells, and (D) gp240-positive and -negative cell lines. scFvMEL/rGel as a positive control protein.

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: Internalization of IκBα/scFvMEL into antigen-positive and -negative human melanoma cells. (A) IκBα/scFvMEL specifically internalized into gp240-positive cells. Cells were treated with different concentrations of IκBα/scFvMEL for 2 hours. Cell surfaces were washed and stripped to remove excess fusion protein. Whole-cell lysates were analyzed byWestern blot using anti-IκBα Ab. (B) Time course of IκBα/scFvMEL internalized into gp240-positive cells. A375M and T-24 cells were treatedwith 100 nM of IκBα/scFvMEL for the indicated times. Cell surfaces were washed and stripped to remove excess fusion protein. Whole-cell lysates were analyzed by Western blot using anti-IκBα Ab. The arrows demonstrate the migration positions of IκBα/scFvMEL and native IκBα, respectively. (C) Internalization of IκBα/scFvMEL into A375M cells analyzed by pulse chase analysis. A375M and T-24 cells were pretreated with 100 nM of IκBα/scFvMEL for 2.5 hours, then washed with PBS twice, and were added with a fresh medium. After culturing for different times, cells were lysed, and proteins were analyzed by Western blot using anti-IκBα Ab. (D) 1, Internalization of IκBα/scFvMEL into A375M, SK-MEL-5, and T-24 cells was visualized using confocal microscopy (original magnification, x200). 2, Time-dependent internalization of IκBα/scFvMEL into A375M cells was visualized using confocal microscopy (original magnification, x200).

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Western Blot, Migration, Pulse Chase, Confocal Microscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: IκBα/scFvMEL associates with NF-κB and inhibits TNFα-induced NF-κB activation. (A) IκBα/scFvMEL associates with NF-κB. A375M cells were left untreated or were incubated with 100 nM IκBα/scFvMEL overnight and then stimulated with or without 0.5 nM TNFα for 30 minutes. Cells were washed and lysed, and the NF-κB p65 was immunoprecipitated. Immune complexes were resolved in SDS-PAGE, transferred onto a membrane, and sequentially probed with anti-IκBα, anti-p50, and anti-p65 Abs. (B) IκBα/scFvMEL specifically inhibits TNFα-induced NF-κB activation on A375M but not T-24 cells. A375M and T-24 cells were pretreated with 200 and/or 400 nM IκBα/scFvMEL for 24 hours at 37°C and then stimulated with 1 nM TNFα for 20 minutes. Nucleus extracts were then prepared and assayed for NF-κB by EMSA. (C) Time- and dose-dependent inhibition of TNFα-induced NF-κB activation by IκBα/scFvMEL. A375M cells were preincubated with 400 nMIκBα/scFvMEL for the indicated times, or A375Mcells were treated with different doses (5, 25, 50, 100, 200, or 400 nM) of IκBα/scFvMEL for 24 hours and then induced by 1 nM TNFα for 20 minutes, and nucleus extracts were examined for NF-κB activation by EMSA. NT indicates not treated.

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Activation Assay, Incubation, Immunoprecipitation, SDS Page, Membrane, Inhibition

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: IκBα/scFvMEL inhibits TNFα-induced nuclear translocation of p65. (A) A375M cells were either untreated or pretreated with 200 nM IκBα/scFvMEL for 24 hours and then treated with 1 nM TNFα for the indicated times. Nucleus extracts were prepared and analyzed by Western blot using Abs against p65 and p50. As a nuclear protein loading control, themembrane was blotted with anti-poly (ADP-ribose) polymerase (PARP) Ab. (B) Immunocytochemical analysis of NF-κB p65 localization. A375M and T-24 cells were not treated or pretreated with 200 nM IκBα/scFvMEL for 4 hours, then stimulated with 1 nMTNFα, and subjected to immunocytochemistry analysis. NF-κB p65 were stained red and counterstained for nuclei with Hoechst 33342 (50 ng/ml) for 5 minutes.

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Translocation Assay, Western Blot, Control, Immunocytochemistry, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: IκBα/scFvMEL inhibits TNF-induced expression of NF-κB-dependent gene. Cells were transiently transfected with a NF-κB-SEAP plasmid and then pretreated with the indicated concentration of IκBα/scFvMEL and/or curcumin for 4 hours and then coincubated with 1 nM TNF for 24 hours. Cell supernatants were collected and assayed for SEAP activity: (A) A375M, (B) SK-MEL-5, and (C) T-24. (D) IκBα/scFvMEL inhibits TNFα-induced expression of NF-κB-dependent antiapoptotic protein Bcl-2 and upregulates the expression of proapoptotic protein Bax. A375M cells were incubated with 400 nM IκBα/scFvMEL for 24 hours and then treated with 1 nM TNFα for the indicated times. Whole-cell lysates were prepared and analyzed by Western blot analysis using the indicated Abs. NT indicates not treated.

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Expressing, Transfection, Plasmid Preparation, Concentration Assay, Activity Assay, Incubation, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: Treatment with IκBα/scFvMEL sensitizes gp240 antigen-positive melanoma cells to ionizing radiation. (A) Treatment with IκBα/scFvMEL specifically blocks constitutive and radiation-induced NF-κB activity in gp240 antigen-positive A375M cells but not on gp240 antigen-negative TXM-1 melanoma cells. A375M, A375SM, and TXM-1 cells were left untreated, exposed to 4 Gy, and pretreated with 300 nM IκBα/scFvMEL for 2.5 hours or a combination of IκBα/scFvMEL and radiation. Cells were harvested 2 hours after irradiation for analysis by EMSA. (B) Decrease in levels of Bcl-2 and Bcl-xL in A375M but not TXM-1 cells after treatment with 200 nM IκBα/scFvMEL. Western blot analysis for Bcl-2 and Bcl-XL in A375M and TXM-1 cells treated with 200 nM IκBα/scFvMEL for 4 hours. (C) Radiosensitization by IκBα/scFvMEL was based on clonogenic cell survival assays. A375M and TXM-1 cells were pretreated with IκBα/scFvMEL (300 nM for 2 hours), the drug was washed off, and cells were irradiated at various doses and plated for clonogenic cell survival assay. Observed sensitizations were statistically significant at 2-, 4-, and 6-Gy dosage groups on A375M cells (P < .05; the results were analyzed from three experiments). No statistically significant sensitization was observed in gp240 antigen-negative TXM-1 cells (P > .05). Treatment of cells with the construct alone had no effect on survival (data not shown).

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Activity Assay, Irradiation, Western Blot, Clonogenic Cell Survival Assay, Construct

Journal: Neoplasia (New York, N.Y.)

Article Title: Characterization and Mechanistic Studies of a Novel Melanoma-Targeting Construct Containing I?Ba for Specific Inhibition of Nuclear Factor-?B Activity 1 2

doi:

Figure Lengend Snippet: IκBα/scFvMEL demonstrates antitumor activity in a xenograft melanoma model. (A) Nude mice bearing human melanoma (A375M) tumors were treated i.v. with either saline (control) or IκBα/scFvMEL (100 mg/kg; total dose) for 10 consecutive days (arrows). The treatment of mice bearing established (30–50 mm3) tumors with IκBα/scFvMEL at a total dose of 100 mg/kg resulted in potent tumor suppression and complete tumor regression of all lesions. In contrast, all mice treated with either saline showed rapid tumor growth. (B) Intratumor activities of several signaling events downstream of NF-κB including Bcl-2 and Bcl-xL were assessed by Western blot. (C) IκBα/scFvMEL is localized or internalized in tumor tissues. Twenty-four hours after the last dose, animals were killed, and tumor tissues were removed, fixed, and stained by immunohistochemical staining for IκBα/scFvMEL detected by either anti-scFvMEL Ab. Localization and internalization of IκBα/scFvMEL were observed in tumor tissues in the treatment group but not in the control group.

Article Snippet: A375M xenograft model Athymic ( nu/nu ) mice, 4 to 6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis, IN).

Techniques: Activity Assay, Saline, Control, Western Blot, Staining, Immunohistochemical staining

Journal: Cell Research

Article Title: Cortical branched actin determines cell cycle progression

doi: 10.1038/s41422-019-0160-9

Figure Lengend Snippet: Cell transformation is generally associated with insensitivity to Arp2/3 inhibition. a Various immortalised cell lines from human breast were treated with the Arp2/3 inhibitor, CK-666, or with the inactive control, CK-869, at the indicated concentrations. These immortalised human breast cell lines stopped cycling in response to Arp2/3 inhibition, in a dose-dependent manner. Mouse Embryonic Fibroblasts (MEF) behaved the same. b Various mammary carcinoma cell lines and the melanoma cell line A375M were insensitive to Arp2/3 inhibition. c siRNA mediated inactivation of the tumour suppressor genes RB1 and CDKN1A , which encodes the CDK inhibitory protein p21 WAF1/CIP1 , prevents the cell cycle arrest due to Arp2/3 inhibition. A similar effect is seen in MCF10A cells knock-out for CDKN1A , and mouse embryonic fibroblasts knock-out for all three genes encoding retinoblastoma proteins. Data are mean ± s.e.m of five technical repeats ( a – c ). One experiment representative of 2 biological repeats is displayed, Mann–Whitney test at 200 µM of drug for a – c (middle and right), twoway ANOVA followed by Sidak’s test for c (left). For b and c , no significant difference between groups was found

Article Snippet: All cell lines derived from human breast were from the collection of breast cell lines organised by Thierry Dubois (Institut Curie, Paris), A375M, WM1960, IGR1 were from Lionel Larue (Institut Curie, Orsay), 83 T, 104 T were from Yardena Samuels (The Weizmann Institute of Science, Rehovot), MEF triple knockout for Rb were from Julien Sage (Stanford University of Medicine), MCF10A p21 −/− were from Ben Ho Park (Johns Hopkins School of Medicine, Baltimore).

Techniques: Transformation Assay, Inhibition, Control, Knock-Out, MANN-WHITNEY