Original article
Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

https://doi.org/10.1016/j.biopha.2012.04.008Get rights and content

Abstract

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Pho-s has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma.

Introduction

Melanomas are malignant skin cancers and their incidence has been constantly increasing during the last decades affecting about 150,000 new patients per year in the world [1]. They are fast growing, multiresistance tumors with a high capacity for metastasis, which characterizes them as very aggressive cancers. Although early diagnosis and surgical resection of melanomas enable the cure in a high percentage of cases, melanoma metastasis still presents a very poor prognosis mainly because of its resistance to chemotherapic agents [2], [3]. Due to the low availability of chemotherapeutic agents to treat melanoma, dacarbazine, which has been used for 30 years now, remains the standard drug even with a response rate averaging only 15–20% [4]. Until recently, the treatment of patients with advanced melanoma was unsuccessful. However, the situation has begun to change in the last years. The careful molecular characterization of tumors has been extremely important in the discovery of new drugs against melanoma [5], [6]. On the other hand, this strategy is much unpredictable and expensive, and it is not always possible to find new effective and selective promising molecules [7]. This scenario encourages the development of more efficient and cheaper approaches in the search for new compounds for the treatment of cancer.

Presently, the synthetic alkyl-lysophospholipid analogs (ALPs) constitute a novel class of compounds, namely antineoplastic phospholipids (APs), with promising anticancer activity. This class includes clinically relevant drugs such as edelfosine, miltefosine and perifosine [8]. The most important characteristic of the action mechanism of APs is that they do not target DNA. They easily insert into the lipid bilayer, which might interfere with lipid metabolism and signal transduction, inducing apoptosis [9].

With the recent advent of APs to the treatment of malignant tumors, we investigated whether the central precursor in the biosynthesis of phospholipids has antitumor effects. The primary amine phosphoethanolamine (PEA), a lipid involved in phospholipid turnover, is a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine [10]. We previously reported that the synthetic phosphoethanolamine (Pho-s) is highly cytotoxic to a wide variety of tumor cells, including the human melanomas SK-MEL-28 and MEWO and the murine melanoma B16-F10, by inducing apoptosis [11].

The aims of the present study are to investigate the mechanisms of Pho-s mediated mitochondria-dependent apoptosis in tumor cells. In the present study, we provide experimental evidence to support the hypothesis that Pho-s has high antitumor activity against melanoma.

Section snippets

Chemicals and antibody

Synthetic Pho-s was synthesized and supplied in the Laboratory of Chemistry and Polymers Technology, University of Sao Paulo, Sao Carlos, Brazil. B16-F10 cells (number CRL-6475) were purchased from the American Type Culture Collection (ATCC, Baltimore, MD, USA). The antibody against Caspase-3 was obtained from Biotium Inc. (Hayward, CA, USA). Propidium iodide (PI), JC-1 (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), Methylthiazolyldiphenyl-tetrazolium bromide and

Phosphoethanolamine inhibits cell proliferation of B16-F10 cells

In previous report, we demonstrated cytotoxic effect of Pho-s on melanoma cells. In this work, we started by verifying the antiproliferative effects of Pho-s on B16-F10 cells at lower concentrations, after 96 h of treatment. We observed that Pho-s inhibits cell proliferation of B16-F10 cells in vitro in a dose-response manner. Moreover, we showed that it does not induce changes in the cell morphology (Fig. 1A). The reduction in cell proliferation by Pho-s was significant (***P < 0.001) at

Discussion

Metastatic melanomas are the most therapeutically challenging malignancies. Its disappointing treatment results evidence the urge for the development of better strategies and new compounds [12], [13]. In this regard, APs have pharmacological properties involving the inhibition of multiple pathways, leading to apoptosis, making these compounds attractive candidates for malignant tumor therapy [14], [15], [16].

In the present study, we investigated the antiproliferative and antitumor effects of

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgements

This work was supported by a Sao Paulo Research Foundation – (FAPESP) grant (2007/50571-3; Doctorate and Master Fellowship 2008/56089-1; 2010/50220-9).

References (21)

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