ressed in NIH3T3 cells 0 . To elucidate how PQIP affected downstream signaling, we investigated the Akt and ERK/ pathways upon IGF-I or insulin treatment in cancer cells. As shown in Fig. b, MCF-7 cells have relatively low basal phosphorylation of ERK/ and Akt. After IGF-I treatment, ERK/ and Akt phosphorylation was dramatically increased. Insulin parthenolide stim- ulated the phosphorylation of ERK/ and Akt to a lesser extent. Pre-treatment of cells with PQIP dose-dependently inhibited phosphorylation of IRS- and IRS-, Akt and ERK/. Because insulin signaling was weaker than IGF-I induced signaling, 0. l M of PQIP was able to efficiently block downstream effects of insulin. LCC6 is a Carboplatin derivative 3 We further evaluated the effect of PQIP on anchorage- independent growth.

MCF-7 cells were grown in soft agar with % FBS plus IGF-I, in the absence or presence of increasing concentration of PQIP. As shown in Fig. b, PQIP dose-dependently inhibited anchorage-independent growth with or without IGF-I stimulation. IGF-I is a well- known mitogen that stimulates G to S phase progression 3 . To investigate the ability of PQIP to interfere with IGF- I mediated cell cycle progression, we analyzed MCF-7 cell cycle nisoldipine 63675-72-9 distribution by flow cytometry. Serum-starved MCF-7 cells were treated with PQIP (0.3 l M) in the absence or presence of IGF-I for 4 h, and cell cycle progression was analyzed. As shown in Fig. c, about 0% of serum- deprived cells were found in the S phase and IGF-I induced a significant increase of S phase cell population; PQIP completely inhibited the IGF-I stimulated S phase pro- gression. Together these results show that PQIP is a potent inhibitor of IGF-I dependent cell proliferation in vitro.

PQIP induced autophagy, but not apoptosis To investigate potential mechanisms by which PQIP inhibits cell proliferation, we studied whether PQIP induced cell apoptosis in the presence of IGF-I. We have previously shown that monoclonal antibodies directed 4 Breast Cancer Res Treat adherent and non-adherent cells were collected and sub- jected to Western blotting analysis to detect the cleaved fragment of poly (ADP) ribose buy nisoldipine polymerase (PARP), a caspase substrate. As shown in Fig. 3 a, increased concen- tration of PQIP failed to induce PARP cleavage in MCF-7 and LCC6 cells while the monoclonal antibody AVE64 did (data not shown and 3 ). In addition, annexin V did not increase when MCF-7 and LCC6 cells were treated with PQIP (data not shown). These results suggest that PQIP does not induce cell apoptosis. PQIP inhibited IGF-I and insulin-induced Akt signaling. The mammalian target of rapamycin (mTOR) is a substrate for Akt and a key regulator of autophagy 4 , so we examined whether PQIP led to the activation of autophagy. The LC3 protein is widely used to detect autophagy. We therefore examined the presence of cleaved LC3 (LC3-II) and the localization of LC3 in PQIP treated cells. The amount of LC3-II fragmentation is correlated with the number of autophagosomes 5 . As shown in Fig. 3 b, 0.3 l M PQIP time-dependently increased the levels of LC3-II fragments. To support the specific induction of autophagy by inhibition of IGFR activation, we also observed an increase in autophagy with the anti-IGFR antibody, AVE-64.

While both drugs induced autoph- agy, PQIP resulted in a more rapid appearance of LC3-II fragments. To confirm PQIP and AVE-64 induced autophagy, we performed cognitive performance immunofluorescent staining for LC3 to detect the formation of autophagosomes. As shown in Fig. 3 c, PQIP and AVE-64 induced the formation of auto- phagosomes, indicating that PQIP treatment was associated with increased autophagy. PQIP enhanced the cytotoxicity of DOX in vitro Previous results from our laboratory have shown that anti- IGFR antibodies enhanced the cytotoxicity of DOX in a sequence-dependent manner. Only DOX followed by antibody significantly enhanced DOX’s effects in vitro and in vivo 5 . Herein, we examined whether PQIP could enhance the cytotoxicity of DOX using an anchorage- F