FIGURE 2.

Anti-myeloma activity of K-7174 in vivo. NOD/SCID mice were inoculated subcutaneously with 1 × 10⁷ cells of U266 or 3 × 10⁷ cells of RPMI8226 into the right thigh. Treatments were started on day 0, when tumors were measurable. Caliper measurements of the longest perpendicular tumor diameters were performed on alternate days to estimate the tumor volume (mm³) using the following formula: 4/3π × (width/2)² × (length/2). A, mice were treated with 75 mg/kg of K-7174 intraperitoneally once daily for 14 days (n = 4) or the vehicle (3% DMSO in 0.9% NaCl) alone (Control) on the same schedule (n = 4). The y axis shows the average tumor volume in inoculated mice on the indicated days. The mean ± S.D. (bars) are shown. p values were calculated by one-way analysis of variance with the Student-Newman-Keuls multiple comparisons test. B, representative photographs of NOD/SCID mice inoculated with U266 cells on days 0 and 14 (original magnification: ×2). Inoculated tumors are indicated in circled regions. C, mice were treated with 50 mg/kg of K-7174 post-orally once daily for 14 days (U266 + K-7174, closed circle; n = 3, RPMI8226 + K-7174, closed triangle; n = 3), or the vehicle (3% DMSO in 0.9% NaCl) alone on the same schedule (U266 + control, open circle; n = 4, RPMI8226 + control, open triangle; n = 3). The y axis shows the average tumor volume in inoculated mice (bars indicate S.D.). p values were calculated by one-way analysis of variance with the Student-Newman-Keuls multiple comparisons test. Asterisks indicate p < 0.05 against the vehicle control. D, representative photographs of NOD/SCID mice inoculated with RPMI8226 cells on days 0 and 14 (original magnification: ×2). Inoculated tumors are indicated in circled regions. E, NOD/SCID mice were inoculated subcutaneously with 1 × 10⁷ cells of U266 into the right thigh. When tumors were measurable, mice were treated with 50 mg/kg of K-7174 post-orally once daily (K-7174; n = 2) or the vehicle (3% DMSO in 0.9% NaCl) alone (Control; n = 2) for 3 days. We resected inoculated tumors and sorted human CD138-positive cells using a FACSaria flow cytometer for immunoblot analysis of cellular protein ubiquitination (Ubiq) and CD138 (hCD38) expression. We used lysates of U266 cells treated with 5 μm K-7174 (U266 + K-7174) or the vehicle control (U266) for 3 days in vitro as controls. The signal intensities were quantified by densitometry, normalized to those of the corresponding CD138, and shown as relative values setting the vehicle control (U266) to 1.0.