tion criteria (AIC and BIC). Statistical analysis on tumor volume was conducted using SAS 9. (SAS Institute Inc., Cary, NC, USA). A P value \ 0.05 indicated statis- tical significance. Tumor growth in athymic mice Into the mammary fat pad of the mice (Foxnnu strain from naratriptan Harlan Sprague–Dawley, Indianapolis, IN, USA), 5 9 0 6 LCC6 cells in serum-free Iscove’s modified essential medium were injected. Tumor growth was measured every 3 days, and tumor volume was estimated from bidirectional measure- ments using the formula length 9 breadth /. When the tumors reached palpable mass, the mice were randomized by tumor volume into six groups of five animals each. The mice were treated with PBS, DOX (3 mg/kg/week, ip), OSI-906 ( OSI ) (30 mg/mouse/week, orally).

DOX and OSI simulta- neously, DOX followed by OSI, or OSI followed by DOX,  CC-5013 respectively. Treatment was given weekly for 4 weeks. IGF-I signaling in OSI-906 treated xenograft tumors LCC6 cells were injected into the mammary fat pad of female athymic mice. When tumors reached a volume of Results PQIP inhibited both IGF-I and insulin signaling The MCF-7 breast cancer cell line expresses both IGFR and InsR. IGF-I and insulin has been reported to stimulate the growth of this cell line 8 . To assess whether PQIP inhibits both IGFR and InsR, MCF-7 cells were treated with either IGF-I or insulin, and with increasing concen- trations of PQIP. After immunoprecipitation with anti- IGFR antibody, the phosphorylation of IGFR upon IGF-I treatment was examined. As shown in Fig. a, IGF-I stim- ulated the phosphorylation of IGFR, and PQIP inhibited the phosphorylation of IGFR in a dose-dependent manner. Similarly, PQIP dose-dependently inhibited the phosphor- ylation of InsR upon insulin stimulation.

However, PQIP was about three-fold more potent toward inhibiting the 3 3 Breast Cancer Res Treat of the MDA-MB-435 cell line. While there has been some debate regarding the origin of these cells 9 , 0 , IGFR has been shown to play a critical role in the proliferation and metastasis 6 , . Recently, we have also shown that downregulation of InsR in these AMN-107 Src-bcr-Abl inhibitor cells inhibited cancer cell proliferation and metastasis . Therefore, LCC6 cells provide a good model to assess the anti-tumor effects of PQIP. ERK/ is constitutively active in these cells thus was not regulated by ligands or PQIP (Fig. b, lower panel). In contrast, the phosphorylation of Akt was acti- vated by IGF-I and to a lesser extent, by insulin. PQIP dose-dependently inhibited the phosphorylation of Akt in LCC6 cells. Our results suggest that PQIP is a potent inhibitor against both IGF-I and insulin signaling in MCF-7 and LCC6 cells. PQIP inhibited MCF-7 proliferation and progression into S phase .

To assess the activity of PQIP on cell proliferation, MCF-7 cells were treated with or without IGF-I, and increasing concentration of PQIP. The IC 50 concentration for PQIP was determined using MTT assays. As shown in Fig. a, MCF-7 cells treated with PQIP AMN-107 641571-10-0 exhibited a dose-dependent decrease in monolayer growth compared with untreated cells. The IC 50 of PQIP was in the submicromolar range in the presence of IGF-I, consistent with the IC 50 measured in NIH 3T3 fibroblasts and GEO human colorectal cancer cells 0 . Fig. PQIP inhibited both IGF-I and insulin signaling. a Serum- starved MCF-7 cells were treated with or without l M PQIP, or with increasing concentration of PQIP (0.03, 0., 0.3, and l M) in the presence of 5 nM IGF-I or 0 nM insulin. Cells were lysed, and the IGFR or InsR was immunoprecipitated and blotted with seniors phospho- tyrosine antibody. b Serum-starved MCF-7 or LCC6 cells were treated with or without l M PQIP, or with increasing concentration of PQIP (0.0, 0.03, 0., 0.3, and l M) in the presence of 5 nM IGF- I or 0 nM insulin. Cellular lysates were immunoblotted for IGFR and InsR, and total and phospho-ERK/, Akt phosphorylation of IGFR than that of InsR, consistent with previous reports that IGFR is more sensitive to PQIP than InsR when overexp