FIG 4.

ASM inhibitors abrogate K-Ras signaling and function in vitro and in vivo. (A) MDCK cells stably expressing mGFP–K-RasG12V were treated with vehicle (DMSO) or 10 μM imipramine, desipramine, amitriptyline, or nifedipine for 48 h. Following treatment, whole-cell lysates were prepared and ppERK levels were measured by quantitative Western blotting. ppERK levels were normalized to the control sample. Significant differences were assessed by using Student's t tests (*, P < 0.05) (n = 3). (B) A panel of wild-type (BxPC-3, KLE, NCI-H508, and NCI H1975) or oncogenic mutant K-Ras-expressing (MiaPaCa-2, MOH, MPanc96, Hec-1a, Hec-1b, SK-CO-1, SW948, and NCI H23) pancreatic, endometrial, colon, and lung tumor cells were seeded in 96-well plates and treated for 72 h with vehicle (DMSO) or various concentrations of imipramine. The number of viable cells was quantified using the CyQuant cell proliferation assay kit (Molecular Probes). (C) nu/nu mice implanted with BxPC-3 or MIAPaCa-2 tumors were treated with vehicle (DMSO) or fendiline (12.5 mg/kg) intraperitoneally, and tumor sizes were measured with an external caliper every 3 to 4 days. Time and fendiline treatment affected MiaPaCa2 xenograft growth (P = 0.027 by 2-way ANOVA). Posttest analysis, using the Bonferroni correction for multiple comparisons, showed that xenografts treated with fendiline were significantly smaller on days 28, 32, and 35 (*, P < 0.05; **, P < 0.01; ***, P < 0.001). No significant effects due to drug treatment were observed in BxPC-3 tumors.