Effects of the microtubule stabilizing agent peloruside A on the proteome of HL-60 cells

Abstract

Peloruside A, isolated from the marine sponge Mycale hentscheli, has a similar mechanism of action to paclitaxel (Taxol®), a drug used clinically to treat tumors of the breast, ovary and lung. Paclitaxel and peloruside stabilize the polymerized form of tubulin and arrest cells in G₂/M of the cell cycle. We have therefore used two-dimensional electrophoresis of proteins to examine the effect of peloruside A on the human HL-60 promyeloid leukemic cell line. Our goals included investigation whether affected proteins could be mapped onto pathways that predicted the cellular effects of this compound. In response to 100 nM peloruside A treatment for 24 h, seventeen identified proteins showed significant change in abundance with fourteen increases and three decreases. Use of Ingenuity Pathways Analysis confirmed that peloruside A affected pathways consistent with the known effects on microtubules and apoptosis. Our results also indicate a potential role of c-Myc in the cellular actions of peloruside consistent with an induction of aneuploidy seen at low concentrations of peloruside.

Paclitaxel effects on the proteome of HL-60 promyelocytic leukemic cells: comparison to peloruside A

“…Both compounds were stored as a 1 mM solution in absolute ethanol at −20°C. HL-60 human promyeloid leukemic cells, a gift from Dr. Michael Berridge (Malaghan Institute of Medical Research, Wellington, New Zealand) were cultured in RPMI-1640 medium with 10% FCS and penicillin/streptomycin as previously described [6]. Drug effects on cell growth and cell cycle block were determined as previously reported [6].…”

“…Electrophoresis, tryptic digestion, mass spectrometry, and database analysis One-dimensional (1DE) and two-dimensional electrophoresis (2DE), CyDye labelling of protein, differential in-gel electrophoresis (DIGE), tryptic digestion of electrophoresis spots, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry were carried out as previously described [6]. For DIGE, isoelectric focusing included two pH ranges: pH 4-7 and pH 6-11.…”

“…The secondary antibody was anti-mouse Ig-Cy5 (GE-Healthcare, 1:5000). The Western blotting protocol was as previously described [6].…”

“…A good approach to do this is to use a proteomic approach that employs separation of proteins by 2DE, followed by tryptic digestion, mass spectrometry, and bioinformatics to identify and analyze the specific protein changes [5]. In a prior DIGE study in our laboratory, Pel at a concentration of 100 nM for 24 h was shown to alter 17 proteins, with 14 increasing in abundance and three decreasing in abundance [6]. Nine more proteins were seen to change but could not be identified because of low abundance or inconsistent database matches.…”

“…However, only one study, which did not use DIGE technology, directly investigated the effects of Ptx treatment on the cell proteome [10]. The aim of the present study was to carry out a proteomic analysis with Ptx identical to that carried out with Pel [6] using Ptx at an equipotent dose and comparing the responses of these two microtubule-stabilizing agents to ascertain if their activity profiles differed. Such an approach could identify secondary targets of the drugs and downstream effects that could aid in the understanding of the complex responses and side effects seen when the drugs are delivered in vivo.…”