Mechanistic Insight Into Taxol-Induced Cell Death
Total Page：16
File Type：pdf, Size：1020Kb
Oncogene (2008) 27, 4580–4591 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Mechanistic insight into taxol-induced cell death F Impens1,2, P Van Damme1,2, H Demol1,2, J Van Damme1,2, J Vandekerckhove1,2 and K Gevaert1,2 1Department of Medical Protein Research, VIB, Ghent, Belgium and 2Department of Biochemistry, Ghent University, Ghent, Belgium We analysed the involvement of proteases during taxol- One class of chemotherapeutic drugs that induce such mediated cell death of human A549 non-small-cell lung alternative forms of PCD is microtubule-stabilizing carcinoma cells using a proteomics approach that specifi- agents with their prototypical representative taxol cally targets protein N termini and further detects newly (paclitaxel). Taxol and derivatives are used as potent formed N termini that are the result of protein processing. drugs against several solid tumors. Although their Our analysis revealed 27 protease-mediated cleavages, cytotoxic mechanism depends on cell type, concentra- which we divided in sites C-terminal to aspartic acid (Asp) tion and exposure duration, in most studies with and sites C-terminal to non-Aspresidues, as the result of clinically relevant taxol concentrations (10–200 nM), caspase and non-caspase protease activities, respectively. apoptosis is induced by blocking the mitotic spindle Remarkably, some of the former were insensitive to potent and a G2/M arrest (Schiff and Horwitz, 1980; Torres pancaspase inhibitors, and we therefore suggest that and Horwitz, 1998; Blagosklonny and Fojo, 1999; Zhao previous inhibitor-based studies that report on the caspase- et al., 2005). The signaling pathways leading to cell independent nature of taxol-induced cell death should be death have been extensively studied (Blagosklonny and judged with care. Furthermore, many of the sites C-terminal Fojo, 1999; Zhao et al., 2005) and recently several to non-Aspresidues were also uniquely observed in a model papers on the proteases involved were published. It is of cytotoxic granule-mediated cell death and/or found by now clear that taxol triggers apoptosis by both caspase- in vitro cataloging human l-calpain substrates using a dependent (Park et al., 2004; Ehrlichova et al., 2005; Li similar proteomics technique. This thus raises the hypothesis et al., 2005; Lu et al., 2005; Day et al., 2006; Janssen that killing tumor cells by chemotherapy or by immune cells et al., 2007; Pineiro et al., 2007) and caspase-indepen- holds similar non-Asp-specific proteolytic components with dent pathways (Broker et al., 2002, 2004; Huisman et al., strong indications to calpain activity. 2002; Ofir et al., 2002). One of the main supporting Oncogene (2008) 27, 4580–4591; doi:10.1038/onc.2008.96; observations for the latter is the failure of the published online 14April 2008 pancaspase inhibitor zVADfmk (N-benzyloxycarbonyl- Val-Ala-Asp(O-Me) fluoromethyl ketone) to protect Keywords: protease substrates; caspase-like activity; against taxol-induced apoptosis (Broker et al., 2002; taxol; cytotoxic granule Huisman et al., 2002). Here, we identified protease substrates targeted during taxol treatment of human A549 non-small-cell Introduction lung carcinoma cells using the peptide-centric proteo- mics technique known as N-terminal COFRADIC Chemotherapeutic agents kill tumor cells by different (COmbined FRActional DIagonal Chromatography). forms of programmed cell death (PCD). Next to This method specifically isolates (neo-)N-terminal pep- apoptosis with activation of the caspase cascade as the tides from digested proteomes before identification by most studied form, alternatives such as ‘apoptosis-like tandem mass spectrometry (Gevaert et al., 2003) and has PCD’ and ‘necrosis-like PCD’ have been described proven to be a powerful tool for substrate degradome (Leist and Jaattela, 2001). These modes of PCD are analysis (Lopez-Otin and Overall, 2002; Van Damme characterized by an essential activity of proteases other et al., 2005; Vande Walle et al., 2007): not only the than caspases during the onset or execution phase of cell protein substrates but also the exact cleavage sites death. For example, cathepsins (Foghsgaard et al., 2001; are simultaneously assigned, providing links to the Bidere et al., 2003; Broker et al., 2004), calpains functional implication of the cleavage events and to (Karmakar et al., 2007; Pineiro et al., 2007) and yet the type(s) of protease(s) creating the cleavage patterns. uncharacterized proteases (de Bruin et al., 2003) were reported to play key roles in different cell death models. Results Correspondence: Professor Dr K Gevaert, Department of Biochem- istry, Faculty of Medicine and Health Sciences, Ghent University, Treatment with taxol results in detachment and apoptosis A Baertsoenkaai 3, B-9000 Ghent, Belgium. E-mail: [email protected] of G2/M-arrested A549 cells Received 12 September 2007; revised 18 February 2008; accepted 5 Twenty-four hours after induction with 200 nM taxol, a March 2008; published online 14April 2008 fraction of A549 cells had detached from the culture Substrate degradomics of taxol-treated A549 cells F Impens et al 4581 dish and died during the next 24h, whereas a significant all parameters; however, the remarkable changes fraction of cells remained attached and did not show any between 24and 48h observed for detached cells as a apoptotic alterations, even after 48 h (Figure 1). Cell result of the execution phase of cell death are not cycle analysis by flow cytometry revealed that detach- present, pointing to stress conditions without triggering ment and associated cell death was the result of a mitotic cell death. Together, these observations clearly indicate arrest in the G2/M phase. This was not the case for the need to distinguish between detached and adherent adherent cells, where next to a limited increase in G2/M- A549 cell populations for further substrate degradomics. phase cells, a significant fraction of the cell population resided in other phases of the cell cycle (Figure 1). Besides the fact that the latter stopped dividing and that Lysosomal membrane permeabilization and activation stress granules were formed, no signs of PCD were of caspase-3 and calpains occur only in detached cells present. Next to caspases (Park et al., 2004; Ehrlichova et al., Time course flow cytometry experiments (Figure 2) 2005; Li et al., 2005; Lu et al., 2005; Day et al., 2006; further confirmed that detached cells were dying by Janssen et al., 2007; Pineiro et al., 2007), involvement of apoptosis, as a high proportion of these cells stained calpains (Pineiro et al., 2007) and cathepsin-B (Broker positive for annexin V and showed a hypoploid (sub- et al., 2004) was described in taxol-induced apoptosis. G1) DNA content at early time points (p24h). A large We compared taxol-treated detached and adherent fraction of these cells became positive for propidium A549 cells for activation of caspase-3 and calpains iodide (PI) only at late time points (X48 h). In adherent on immunoblots (Figure 3a). Lysosomal membrane cells, we measured a rather slow increase over time for permeabilization (LMP) and intracellular calcium control 24h 200 nM taxol 48h 200 nM taxol adherent cells adherent cells 1400 1400 1400 1120 1120 1120 840 G1 840 840 counts 560 S counts 560 counts 560 G2/M 280 280 sub-G1 280 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 red fluorescence red fluorescence red fluorescence detached cells detached cells 1400 1400 1120 1120 840 840 counts 560 counts 560 280 280 0 0 100 101 102 103 104 100 101 102 103 104 red fluorescence red fluorescence Figure 1 Taxol induces detachment and cell death of A549 cells suffering from mitotic arrest. Results of the analysis of the cell cycle by flow cytometry are shown as overlay histograms (open line) with control cells (filled line). Microscopic analysis revealed that detached cells are present after 24h taxol treatment and further die during the next 24h. In contrast to cells that remain adherent, detached cells are exclusively present in the G2/M phase of the cell cycle after 24h and show a high sub-G1 population after 48h, indicating cell death by apoptosis. Oncogene Substrate degradomics of taxol-treated A549 cells F Impens et al 4582 detached cells adherent cells not shown). Activation of the classical calpains (m- and m-calpain) was initiated 24h after taxol treatment and 100 was clearly visible after 48 h as shown by the formation of smaller autolytic fragments of the common small 75 calpain subunit (Wood and Newcomb, 1999). Flow cytometry revealed that the activation of calpains was accompanied by an increase of intracellular calcium 50 concentration. Finally, by measuring AO fluorescence, we observed LMP between 24and 48h in detached cells. 25 No evidence for the activation of proteases involved in cell death or LMP in adherent cells was obtained, in line with the fact that no PCD was observed in these 0 control 8h 16h24h 48h 72h cells (Figure 2). Based on these results, we screened for protease substrates in 48 h taxol-treated detached 100 A549 cells. 75 Screening by N-terminal COFRADIC in taxol-treated detached A549 cells identified 27 processed sites in 50 22 proteins Two proteomic screens were performed using N-terminal COFRADIC (Gevaert et al., 2003). In the % PI positive cells25 % annexin V positive cells first experiment, we post-metabolically labeled the 16 samples at the peptide level with light ( O2, control) 18 0 or heavy ( O2, taxol-treated) isotopes, introducing a control 8h 16h24h 48h 72h mass difference of 4Da (Staes et al., 2004). In the second 100 screen, cells were metabolically labeled during cell culture by SILAC (stable isotope labeling by amino 12 acids in cell culture) (Ong et al., 2002) with light ( C6, 75 13 taxol-treated) or heavy ( C6, control) arginine, introdu- cing a mass difference of 6 Da.