Desipramine-induced apoptosis in human PC3 prostate cancer cells: Activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2+]i elevation
Introduction
In addition to its clinical effect in treating psychological disorders (Mayers and Baldwin, 2005), desipramine has been shown to exert different effects on many cell types in vitro, such as induction of Ca2+ influx and cytotoxicity in PC3 human prostate cancer cells (Huang et al., 2007), inhibition of activity-dependent sodium channel (Lenkey et al., 2006), and inhibition of Na+/H+ exchanger in human submandibular cells (Choi et al., 2006). Desipramine is commonly used as an inhibitor for acid ceramidase (Elojeimy et al., 2006). Furthermore, desipramine was shown to cause apoptosis in human HT29 colon carcinoma cells (Arimochi and Morita, 2006), and to increase cytosolic free Ca2+ levels ([Ca2+]i) and kill cells in renal tubular cells (Ho et al., 2005). Other actions of desipramine include decreasing tumor necrosis factor-alpha production (Reynolds et al., 2005), downregulating the cortical alpha1- and alpha2-adrenoceptors (Subhash et al., 2003), inhibiting cytochrome P450 enzymes (Shin et al., 2002) and neuronal/skeletal ATP-Ca2+ pump of endoplasmic reticulum (Couture et al., 2001, Soler et al., 2000), suppressing plasma membrane Ca2+ pump (Plenge-Tellechea et al., 1999), activating steroid transporters (Pariante et al., 2001) and GABAergic systems (Asahi and Yonehara, 2001), and altering protein kinase C activity (Morishita and Watanabe, 1997), etc.
The term “apoptosis” was coined in 1972 by John Kerr who observed that certain dying cells shared several morphologic properties. All the criteria used to describe apoptotic cells were morphological and included condensation and margination of chromatin, cytoplasmic vacuolization, cellular shrinkage, increase in cellular density, nuclear fragmentation, and apoptotic body formation (Kerr et al., 1972). Cysteine aspartate specific proteases (caspases) families, which have highly specific proteases activity and can cleave proteins exclusively after aspartate residues, are well-known main actors of the apoptotic proteolytic cascades. Such as caspase-8 is an initiator or activator in caspase cascades; caspase-3 is an effector in caspase cascades; and caspase-12 is a cytokine processor in caspase cascades (Coffey et al., 2001, Hengartner, 2001, Thornberry and Lazebnik, 1998). Caspase-3 is a key protease activated during the early stages of apoptosis (Nakagawa and Yuan, 2000).
Alteration in [Ca2+]i is a crucial regulator of many cellular events (Berridge, 2006). To allow a precise regulation of [Ca2+]i and many signaling pathways by Ca2+, cells have numerous mechanisms by which to modulate [Ca2+]i both globally and at the subcellular level (Berridge, 2005). Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved serine-threonine kinases that participate in cell differentiation, cell growth and survival (Sharma et al., 2005). Extracellular signal-regulated kinases (ERK), stress activated protein kinase c-Jun/N-terminal kinases (JNK) and p38 are three major components of MAPKs. Signal transduction involves sequential phosphorylation of a tripartite kinase module, culminating in an activated MAPK. Activated MAPK may stay in the cytoplasm to phosphorylate structural proteins or translocate to the nucleus, where it can activate transcription factors involved in DNA synthesis and cell division. MAPKs have been suggested to play a pivotal role in apoptosis (Cross et al., 2000, Reddy et al., 2003). Therefore, a [Ca2+]i rise and subsequent apoptosis is tightly related. However, not all types of apoptosis are caused by a Ca2+ signal.
We have previously reported that in PC3 human prostate cancer cells desipramine-induced cell death that was worsened by chelating cytosolic Ca2+ with BAPTA/AM (Huang et al., 2007). The goal of this study was (1) to explore whether apoptosis accounted for desipramine-induced cell death, (2) to examine the involvement of ERK, JNK and p38 mitogen-activated protein kinases, and (3) to investigate the mechanism underlying the cytotoxic effect of BAPTA/AM.
Section snippets
Materials
The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). BAPTA/AM was from Molecular Probes (Eugene, OR, USA). Desipramine, propidium iodide (PI), dimethyl sulfoxide (DMSO) and other reagents were from Sigma–Aldrich (St. Louis, MO, USA).
Cell culture
PC3 cells were obtained from American Type Culture Collection and were cultured in Dulbeco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were kept at 37 °C in
Effect of desipramine on apoptosis of PC3 cells
Previous data showed that desipramine-induced concentration-dependent inhibition of viability of PC3 cells at concentrations between 10 and 800 μM (Huang et al., 2007). The possibility of involvement of apoptosis in the cell death was explored. As shown in Fig. 1A, apoptosis (measuring the increase in subdiploidy nuclei appeared in cells) occurred in cells treated with 10–800 μM desipramine in a concentration-dependent manner. Caspase-3 is thought to function as an executioner of apoptosis and
Discussion
The major novel finding of the present study was that desipramine caused death of human PC3 prostate cancer cells via evoking apoptosis with the participation of JNK pathway, and [Ca2+]i rises played a preventive role in the process. We studied the effect of desipramine on cytotoxic markers such as cell viability, subdiploidy nuclei and activation of caspase-3 (Hengartner, 2001), and found that desipramine-induced cell death was accompanied by DNA fragmentation as detected by propidium iodide
Acknowledgement
This work was supported by grants from Veterans General Hospital Kaohsiung (VGHKS97-071) to CR Jan.
References (46)
- et al.
Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon carcinoma cells
Eur. J. Pharmacol.
(2006) - et al.
Involvement of GABAergic systems in manifestation of pharmacological activity of desipramine
Jpn. J. Pharmacol.
(2001) Calcium microdomains: organization and function
Cell Calcium
(2006)- et al.
The role of MAPKs in adipocyte differentiation and obesity
Biochimie
(2005) - et al.
c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis
Biochim. Biophys. Acta
(2007) - et al.
Serine/threonine protein kinases and apoptosis
Exp. Cell Res.
(2000) - et al.
New insights on the use of desipramine as an inhibitor for acid ceramidase
FEBS Lett.
(2006) - et al.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
(1985) - et al.
Effect of desipramine on Ca2+ levels and growth in renal tubular cells
Cell. Signal.
(2005) - et al.
Desipramine-induced Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells
Toxicol. In Vitro
(2007)