Fig. 3: NCT-58 induces apoptosis and inhibits tyrosine kinase activity of HER2 in trastuzumab-resistant cells.

A NCT-58 (0.1–20 μM) reduced cell viability in trastuzumab-resistant JIMT-1 and MDA-MB-453 cells (***p < 0.001, n = 4). B Morphological changes in these cells as seen through phase-contrast microscopy in the presence or absence of NCT-58 (10 μM, 72 h). C, D NCT-58 treatment (2–10 μM, 72 h) resulted in a significant induction of apoptosis in JIMT-1 cells, as evidenced by increased sub-G1 accumulation (C, ***p < 0.001, n = 3) and annexin V/PI-positive cells (D, ***p < 0.001, n = 3). E Effects of NCT-58 (2–10 μM, 72 h) on expression of PARP, cleaved-PARP, cleaved-caspase-3, cleaved-caspase-7, and survivin in JIMT-1 cells. F Influence of NCT-58 (2–10 μM, 72 h) on protein contents of HSP70 and HSP90 in JIMT-1 cells. G Immunoblot analyses of HER2, phospho-HER2, p95HER2, phospho-p95HER2, EGFR, phospho-EGFR, HER3, phospho-HER3, Akt and phospho-Akt (Ser473) expression in JIMT-1 cells following exposure to NCT-58 (2–10 μM, 72 h). H Effects of NCT-58 (2–10 μM, 72 h) on Ras, Raf, phospho-Raf, Mek, phospho-Mek, Erk, and phospho-Erk protein expression in JIMT-1 cells. I Immunoblot analyses of HER2, p95HER, phospho-HER2, and phospho-p95HER2 in both HER2- and p95HER2-overexpressing MDA-MB-231 cells. J Immunofluorescence analysis of HER2 and p95HER2. Cells were immunostained with ECD-HER2 (green) or ICD-HER2 (green, CB11) antibody and counterstained with DAPI (blue). Vimentin (red) was stained to demonstrate cellular features in MDA-MB-231 cells. K The levels of p95HER2, phospho-p95HER2, Akt, and phospho-Akt were downregulated in p95HER2-overexpressing MDA-MB-231 cells after exposure to NCT-58 (2–10 μM, 72 h).