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Ether fosfolipid PNAE against the tumour cells:
Prevention and therapy of metastases
Extracts from the professional publication written by RNDr. Jindřich Kára DrSc. "Ether fosfolipid PNAE against the tumour cells: Prevention and therapy of metastases", published by AREKO in May 2000 on the occasion of the international oncological symposium held on 5 and 6 May 2000 in Prague - Pruhonice under the auspices of "Liga proti rakovine" Prague.
2.1. Ether-phospholipids with an antitumour effect inhibit the activity
of protein kinase C and induce a selective destruction of tumour cell membranes.
Antitumour effects of ether phospholipids have been described in numerous publications (21-35). Synthetic lysophosphatidylcholine (lysolecithine) analogue, 1-0-octadecyl-2-methoxy-rac-glycero-3-phosphocholine (ET-18-OCH3) has been studied the most often (21-28). This preparation selectively destructs tumour cells and its effect is manifested by these cell membranes destruction, as it has been proven by electron microscopy (24-26). ET-18- OCH3 preparation (Edelfosine) also increases the immunological protection of organism, activates the antitumour activity of macrophages, and has a significant effect on tumour metastases in experimental models (21-24, 48). Also, another synthetic lysolecithine analogue has similar antitumor effects, thioether 1-hxadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine, (BM 41.440, Ilmofosine) (28). Even another synthetic phosphatidylcholine analogue with antitumour effect has been prepared (27, 32). Clinical tests of these preparations, especially Edelfosine and Ilmofosine, have proven the therapeutic effect of these preparations (27, 28, 50) but also secondary toxic effects on organism limiting their clinical utilisation.
Discovery of a novel alkyl-phospholipid with a selective antitumor activity (29), 1-0-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl) ethanolamine (PNAE), which is a natural derivative of phosphatidylethanolamine and has no secondary toxic effects on organism during peroral and also parenteral administration (29-35), opens the new prospect of clinical utilisation of this nontoxic ether-phospholipid, especially in the prevention of human tumour metastases to various organs, mainly to liver (33, 35). In contrast to synthetic preparations (ET-18-OCH3 and BM 41.440), which represent a mixture of racemic stereoisomers, the PNAE (also PNAE(s)) preparation has a natural stereoconfiguration and does not contain any unnatural groupsin the structure of its molecule. Therefore, PNAE and its metabolites are not toxic for organism and thus it is remarkably beneficial for a long-term peroral administration and prevention of human tumour metastases (33, 35). Development and preparation of semisynthetic ether-phospholipid PNAE(s) have been described (30, 34, 35), antitumor activity of PNAE(s) in vitro and in vivo (30, 31), pharmacokinetics and metabolism in vivo (33) have been published (see further in part 5).
2.2. Iduction of apoptosis in cancer cells by ether-phospholipids
Apoptosis is the form of cell death characterised by morphological and biochemical changes such as the condensation of chromatin, the fragmentation of DNA caused by endonuclease activation (57, 58, 69). Stimuli, which increase the fluidity of cellular membranes of tumour cells (eg. hyperthermia or 5% ethanol) (70), or ether-phospholipid ET-18--OCH3 acting (57, 58), can induce an apoptotic death of a tumour cell. Ether-phospholipids induce apoptosis only in tumour cells but not in normal physiological cells (58). There are regulation genes in the cell, expression of which can block the apoptotic death (eg. mitochondrial protein Bcl-2) (58, 71, 72). Probably all antitumour ether-phospholipids induce apoptosis in cancer cells. M. Modolell et al. (58) on the basis of their experiments conclude that the induction of apoptosis by ether-phospholipid ET-18--OCH3 includes three main stages as follows:
- a) ether-phospholipid bond on the surface of cellular membrane;
- b) incorporation of ether-phospholipid to the cell;
- c) release of a signalling mechanism for apoptosis which can be stopped by an enhanced concentration of protein Bcl-2.
The matter of the fact are parameters which can be different in different types of cancer cells. This also explains the different sensitivity of cancer cells to the cytotoxic effect of ether-phospholipids.
5.1. Natural, nontoxic ether-phospholipid PNAE
plasmanyl-(N-acyl)-etanolamine, its chemical structure, antitumor activity
in vitro and in vivo and mechanism of its effect.
The novel phospholipid was identified as 1-0-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)- ethanolamine or pasmanyl-(N-acyl) ethanolamine (PNAE) (29).
The structure of the main molecular species of PNAE is 1-0-oktadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl) ethanolamine (29, 30) (Fig. 1).
Fig.1: 1-0-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine or plasmanyl-(N-acyl)-ethanolamine, PNAE (Kára et. al. 1986).
R-oktadecyl; R´-CO-oleoyl; R´´-CO-palmitoyl
The structure of PNAE suggested by us was also proven by prof. Dr. H. K. Mangold et al. (103) who prepared PNAE preparations with the use of synthetic alkyl-ethers of glycerol and biotechnology of plant cells cultures. By this specific semisynthesis they prepared derivatives with a different length of the 1-0-alkyl chain (1-0-tetradecyl, 1-0-hexadecyl and 1-0-(Z)-9'-octadecenyl) - PNAE which have a similar antitumour activity as our semisynthetic alkyl-phospholipid PNAE(s) (103). These results again prove the rightness of the chemical structure of PNAE (29, 30).
The antitumour activity of pure alkyl-phospholipid PNAE was tested in tissue cultures of human tumour cells (HEp-2 a T24 line) by the quantitative measurement of radioactive thymidine-3H incorporation into the cellular DNA at growing microgram concentrations of PNAE in 1 ml of the culture medium (29).
Alkyl-phospholipid PNAE has the selective cytolytic activity on human tumour cells of HEp-2 and T24 lines (human bladder carcinoma) in tissue cultures at the concentration of 50 µg of PNAE/ml and inhibits the proliferation of these tumour cells even at the concentration of 2 µg of PNAE/ml, but does not inhibit the biosynthesis of DNA in normal cells at 50 times higher concentration in the tissue culture of human fibroblasts (29, 30), (Fig. 2). This admirably selective activity of PNAE only against tumour cells indicates the great therapeutic index of PNAE and its nontoxicity for organism and also the growth of human rectum carcinoma cells resistant to fluorouracil in athymic nude mice was significantly inhibited at s. c. doses 4,5 mg of PNAE/mouse during 21 days (P. Poučková et al.) (100).
Similarly like synthetic alkyl-phospholipids, lysophosphatidyl choline analogue (24-26), alkyl-phospholipid PNAE causes selective destruction of cellular membranes of human tumour cells (30, 35). In cooperation with Dr. Z. Pelcbauer (Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague) and with the help of electron-scanning microscopy, the destructive effect of alkyl-phospholipid PNAE on cellular membranes of human tumour cells HEp-2 was proven in 24-28 hours of these cells incubation in tissue cultures at 50 µg of PNAE/ml (30, 35), (see Fig. 3, 4, 5).
Fig. 2: Selective tumoricidal effect of the alkyl-phospholipid PNAE on human tumor cells HEp-2 in culture, without a cytostatic effect on diploid human fibroblasts (LEP line) on the same conditions in a parallel experiment.
The HEp-2 cells and LEP cells were incubated in the cultural micro-plates in media containing the indicated concentration of alkyl-phospholipid PNAE during 48 hours. After the removal of media with PNAE, thymidine-6-3H into DNA of HEp-2 cells and LEP cells was measured. The proliferation of HEp-2 tumour cells was stopped at the concentration of PNAE 12,5 µg/ml, while normal LEP fibroblast growth was not stopped by alkyl-phospholipid PNAE even at the 10 times higher concentration (125 µg/ml).
1 - incorporation of thymidine -6-3H into DNA of HEp-2 tumour cells.
2 - incorporation of thymidine -6-3H into DNA of LEP fibroblasts.
The selective effect of alkyl-phospholipid PNAE on tumour cell membranes was also confirmed by Dr. A. Kotyk (Institute of Physiology, Czech Academy of Sciences, Prague) who, together with his colleagues, experimentally proved the enhanced permeability of cellular membranes of tumour cells exposed to the alkyl-phospholipid PNAE activity. Diffusion of 2-deoxy-D-glukosa labelled by radioisotope 3H through the membranes of these cells significantly increased in comparison with the control tumour cells which were not incubated in the presence of PNAE. The permeability of cellular membranes of normal physiological cells was on the same conditions of incubation with PNAE changed (101, 102).
An important role in the antitumour and antimetastatic effect of PNAE can also be played by the inhibitory effect of PNAE on the enzymatic activity of protein kinase C, which has been experimentally proven (39). With regard to the importance of protein kinase C for the metastatic capacity of tumour cells (11, 18, 19), a long-term in vivo activity of alkylphospholipid PNAE on protein kinase C of tumor cells can have a significant preventive effect against the dissemination of tumour cells in organism and against the genesis of metastases, especially in liver, where PNAE is the most cumulating during a repeated application (33), (see Tab. 1).
Fig. 3: Electron-scanning microphotograph of the human HEp-2 tumour cell in the tissue culture. Microvilli on the cell surface typical of a living cell.
Fig. 4: Electron-scanning microphotograph of the HEp-2 cell treated with PNAE (50µg/ml) for 24 hours. Holes in the cellular membrane may be seen.
Fig. 5: Electron-scanning microphotograph of the HEp-2 cell treated with PNAE (50µg/ml) for 48 hours in culture. The cellular membrane is significantly damaged, the cell is dead.
Tab. 1: Distribution of the radioactivity of 14C-PNAEs and 14C-labelled metabolites following repeated i.v. injections in Mc11-tumor-bearing BDF1 mice (Kára J. et al., 33).
* A daily intravenous injection of 0.9 mg of 14C-PNAEs (25,6x106 dpm) was administered to each of five BDF1 mice bearing Mc11 sarcoma. Radioactivity in animals' organs was determined 24 hours after the last injection. The highest accumulation of 14C-PNAEs and 14C-metabolites after repeated i.v. doses was found in mouse liver. Other metabolic details, pharmacokinetics and metabolism of 14C-PNAEs are stated in the publication of J. Kára et al. (33).
5.3. A novel technology of PNAE-containing Ovosan production.
Recently we have developed a novel technology of the production of Ovosan preparation containing a high percentage of ether-phospholipids PNAE. Ovosan preparation in the form of gelatine capsules contains suspension of egg phospholipids with 30% PNAE and 60% egg lecithin (phosphatidylcholine) in plant oil.
5.4. Possibility of liver metastases prevention in patients
after the operation of colorectal adenocarcinoma by a long-term peroral application of the preparation containing PNAE.
Our conception of a metastases prevention by a long-term application of nontoxic alkyl-phospholipid PNAE is following in the path of the strategy of metastases prevention by an American preparation caboxyamidotriazole (CAI) proposed by E.C. Kohn and L.A. Liotta (95). The advantage of PNAE and our Czech PNAE-containing preparation in comparison to CAI is a complete absence of side toxic effects which, by contrast, appear in patients during a long-term peroral application of the synthetic CAI preparation (94). A high selectivity of the tumour effect of PNAE is also the advantage of this natural ether-phospholipid.
Prevention of metastases can undoubtedly give a better prospective for the survival of the patients than any other therapy of metastases already existing in liver. It is better to prevent than to treat.
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