Combination of PD325901 plus ATO inhibits human plasmacytoma growth in immunodeficient NOD-SCID mice. (A) When tumor size reached 200 mm3, mice were randomly assigned (n = 10/group) to receive vehicle alone, PD325901 (orally), ATO (intraperitoneally), or PD325901 plus ATO at the indicated doses on a 5-days-a-week schedule for 21 days. Results are tumor volume (mm3), mean plus or minus SD, plotted against time. A significant delay in tumor growth in PD325901 plus ATO-treated mice was noted compared with vehicle-treated control mice (P < .001 Hsu MCB test). Inset shows tumors resected from control (vehicle) and PD325901 (10 mg/kg) plus ATO (2.5 mg/kg)–treated mice after 21 days of treatment (end point). RPMI 8226-derived tumors and BM from representative untreated or PD/ATO-treated mice were analyzed by hematoxylin and eosin staining (original magnification ×4 for tumor and ×10 for BM). (B) Kaplan-Meier survival curve was evaluated from the first day of treatment until death (mice were killed when tumors reached 2 cm3 in volume) using JMP version 7.0 statistical software (SAS Institute, Cary, NC). Survival was significantly prolonged in PD/ATO-treated animals versus control (P = .001 after Bonferroni correction). The black bar on the abscissa represents the 21-day period of treatment. (C) After 3 days of treatment, mice from each treatment group were humanely killed, and the tumors were removed for assay. RPMI 8226-derived tumors were analyzed by immunostaining for cleaved caspase-3 (Cell Signaling Technology), phospho-ERK (Cell Signaling Technology), and Ki-67 (NCL-L-Ki67-MM1, Novocastra, Newcastle, United Kingdom; original magnification ×20). The microphotographs shown are representative of similar observations in 3 different mice receiving the same treatment. (D) Tumor tissues from mice treated for 3 and 21 days were harvested and processed, and lysates were analyzed by immunoblotting analysis using rabbit polyclonal anticleaved caspase-3, rabbit polyclonal antiphospho-p44/42 ERK (Thr202/Tyr204), and rabbit polyclonal anti-p44/42 ERK, all provided by Cell Signaling Technology. Antiactin immunoblotting was performed as loading control. CTR indicates vehicle; PD, PD325901; ATO, arsenic trioxide.
