Hexadecylphosphocholine causes rapid cell death in canine mammary tumour cells

https://doi.org/10.1016/j.ejphar.2004.09.015Get rights and content

Abstract

Hexadecylphosphocholine (HePC, Miltefosine) is an antitumour phospholipid and known inducer of apoptosis in human breast cancer cells. The mechanism underlying the induction of cell death by HePC, however, is not clear yet. In this study, we have investigated the cytotoxic effects of HePC on canine mammary tumour cells (CMTs) in vitro. Upon addition of HePC, CMTs rapidly exhibited several features that resembled apoptotic cell death. Cells showed externalisation of phosphatidylserine, a hallmark of apoptosis, within 5 min after addition of HePC at concentrations as low as 10 μM. Furthermore, rapid swelling of mitochondria was observed. Rounding and detachment of cells followed within 30 min. However, fragmentation of nuclear DNA could not be observed. Overall, HePC was shown to induce a type of cell death in CMTs that in some aspects resembles apoptosis, though the process proceeds much more rapidly than reported for other tumour cell lines.

Introduction

Tumours of the mammary glands are the most frequently occurring type of tumours in both women and female dogs. Similarities in biological behaviour, histology and epidemiology have been shown to occur between these two species (Hellmen, 1992, MacEwen, 1990, Van Leeuwen et al., 1996).

Tumourigenesis in the dog appears to be dependent on the hormonal state of the animal: while dogs that have been spayed before 2 years of age hardly even develop mammary tumours, the incidence of mammary tumours in dogs spayed after this age or non-spayed dogs is extremely high (Gourley, 2000, Perez Alenza et al., 2000, Schneider et al., 1969). Both oestrogen and progesterone have been shown to play an important role during development of canine mammary tumours (Rutteman et al., 1988). While receptors for both of these hormones are expressed at relatively high levels in normal mammary tissue, levels drop in malignant tumours. Metastases of mammary tumours often even fail to express oestrogen receptors (Sheikh et al., 1994).

The absence of oestrogen receptors in tumour cells is correlated with an increased resistance against some of the more frequently used chemotherapeutic agents, like e.g. tamoxifen (Osborne, 1999). Therefore, new cytotoxic agents have been developed that act through different targets like e.g. DNA replication and lipid signalling. The latter pathway is targeted by synthetic antitumour phospholipids (APLs), like e.g. hexadecylphosphocholine (HePC; Miltefosine) and 1-O-octadecyl-2-O-methyl-rac-3-glycero-3-phosphocholine (ET-18-OMe; Edelfosine).

HePC and ET-18-OMe are analogues of lysophospholipids and act as antitumour agents through inhibition of invasiveness (Ruiter et al., 2001, Storme et al., 1985) and induction of apoptosis (Ruiter et al., 2001). For both compounds it has been proposed that they enter the cell by means of receptor-independent endocytosis rather than diffusion across the plasma membrane (Bazill and Dexter, 1990, Geilen et al., 1994, Van der Luit et al., 2003). Alternatively, evidence exists that APLs can directly be absorbed into the outer leaflet of the plasma membrane and subsequently be turned to the inner leaflet (Arthur and Bittman, 1998, Fleer et al., 1987, Fleer et al., 1993, Leroy et al., 2003).

Though its exact mode of action is still largely unknown, HePC has been shown to inhibit CTP:phosphocholine cytidylyltransferase (CCT, EC 2.7.7.15) (Wieder et al., 1993) and protein kinase C (PKC) activities (Uberall et al., 1991), whereas it activates phospholipase D (PLD) (Wieder et al., 1996). ET-18-OMe has been shown to inhibit phosphatidylinositol-specific phospholipase C (PI-PLC) (Powis et al., 1992) and CCT activities (Boggs et al., 1995). Based on their relative low toxicity towards normal tissue in comparison with their high toxicity in tumour cells, HePC and ET-18-OMe have been used in several clinical trials, amongst others during treatment of skin metastases of breast cancer (Leonard et al., 2001).

In the present study, the cytotoxic effects of HePC on canine mammary tumour cells (CMTs) have been examined in more detail. Cell morphology, nuclear DNA fragmentation, mitochondrial integrity and externalisation of plasma membrane phosphatidylserine (PS) were investigated.

Section snippets

Cell line and culture

The canine mammary tumour cell line CMT-U335 originates from a spontaneous primary canine mammary osteosarcoma, as has been described previously by Hellmen (Hellmen et al., 2000). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich; Zwijndrecht, The Netherlands) containing 5% fetal bovine serum (Invitrogen; Breda, The Netherlands) in a humidified atmosphere of air containing 5% CO2 at 37 °C. Chinese hamster ovary (CHO)-K1 cells and Madin-Darby canine kidney (MDCK)

HePC induces detachment of canine mammary tumour cells

Synthetic APLs like HePC have been shown to induce apoptosis in a number of different cell lines and several different mechanisms have been proposed to be involved in this process (Ruiter et al., 2001, Storme et al., 1985). The onset of apoptosis was reported to occur between 3 and 6 h after addition of the antitumour phospholipid (APL) (Ruiter et al., 1999, Van der Luit et al., 2002) and was found to be maximal at 6–36 h (Ruiter et al., 1999, Van der Luit et al., 2002, Vrablic et al., 2001),

Discussion

HePC at micromolar concentrations induces cell death in canine mammary tumour cells, a process which in some aspects resembles apoptosis.

CMT-U335 cells rapidly detach from the culture dish after addition of HePC, possibly due to modulation of cell attachment molecules. It has been described that ET-18-OMe, a related alkylphospholipid, modulates the adherens junction-E cadherin complex in human mammary carcinoma cell lines MCF-7/6 and MCF-7/AZ (Steelant et al., 2001). ET-18-OMe restored the

Acknowledgements

The authors would like to thank Dr. Eva Hellmen (Uppsala University, Uppsala, Sweden) for providing the CMT-U335 cell line, Dr. Chris van de Lest for help with statistical analysis and Ing. Anko de Graaff and Dr. Jack Valentijn of the Centre for Cellular Imaging (CCI, Faculty of Veterinary Medicine, Utrecht University) for excellent technical assistance and useful discussions.

References (43)

  • C. Cabaner et al.

    Induction of apoptosis in human mitogen-activated peripheral blood T-lymphocytes by the ether phospholipid ET-18-OCH3: involvement of the Fas receptor/ligand system

    Br. J. Pharmacol.

    (1999)
  • J.G. Culvenor et al.

    Characteristics of plasma membrane isolated from a mouse T lymphoma line: comparison after nitrogen cavitation, shearing, detergent treatment and microvesiculation

    J. Cell. Biochem.

    (1982)
  • M. Enari et al.

    A caspase-activated DNAse that degrades DNA during apoptosis, and its inhibitor ICAD

    Nature

    (1998)
  • I. Eue

    Growth inhibition of human mammary carcinoma by liposomal hexadecylphosphocholine: participation of activated macrophages in the antitumor mechanism

    Int. J. Cancer

    (2001)
  • E.A.M. Fleer et al.

    Metabolism of ether phospholipids and analogs in neoplastic cells

    Lipids

    (1987)
  • E.A.M. Fleer et al.

    Investigations on the cellular uptake of hexadecylphosphocholine

    Lipids

    (1993)
  • J. Gourley

    Incidence and prognosis of canine mammary tumours

    J. Small Anim. Pract.

    (2000)
  • D.R. Green et al.

    Mitochondria and apoptosis

    Science

    (1998)
  • E. Hellmen

    Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

    In Vitro Cell. Dev. Biol.

    (1992)
  • E. Hellmen et al.

    Expression of different phenotypes in cell lines from canine mammary spindle-cell tumours and osteosarcomas indicating a pluripotent mammary stem cell origin

    Breast Cancer Res. Treat.

    (2000)
  • S. Krahling et al.

    Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages

    Cell Death Differ.

    (1999)
  • Cited by (7)

    View all citing articles on Scopus
    View full text