Abstract 4576: Quantitative proteomic analysis of 2-({4-[4-(acridin-9-ylamino)phenylthio]phenyl}(2-hydroxyethyl)amino)ethan-1-ol (CK0402) modulated protein expression in breast cancer SKBR-3 cells

Abstract

Abstract 2-({4-[4-(acridin-9-ylamino)phenylthio]phenyl}(2-hydroxyethyl)amino)ethan-1-ol (CK0402) was selected as potential anticancer agent among the series of sulfur-containing 9-aminoacridine analogs previously synthesized by our group. CK0402 is a topoisomerase II inhibitor and has been shown to exert anti-cancer activities in both in vitro and in vivo assays. Our previous studies demonstrated that CK0402 exhibited highest activity in SKBR-3 cells among a panel of established human breast cancer cell lines; moreover, CK0402-induced responses of G2/M arrest, apoptosis and autophagy were stronger in ER-(−)/HER2-(+) SKBR-3 cells than in ER-(+)/HER2-(−) MCF-7 cells. These findings suggest that CK0402 may be effective against HER-2 (+) breast cancer. The objective of this study is to identify molecular makers in response to CK0402 treatment in HER-2 (+) cells. These treatment markers may be important for clinical application since they can be used to avoid lethal toxic side effects or unnecessary treatment to insensitive patients. To this end, we analyzed the global protein expression modulated by CK0402 treatment in SKBR-3 cells using LC-MS/MS with a quantitative MS-based iTRAQ labeling method. SKBR-3 cells were exposed to various concentrations of CK0402 for 24 and 72 hrs; in which, 2 plates of cells received control vehicle, 3 plates of cells received 1 μmol/L, and another 3 plates of cells received 5 μmol/L of CK0402. The cell lysates were denatured, reduced, alkylated, and then digested with trypsin. The resulting peptides were labeled with 8-plex iTRAQ™ reagents, fractionated by cation exchange, and subjected to a LC-MS/MS analysis. The levels of differentially expressed proteins between control and treated sample were compared using student's t-test. Selected proteins were then validated by Western blot analysis. We found that that a total of 219 proteins were differentially expressed in SKBR-3 cells (p < 0.05) after treated with 5 μmol/L of CK0402 for 72 hours. Among those, the levels of S100 proteins such as A7 and A8/A9 were increased 4.7- and 3.2-fold, respectively; in addition, the increases of these proteins were in a dose- and time- dependent manner, which is consistent with the growth inhibition effect of CK0402 in SKBR-3 cells. These results suggest that the S100 A7 and A8/A9 proteins are responders of CK0402 treatment in SKBR-3 cells. Current mechanistic studies are focusing on determining the roles of these identified proteins in the anticancer activities or toxic side effects of CK0402. The S100 proteins together with other proteins identified in this study could be used as effective indicators of the clinical response of patients to CK0402 treatment in future clinical studies. : Support: Susan G. Koman Breast Cancer Foundation Grant BCTR0707876 and Penn State HMC Dean's Feasibility Grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4576.