affinity  (R&D Systems)

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Interaction of survivin with SMAC, identification of the binding site that as peptide interferes with survivin assembly into dimers and higher oligomeric states (A) Purified survivin used in this study was purchased from Sino Biological (Beijing, China, Cat. SINO-10356HNCE). (B) Survivin binding to itself and to SMAC. Purified survivin was fluorescently labeled as described in the materials and methods. Kd for survivin and SMAC are 100 and 200 nM, respectively. (C) Identifying the SMAC binding site in survivin. A glass-bound peptide array consisting of overlapping peptides derived from 11 selected SMAC-interacting proteins was incubated overnight with purified SMAC (0.8 μM) and then blotted with anti-SMAC antibodies (1:1000), followed by incubation with HRP-conjugated secondary antibodies and detection using a chemiluminescence kit. Dark spots indicate the binding of SMAC to peptides derived from SMAC-interacting proteins, such as with spots 1G12 and 1E23, derived from BIRC2 and 1H13 derived from survivin. A representative blot of three independent experiments is shown. (D) Overlapping sequences of peptides derived from BIRC5 in the glass-bound peptide array. The sequence of the peptide that interacted with SMAC is in red, and the peptides located before and after appearing in black. The peptide localization in dimeric survivin in each subunit of the homodimers with the proposed stabilized interacting residues Phe93, Glu94, Glu95, Leu96, Thr97, Leu98, Gly99, Phe101, and Leu102, which were all present in the peptide (in blue, and Leu98 and Phe101 highlighted in yellow). (E) Dimeric survivin crystal structure, (PDB_ 1e31 ), in the red the peptide location the image was prepared using UCSF Chimera. (F) Survivin interaction with the indicated concentrations of the survivin 1H13-derived peptide analyzed using the MST method. Fluorescently labeled purified survivin (650 nM) was incubated for 30 min at 37°C with the peptide (2–30 μM), and thermophoresis was measured as described in (B). Kd = 2 ± 0.05 μM ( n = 3). (G) Purified survivin (1 μg/mL) was incubated in the absence and presence of an 1H13 peptide (30 or 60 μM) in PBS, pH 8.3 (15 min, 30 o C), and then incubated with EGS (50 or 100 μM, 15 min, 30 o C). The reaction was terminated by adding sample buffer, followed by SDS-PAGE (gradient gel 4%–20% acrylamide), followed by immunoblotting with anti-survivin antibodies The positions of the survivin monomers, dimers, trimers, tetramers, and higher oligomers are indicated. (H–K) The levels of survivin dimers, trimers, tetramers, and multimers were quantified using ImageJ software and presented relative to their levels in the EGS-untreated samples and their levels in the presence of the peptide compared to its absence (shown in blue). (L and M). IH13 peptide (60 μM) in PBS, pH 8.3 was incubated with EGS (50 μM, 15 min, 30 o C), followed by SDS-PAGE (gradient gel 4%–20% acrylamide) and Coomassie blue staining. The levels of peptide dimers, trimers, tetramers, and multimers are indicated (L). Peptide levels (monomeric) before and after crosslinking (M).

Article Snippet: Purified human recombinant SMAC (10339-H08E) and human survivin (10356-HNCE) were produced by Sino Biologicals (Wayne, PA).

Techniques: Binding Assay, Purification, Labeling, Peptide Microarray, Derivative Assay, Incubation, Sequencing, SDS Page, Western Blot, Software, Staining

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: Purified human recombinant SMAC (10339-H08E) and human survivin (10356-HNCE) were produced by Sino Biologicals (Wayne, PA).

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: 2/3D-1H13-Nuc decreases survivin, SMAC, and Ki-67 levels, and increases p53 levels A549 cells were seeded on 13-mm glass coverslips, untreated (control) or treated with the 2/3D-1H13-Nuc peptide (20 or 40 μM, 24 h), fixed and subjected to IF using anti-survivin (A and B), anti-SMAC (C and D), anti-Ki-67 (E and F), or anti-p53 antibodies (G and H). The yellow and white arrows point to nuclear p53 and binuclear cells, respectively (G). Confocal microscope images are shown (A, C, E, and G), and quantification of the staining intensity per/cell (120–140 cells analyzed for each sample) is presented (B, D, F, and H). (I and J) Cells treated with the 2/3D-1H13-Nuc peptide for 24 h, harvested, and then subjected to immunoblotting using specific antibodies against p53, Ki-67, or β-actin (I), and band intensities were quantified using ImageJ software (J), or subjected to RT-qPCR for quantification of mRNA levels of p53, Ki-67, or β-actin (K), as described in the Method section. Results represent the means ± SEM ( n = 3); ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Purified human recombinant SMAC (10339-H08E) and human survivin (10356-HNCE) were produced by Sino Biologicals (Wayne, PA).

Techniques: Control, Microscopy, Staining, Western Blot, Software, Quantitative RT-PCR

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: 1H13 peptides targeted to the nucleus, mitochondria, or cytosol inhibit decreased expression of survivin, SMAC, p53, and β-tubulin in tumors Sections of control and tumor-treated mice with the indicated 1H13-peptide were IF-stained with specific antibodies for survivin (A and B), SMAC (C and D), p53 (E and F) or β-tubulin (G and H). Representative confocal images are shown (A, C, E, and G), and staining intensity quantification from three different tumors each (B, D, F, and H) are shown. (I–L) Paclitaxel treatment of a breast cancer xenograft mouse model inhibits tumor growth, stabilizes microtubules, and reduces survivin expression. (I) Athymic female nude mice were s.c. injected with MDA-MB-231 (3×10 6 ), and tumor formation was followed. Upon reaching a volume of 50–100 mm 3 (day 14), the mice were split into two tumor volume-matched groups and were i.v. injected three times a week with the vehicle (NaCl 0.9%) or with paclitaxel (10 mg/kg), followed by measurement of the tumor volume. Tumor volume as a function of time post-cell inoculation is presented. (J–L) 45 days post-cell inoculation, the tumors were dissected, fixed, and embedded in paraffin. Sections of control and paclitaxel-treated tumors were IF-stained for survivin (J and K) or β-tubulin (L) using specific antibodies, and staining intensities were quantified (K) or indicated in the bottom of the image (L). Results represent the means ± SEM ( n = 3), ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗∗ p < 0.0001.

Article Snippet: Purified human recombinant SMAC (10339-H08E) and human survivin (10356-HNCE) were produced by Sino Biologicals (Wayne, PA).

Techniques: Expressing, Control, Staining, Injection

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Proposed mechanisms for the multiple effects of the survivin-derived peptide in tumors: a possible new multi-target treatment for cancer schematic of the mechanism of action of the multiple effects of the survivin/BIRC5-derived peptide on cancer hallmarks: Survivin/BIRC5 dimer interacting with the peptide leading to dissociation of the dimer. This leads to (A) Apoptosis—survivin binding to SMAC prevents caspase activation, and thereby apoptosis, (B) Altered cell cycle due to decreased tubulin levels and/or checkpoint activation—survivin being part of the CPC and directs the aurora-B kinase to the centromere during mitosis, thereby inhibiting cell proliferation, and (C) Modulating the immune response—increasing the tumor infiltration of CD-8 and activated NK cells and increasing the expression of PD-1 and PD-L1 on cancer cell sub-populations results in their interaction, and leads to cancer cell death, while sparing the immune cells.

Article Snippet: Purified human recombinant SMAC (10339-H08E) and human survivin (10356-HNCE) were produced by Sino Biologicals (Wayne, PA).

Techniques: Derivative Assay, Binding Assay, Activation Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Trk Signaling Inhibition Reduces cSCC Growth and Invasion in In Vitro and Zebrafish Models and Enhances Photodynamic Therapy Outcome

doi: 10.3390/ijms262110434

Figure Lengend Snippet: Trk signaling inhibition by K252a affects cSCC growth and viability. ( a ) Cell viability evaluated in NHK, SCC12 and SCC13 cells after treatment with K252a (100, 200 or 400 nM) at 24, 48 and 120 h by MTT assay. ( b ) TrkA, P-TrkA, Cyclin B1, Survivin, and Pro-caspase-3 expression evaluated by Western blotting at 24 h after K252a (200 nM) treatment; b-actin was used as the loading control. ( c ) Ki-67 mRNA expression evaluated by qPCR; b-actin was used as the housekeeping gene. ( d ) Ki-67 protein expression evaluated by immunofluorescence. Images were taken at 40× magnification. ( e ) FACS analysis and ( f ) % of SubG1 cells. ( g ) TrkA, P-TrkA, AKT, and PCNA expression evaluated by Western blotting at 24 h after K252a (200 nM) treatment, with or without NGF (100 ng/mL); b-actin was used as the loading control. For all experiments, statistical analysis was performed using two-way ANOVA. The results are represented as mean ± SD. p -values are indicated as the following *: 0.01 < p < 0.05; **: 0.001 < p < 0.01; *** 0.0001 < p < 0.001; **** p < 0.0001.

Article Snippet: Rabbit anti-human Survivin , Cell Signaling Technology (Danvers, MA, USA) , 1:1000.

Techniques: Inhibition, MTT Assay, Expressing, Western Blot, Control, Immunofluorescence