NuBCP-9 suppresses H460 paclitaxel resistant lung cancer cell growth in a xenograft zebrafish model. (A) Effect of NuBCP-9, NuBCP-9/AA (inactive form) on H460 parental and paclitaxel resistant lung cancer cells after 72 hours of treatment in 1% serum. Two-way ANOVA with Dunnett’s multi comparisons post-test, ^****=P<0.0001. (B) Annexin V staining of H460 parental and paclitaxel resistant cells treated with vehicle or 10 μM NuBCP-9 for 24 hours in 1% serum. Results are the mean±s.d. of three technical replicates. Two-way ANOVA with Dunnett’s multi comparisons post-test, ^***=P<0.001, ^****=P<0.0001. (C) NuBCP-9 induces conformational change in Bcl-2 and exposes its BH3 domain. Parental and Resistant H460 cells were treated for 24 hours with vehicle or NuBCP-9 (10 μM) and immunostained with Bcl-2 BH3 specific antibody (upper panel), or Bcl-2 conformation independent antibody (lower panel) and analyzed by flow cytometry. Shift of peak to the right indicates BH3 domain exposure in upper panel. There is no such shift in the lower panels. (D) Effect of Paclitaxel, Doxorubicin (Doxo), NuBCP-9 and NuBCP-9/AA (inactive form) on multidrug resistant H69AR lung cancer cells after 72 hours of treatment in 1% serum. Two-way ANOVA with Dunnett’s multi comparisons post-test, ^***=P<0.001, ^****=P<0.0001. (E) Annexin V staining of H69AR multidrug resistant cancer cells treated with vehicle, doxorubicin (100 nM) or NuBCP-9 (10 μM) for 36 hours. Results are the mean±s.d. of three technical replicates. Unpaired student t-test, ^**=P<0.01. (F) NuBCP-9 reduces growth of multidrug resistant lung cancer cell 3D spheroids. H69AR spheroid cultures were grown for 48 hours and were then treated for 72 hours with vehicle, NuBCP-9 (6 and 10 μM). Percentage viability is calculated relative to vehicle treatment. Unpaired student t-test, ^****=P<0.0001. (G) Representative images of zebrafish xenograft 1-day post injection (dpi) and 4 dpi. Red indicates dyed H460 resistant cells. (H) NuBCP-9 suppresses growth of paclitaxel resistant H460 cells in a zebrafish xenograft model. Growth of H460 paclitaxel resistant cells in xenograft zebrafish model, pre-treated with vehicle or NuBCP-9 (10 μM) for 6 hours prior to injection into zebrafish. Paclitaxel-resistant H460 cells were pre-treated with NuBCP-9 to ensure delivery of the peptide and avoid absorption issues in zebrafish embryos. n= 34 for vehicle and n = 27 for NuBCP-9. Results are the mean±SEM of two independent experiments. Students t-test, ^*P<0.05.