Research Paper Volume 12, Issue 22 pp 22949—22974

In vitro and in vivo efficacy of the novel oral proteasome inhibitor NNU546 in multiple myeloma

Figure 4. Effects of NNU219, NNU546, bortezomib and ixazomib on the growth of ARH77 xenograft model established in nude mice. (A) 5×106 of ARH77 cells were subcutaneously inoculated into the right flank of nude mice. When the mean tumor volume reached 100-150 mm3, mice were randomized into vehicle group (1% DMSO and 5% HPβCD) and treatment group (bortezomib, 1 mg/kg or NNU219, 0.4 mg/kg), intravenously administered twice weekly for 3 weeks. The measurement was performed using a caliper. Data were presented as mean tumor volume ± SD (n=5; *, p < 0.05). (B) Average tumor weight of mice in the vehicle and treatment groups. Data were showed as mean ± SD (**, p < 0.01). (C) Tumor-bearing mice were orally treated with vehicle, ixazomib (5 mg/kg, BIW) or NNU546 (1 mg/kg, QD or 2 mg/kg, QOD) schedule for 3 weeks. Data were presented as mean tumor volume ± SD (n=5; *, p < 0.05). (D) Average tumor weight of mice in the vehicle and treatment groups. Data were showed as mean ± SD (*, p < 0.05). (E) Left, tumor sections of untreated, Ixazomib or NNU546 treated mice were subjected to immunostaining for apoptosis (TUNEL, green color), proliferation marker Ki-67 (red color) and angiogenesis markers CD31 (green color) and α-SMA (red color) and detected using confocal microscopy (PerkinElmer UltraVIEW Vox, magnification, ×200 or ×400, nine continual fields put together). Right, the tissue sections were screened under a low-power field, and five fields were selected. Each field was analyzed separately to obtain the fraction of apoptotic cells, ki67 positive cells fluorescent areal density and microvessel density. The data is presented as mean ± SD across the vision fields (*, p < 0.05; **, p < 0.01; ***, p < 0.001). SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick end labelling; SMA, smooth muscle actin.