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Arnold S, Kadenbach B

Arnold S, Kadenbach B. inhibited cytochrome c oxidase (CcO, complex IV) activity from chemoresistant Nepsilon-Acetyl-L-lysine but not chemosensitive cells, without influencing additional mitochondrial complexes. Notably, our earlier studies revealed the switch to chemoresistance in glioma cells is definitely accompanied by a switch from your manifestation of CcO subunit 4 isoform 2 (COX4-2) to COX4-1. In this study, chlorpromazine induced cell cycle arrest selectively in glioma cells expressing COX4-1, and computer-simulated docking studies indicated that chlorpromazine binds more tightly to CcO expressing COX4-1 than to CcO expressing COX4-2. In orthotopic mouse mind tumor models, chlorpromazine treatment significantly improved the median overall survival of mice harboring chemoresistant tumors. These data show that chlorpromazine selectively inhibits the growth and proliferation of chemoresistant glioma cells expressing COX4-1. The feasibility of repositioning chlorpromazine for selectively treating chemoresistant glioma tumors should be further explored. < 0.001) in soft agar growth assays (Figure ?(Figure1B).1B). Because CPZ clogged cell proliferation specifically in chemoresistant glioma cells, we investigated whether Nepsilon-Acetyl-L-lysine CPZ blocks cell proliferation in the proportion of TMZ-resistant cells that have GSC properties. As illustrated in Number ?Number1C,1C, when cultured in serum-free tradition medium supplemented with epidermal growth element (EGF) and fundamental fibroblast growth element (bFGF), TMZ-resistant UTMZ cells formed neurospheres ranging from 0.1 to 1 1 mm in diameter. However, when UTMZ cells were cultured in the presence of CPZ, smaller and fewer neurospheres developed, ranging from 2.5 to 10 m in diameter. When cells were plated in an limiting dilution assay, CPZ also inhibited the formation of tumor neurospheres inside a dose-dependent manner (Number ?(Figure1D1D). Open in a separate window Number 1 Effect of CPZ on proliferation of TMZ-resistant cells(A) Effect of CPZ on TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cell proliferation. Cells were treated with CPZ in the indicated concentrations. (B) Anchorage-independent growth, assessed by colony formation of UTMZ cells in semisolid medium. Cells were grown on smooth agar plates for 3 weeks before colonies were visualized microscopically. Remaining panel: Representative micrographs of vehicle-treated (top) and CPZ-treated cells (bottom). Right panel: Quantification of colony formation. Colonies were counted Rabbit Polyclonal to HDAC5 (phospho-Ser259) inside a blinded fashion. Nepsilon-Acetyl-L-lysine (C) Representative micrographs from limiting dilution assays with GSCs treated with PBS or CPZ in the indicated concentrations. (D) Quantification of GSCs in the respective assays in (C). Results represent the average from two self-employed experiments. CPZ inhibits CcO activity CPZ has been reported to target mitochondrial function [39, 40], therefore we tested whether CPZ focuses on the mitochondrial ETC complexes. The activities of complexes I, IICIII, IV (CcO) and V (ATP synthase) were measured in mitochondrial components from TMZ-sensitive U251 and TMZ-resistant UTMZ cells in the presence of differing CPZ concentrations (Number ?(Figure2).2). Although CPZ did not impact complexes I, IICIII, or V (Number 2A, 2B and ?and2D),2D), it significantly decreased CcO activity inside a dose-dependent manner (Number ?(Figure2C)2C) specifically in UTMZ cells. We next investigated the kinetic mechanism of CPZ inhibition of CcO. CPZ lowered the Vmax (870 57 to 375 24 pmol/sec/mg) but not the Km for cyt c. Number ?Number2E2E shows the representative Michaelis-Menten graph, and Number ?Number2F2F shows the representative LineweaverCBurk double-reciprocal plots indicating a non-competitive inhibition of cyt c, having a 50% decrease in Vmax at 2 M CPZ. Open in a separate window Number 2 Effects of CPZ on mitochondrial complexes(ACD) CPZ was tested on mitochondrial components from TMZ-sensitive U251 and TMZ-resistant UTMZ glioma cells to determine the effects Nepsilon-Acetyl-L-lysine on the activity of complex I (A), II-III (B), CcO (complex IV) (C), and complex V (D) of the mitochondrial transport chain. Graphs symbolize the activity level of each complex in the presence of PBS (control) or CPZ (up to 50 M). The results are averages from triplicate determinations from two self-employed experiments. (E) Representative Michaelis-Menten graph depicting the.