Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: Celastrol directly binds to Prdx2 and inhibits its activity. ( A ) Schematic of the procedure for detecting Celastrol-binding proteins. ( B ) Human protein microarrays were probed with biotinylated-Celastrol. Binding was detected by Cy3-labeled streptavidin. Control experiments were carried out with free biotin (green pots showing interacting proteins). ( C ) Pathway enrichment analysis by DAVID. Celastrol showed the strongest binding ability to Prdx proteins. ( D ) Representative Prdx signals from the protein arrays. Signal-to-noise ratios (SNR) are shown (- = free biotin). ( E, F ) The binding affinity of Celastrol with rhPrdx2 and rhPrdx1 were determined by the SPR assay. KD values are shown above the traces. ( G ) Determination of Celastrol binding to rhPrdx2 protein by isothermal titration calorimetry (ITC). ( H ) Molecular interaction between Celastrol and Prxd2 as determined by docking software. Figure showing possible interaction between Celastrol and Cys172 of Prdx2. ( I, J ) Biotinylated-Celastrol interacts with Prdx2 in SGC-7901 cells and tumor tissues from mice implanted with SGC-7901 cells. Lysates were prepared from untreated SGC-7901 cells in culture and untreated mice subcutaneously injected with SGC-7901 cells. Biotinylated-Celastrol (Bio-Cel) was added to streptavidin-agarose beads. Biotin alone was used as a control. Lysates prepared from SGC-7901 cells (I) and tumor tissues from mice (J) were added. Eluent was then loaded on a polyacrylamide gels for immunoblotting. Total lysates were used as an input control. ( K ) Residue Cys172 of Prdx2 protein was mutated to Ala172 and Ser172. Prdx2 variants were probed for Celastrol interaction and compared to wildtype Prdx2, using pull-down assays described in panels I and J. ( L ) Peroxidase activity of rhPrdx2 proteins were monitored by measuring H 2 O 2 levels as described in Methods (n = 3; * P <0.05, ** P <0.01 compared to 0 µM control]. ( M, N ) rhPrdx2 proteins were incubated with Celastrol or Adenanthin for 10 min and peroxidase activity was measured (n = 3; * P <0.05, ** P <0.01, *** P <0.001 compared to control). The IC 50 of compounds against rhPrdx2 activity are shown. ( O ) Peroxidase activity following addition of Celasterol to rhPrdx1 (n = 3; * P <0.05, ** P <0.01 compared to control). IC 50 of Celastrol is shown. ( P ) Cellular Prdx enzyme activity was measured by adding Celastrol to lysate prepared from SGC-7901 cells (n = 3; * P <0.05, ** P <0.01 compared to 0 µM control). The IC 50 value of Celastrol was shown.

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: Activity Assay, Binding Assay, Labeling, SPR Assay, Isothermal Titration Calorimetry, Software, Injection, Western Blot, Incubation

Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: Knockdown of Prdx2 potentiates and overexpression normalizes the activities of Celastrol. ( A ) BGC-823 cells were stably transfected with negative control shRNA plasmid or shRNA targeting Prdx2. Total cell lysates were probed for Prdx2. GAPDH was used as loading control. Densitometric quantification is shown in lower panel. ( B ) Knockdown of Prdx2 in BGC-823 cells by shRNA transfection enhances Celastrol-induced cytotoxicity as determined by the MTT assay. ( C ) Knockdown of Prdx2 by siRNA transfection in BGC-823 cells significantly increased Celastrol-induced apoptotic cells. Cells were exposed to Celastrol at 2 µM for 24 h. Apoptosis was determined by Annexin/PI staining (siRNA = Prdx2 siRNA, NC = negative control siRNA; n = 3; * P <0.05, ** P <0.01 compared to 0 µM Celastrol NC). ( D ) Representative phase contrast images of cells exposed to Celastrol for 24 h after Prdx2 knockdown (scale bar = 40 µm). ( E ) SGC-7901 cells were stably transfected with empty vector (NC) or Prdx2 cDNA (O/E). Lysates were probed for Prdx2 protein levels. GAPDH was used as loading control. Densitometric quantification is shown on right (n = 3; *P<0.05 compared to NC). ( F ) Prdx2 overexpressing cells and vector control transfected cells were exposed to Celastrol for 24 h. Cell viability was measured by MTT assay. IC 50 values are shown. ( G ) PI staining of SGC-7901 cells showing reduced apoptosis (red; arrows) in cells expressing Prdx2 O/E plasmids. Cells were counterstained with DAPI (Blue) (scale bar = 10 µm). ( H ) The quantification of PI fluorescence intensity in cells expressing Prdx2 O/E plasmids (n = 3; * P <0.05 compared to NC).

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: Over Expression, Stable Transfection, Transfection, Negative Control, shRNA, Plasmid Preparation, MTT Assay, Staining, Expressing, Fluorescence

Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: Celastrol inhibits SGC-7901 xenograft tumor growth in vivo by decreasing Prdx enzyme activity and increasing ROS levels. ( A-C ) Effect of Celastrol treatment on gastric cancer growth in mice. Figure showing tumor volumes (A), harvested tumors at 24-day follow-up (B), and tumor weights (C) (** P <0.01, *** P <0.001 compared to vehicle control). ( D ) Graph showing body weights of mice at indicated time points. ( E ) Staining of tumor tissue sections with ROS probe DHE (red). Increased fluorescence intensity is indicative of increased ROS levels (scale bar = 40 µm). ( F ) Prdx activity levels in harvested tumor specimens (* P <0.05, ** P <0.01 compared to control). ( G ) Western blot analysis of cleaved-caspase3, pro-caspase3, Prdx2 and CHOP in tumor tissue lysates. GAPDH was used as protein loading control. ( H ) Quantification of cleaved-caspase3, pro-caspase3, Prdx2 and CHOP levels in tumor tissue lysates (n = 3; * P <0.05, ** P <0.01 compared to control). ( I ) Immunohistochemical staining of tumor specimens for cell proliferation marker Ki-67 and apoptosis marker cleaved-caspase3. Immunoreactivity was detected by DAB chromogen (brown). Sections were counterstained with hematoxylin (blue). Inserts showing higher magnification (scale bar = 50 µm).

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: In Vivo, Activity Assay, Staining, Fluorescence, Western Blot, Immunohistochemical staining, Marker

Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: ROS and Prdx2 mediate Celastrol-mediated inhibition of gastric cancer growth. ( A ) Measurement of tumor volumes at indicated time points following implantation of SGC-7901 cells in BALB/c mice. Mice treated with 1.5 mg/kg Celastrol, NAC with 1.5 mg/kg Celastrol, or 5 mg/kg Cisplatin. NAC was given at 1 g/L in drinking water. Celastrol and Cisplatin treatments were administered every other day. Control mice received vehicle alone. ( B ) Tumor weights for mice in panel A (** P <0.01, *** P <0.001). ( C ) Staining of tumor tissue sections harvested from mice with ROS probe DHE (red) (scale bar = 50 µm). ( D ) Xenograft of Prdx2 shRNA-expressing BGC-823 cells. Mice were treated with 1.5 mg/kg Celastrol every other day (Ctrl shRNA = negative control shRNA). ( E ) Tumor weights for mice in panel D (*P<0.05, ** P <0.01, *** P <0.001). ( F ) Staining of tumor tissue sections harvested from mice in panel D with ROS probe DHE (red) (scale bar = 50 µm). ( G ) Xenograft of Prdx2 cDNA expressing (O/E) SGC-7901 cells. Mice were treated with 1.5 mg/kg Celastrol every other day (Ctrl O/E = negative vector). ( H ) Tumor weights for mice in panel G (* P <0.05, ** P <0.01). ( I ) Staining of tumor tissue sections harvested from mice in panel G with ROS probe DHE (red) (scale bar = 50 µm).

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: Inhibition, Staining, shRNA, Expressing, Negative Control, Plasmid Preparation

Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: Prdx2 is upregulated in human gastric cancer. ( A ) Prdx2 mRNA levels in gastric cancer tissues (T) and patient-matched, tumor-adjacent normal gastric tissues (N). Transcript levels were normalized to β-actin levels. ( B ) Western blot analysis of Prdx2 protein levels in different gastric cancer tissues (T) and patient-matched, adjacent normal gastric tissues (N). GAPDH used as loading control. Data from the remaining 9 samples (n =17) is provided in . ( C ) Representative immunohistochemical staining for Prdx2 (brown) in gastric cancer tissues (T) and adjacent normal gastric tissues (N). Slides were counterstained with hematoxylin (blue) [scale bar = 50 µm]. ( D ) Overall survival of the gastric cancer patients with high or low levels of Prdx2 using probe 215067_x_at. ( E ) Overall survival of the gastric cancer patients with high or low levels of Prdx2 using probe 201006_at.

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells

doi: 10.7150/thno.46728

Figure Lengend Snippet: Schematic illustration of the main findings of the study. Celastrol binds to and inhibits Prdx2 in gastric cancer cells. Inhibition of Prdx2 augments ROS levels and initiates cell death through ER stress and mitochondrial dysfunction.

Article Snippet: Prdx2 overexpression was carried out with Prdx2 cDNA plasmid (pGV492-GFP-O/E-Prdx2; Shanghai GeneChem Co) and using the same protocol as indicated.

Techniques: Inhibition

Journal: Cells

Article Title: Identification of Targets from LRRK2 Rescue Phenotypes

doi: 10.3390/cells10010076

Figure Lengend Snippet: List of functional clusters that were enriched using DAVID.

Article Snippet: PRDX2 plasmid (RC207413) was purchased from Origene.

Techniques: Functional Assay

Journal: Cells

Article Title: Identification of Targets from LRRK2 Rescue Phenotypes

doi: 10.3390/cells10010076

Figure Lengend Snippet: Validation of PRDX2. ( a ) Functional annotation enrichment on downregulated differential transcripts in pathogenic variant reveals significant enrichment of PRDX2 -centred oxidoreductase pathway genes by the DAVID enrichment tool (nominal p -value <0.01). ( b ) MetaCore gene networks on the core nodes of oxidoreductase annotation clusters demonstrate PRDX2 acts as a key player in PTEN , CREB1, and FLRE pathways. ( c ) Histogram plot shows differential FPKM levels across biological fly phenotypes for two PRDX2 isoforms (TCONS_00023592 and TCONS_00023593; FDR p < 0.05). ( d ) Normalized Jafrac1 (Drosophila PRDX2 orthologue) mRNA levels measured by quantitative PCR in RNA isolated from 60-day-old fly heads. Data show mean and s.d. of three independent biological replicates. Significance on the graph: * p < 0.05.

Article Snippet: PRDX2 plasmid (RC207413) was purchased from Origene.

Techniques: Biomarker Discovery, Functional Assay, Variant Assay, Real-time Polymerase Chain Reaction, Isolation