PQIP inhibited MCF-7 cell proliferation and progression into S phase in cell cycle. a Monolayer growth of MCF-7 cells treated with different concentrations of PQIP in the presence or absence of 5 nM IGF-I. b Anchorage-independent growth of MCF-7 cells sodium butyrate treated with different concentration of PQIP in the presence or absence of 5 nM IGF-I in % FBS. c Sub-confluent MCF-7 cells were exposed to either SFM, 5 nM IGF-I, 0.3 uM PQIP or 5 nM IGF-I plus 0.3 uM PQIP for 4 h. Cells were harvested, and cell cycle analysis was performed by flow cytometry. * P \ 0.05 against IGFR induced apoptosis 3 . MCF-7 and LCC6 cells were incubated in serum-free media or % FBS in the absence or presence of PQIP for 4 h. After treatment, both independent growth assay. We also studied whether there was sequence dependence. MCF-7 and LCC6 cells were treated with PQIP in combination with DOX as the fol- lowing schedules:

5 ng/ml (MCF-7), 00 ng/ml (LCC6) DOX; (3) 0. l M PQIP; (4) simulta- neous DOX and PQIP treatment; (5) DOX for 4 h followed by PQIP; (6) PQIP for 4 h followed by DOX. While DOX alone inhibited colony formation in MCF-7 cells, PQIP significantly enhanced the cytotoxicity of DOX under two sequence conditions: (4) simultaneous DOX and PQIP and (5) DOX followed by PQIP (Fig. 4 a). In contrast, PQIP followed by DOX had little Pemetrexed effect on colony 3 5 Breast Cancer Res Treat formation. To confirm the effects of PQIP on chemother- apy in other cancer cells, LCC6 cells were also examined. Unlike MCF-7 cells, PQIP or DOX alone did not signifi- cantly inhibit LCC6 colony formation. However, LCC6 colonies were similarly affected by simultaneous treatment of PQIP with DOX or DOX followed by PQIP as seen in MCF-7 cells (Fig. 4 b).

Therefore, sequencing of PQIP with DOX can significantly enhance the effectiveness over chemotherapy alone in different cancer cells. A PQIP derivative compound, OSI-906, enhanced the effect of DOX in vivo OSI-906 is a derivative compound of PQIP that has purchase Ofloxacin similar inhibitory effects on IGFR and InsR activity, yet has better drug metabolism and pharmacokinetics in vivo . We therefore used OSI-906, not PQIP, to examine sequencing with DOX and IGF-I signaling in vivo. LCC6 cells were injected into the mammary fat pad of athymic mice. When tumors were palpable, mice were pre-treated with or without OSI-906 for 4 h and then treated with IGF- I for 40 min. As shown in Fig. 5 a, IGF-I significantly Fig. 3 PQIP induced autophagy instead of apoptosis. a Cells were serum-starved for 4 h and treated with or without l M PQIP, or with increasing concentration of PQIP (0., 0.3, , 3, and 0 l M) in the presence of IGF-I (MCF-7 cells) or % FBS (LCC6 cells). Twenty-four hours later, cells were lysed and PARP cleavage was assessed by immunoblotting. b MCF-7 cells were treated with 0.3 l M PQIP or 0 l g/ml AVE64. Cells were collected at indicated hours and analyzed by immunoblotting using an LC3 antibody. PC positive control consisted of serum-starved neuro A cellular lysates.

Cells were treated with 0. or l M PQIP, or 0 l g/ml AVE-64 for 4 h with 5% FBS. Cells were order Ofloxacin fixed, and fluorescent immunohistochem- istry was performed to detect endogenous LC3. Representative confocal microscopy images were shown 3 Fig. 4 PQIP and DOX inhibited anchorage-independent growth in a sequence-dependent manner MCF-7. a and LCC6 ( b ) cells with or without PQIP ( a , 0.3 l M; b , l M), DOX ( DOX ) or PQIP plus DOX were mixed with 0.45% agarose and overlaid over 0.8% bottom agar in 6-well plate for 4 h, and then, second treatment were added in the top of agar and continue incubating for 9–0 days. Colonies formed were counted on a portion of the well. Each psychological stress treatment was done in triplicate, and the results are shown as the average number of colonies ± SEM. * P \ 0.05, ** P \ 0.0. The experiment was repeated three times with similar results, and a representative experiment is shown 6 Breast Cancer Res Treat b Fig. 5 OSI-906 enhanced the cytotoxicity of DOX