Journal: Oncology Reports
Article Title: C-terminal HSP90 inhibitor NCT-58 impairs the cancer stem-like phenotype and enhances chemotherapy efficacy in TNBC
doi: 10.3892/or.2025.9018
Figure Lengend Snippet: NCT-58 exerts anti-proliferative effect in TNBC cells by targeting the C-terminal domain of HSP90. (A) Four TNBC cell lines, MDA-MB-231, BT549, Hs578T and 4T1 cells were treated with various concentrations of NCT-58 (0–20 µM) for 72 h. Cell viability was assessed using MTS assay, and IC 50 values were calculated using non-linear regression with a sigmoidal dose-response curve. (B) MDA-MB-231 and 4T1 cells were treated at the indicated concentrations of NCT-58 (0–10 µM) for 72 h. Apoptosis was determined through sub-G1-DNA analysis using flow cytometry. (C) Immunoblot analyses of PARP, cleaved-PARP, caspase-3, cleaved caspase-3, caspase-7 and cleaved caspase-7 protein expression in MDA-MB-231 cells after treatment with NCT-58 (0–10 µM, 72 h). GAPDH was used as an internal loading control. Quantitative graphs of these protein levels. The results are presented as the mean ± SEM of at least three independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post hoc test. (D) Effect of NCT-58 on C-terminal HSP90 binding activity. The inhibitory effect of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 500 µM) on the interaction between HSP90α (C-terminal) and its co-chaperone peptidylprolyl isomerase was determined using an HSP90α (C-terminal) inhibitor screening assay. (E) Influence of NCT-58 on N-terminal HSP90 binding activity. The competitive HSP90α binding activity of HSP90 inhibitors (NCT-58, novobiocin or geldanamycin, 1 µM) with FITC-labeled geldanamycin was determined using an HSP90α N-terminal domain assay. (F and G) Molecular docking analysis of NCT-58 binding to the CTD of HSP90 (PDB ID: 7RY1). (F) The binding pose of NCT-58 at the dimerization interface is displayed as a space-filling model. The α1 chain of HSP90 is rendered in a blue ribbon, and the α2 chain in a pink ribbon. Connolly surface representation of the HSP90 CTD, with NCT-58 modeled within the binding interface (docking score=−9.5). (G) A 2D interaction diagram of NCT-58 with key residues in the HSP90 CTD. Hydrogen bonds and π-anion interactions are indicated by dashed green and blue lines, respectively. (H and I) Comparison of the effects of NCT-58 and the N-terminal HSP90 inhibitor geldanamycin on HSF-1 and HSP70 expression. MDA-MB-231 cells were treated with NCT-58 (300 nM and 10 µM) or geldanamycin (300 nM) for 24 h. Cells were immuno-stained for HSF-1 (red, H) or HSP70 (green, I) using DAPI (nuclei, blue). Images were acquired using a confocal microscope, and quantification of immunofluorescence intensity was performed using ImageJ software. Nuclear HSF1 intensity was expressed as the HSF1/DAPI ratio, and cytoplasmic HSP70 intensity was expressed as corrected total cell fluorescence normalized to DAPI. (J and K) Effect of NCT-58 and geldanamycin on cytotoxicity in non-malignant cells. Normal human mammary epithelial MCF10A (J) and 293 (K) cells were treated with various concentrations (0.1–10 µM) of NCT-58 or geldanamycin for 72 h. Cell viability was determined using MTS assay (***P<0.001). *P < 0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TNBC, triple-negative breast cancer; Gelda, geldanamycin; Novo, novobiocin; CTD, C-terminal domain.
Article Snippet: A C-terminal HSP90α Inhibitor Screening Kit (BPS Bioscience) was used to evaluate inhibition of the interaction between the C-terminal region of HSP90α and its co-chaperone peptidylprolyl isomerase D (PPID) by HSP90 inhibitors, as previously described ( , ).
Techniques: MTS Assay, Flow Cytometry, Western Blot, Expressing, Control, Binding Assay, Activity Assay, Screening Assay, Labeling, Comparison, Staining, Microscopy, Immunofluorescence, Software, Fluorescence
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