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Comparative anti-mitotic effects of lithium gamma-linolenate, gamma-linolenic acid and arachidonic acid, on transformed and embryonic cells
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291 © 1998 Harcouri Brace& Co. Ltd
C o m p a r a t i v e a n t i - m i t o t i c e f f e c t s of lithium g a m m a . l i n o l e n a t e , g a m m a linolenic acid and a r a c h i d o n i c acid, on t r a n s f o r m e d and e m b r y o n i c cells J. C. S e e g e r s , 1 M.-L. Lottering, 1 A. Panzer, 1 P. Bianchi, 2 J. H. S t a r k 2 ~Department of Physiology, University of Pretoda, Pretoria, South Africa 2Flow Cytometry Unit, Faculty of Life Sciences, University of the Witwatersrand, Johannesburg, South Africa
Summary The effects of gamma-linolenic acid (GLA), the lithium salt of gamma-linolenic acid (LiGLA) and arachidonic acid (AA) were compared at doses of 50 t~g/ml for periods of 6 and 24 h on cell cycle progression and apoptosis induction in transformed and in normal cells. In WILCO3 (oesophageal cancer) cells and on primary embryonic equine lung cells, we found LiGLA to be the most effective in apoptosis induction. After 24 h, 94% of the WHCO3 cancer cells and 44% of the primary embryonic equine lung cells exposed to LiGLA were apoptotic. The WILCO3 cancer cells were also very susceptible to the apoptosis-inducing effects of AA (56%) and GLA (44%), whereas the embryonic equine lung cells were much less affected by these two fatty acids. After 6 h exposure to all three compounds, most of the cycling WILCO3 cancer cells were blocked in S-phase. After 24 h treatment, some of the S-phase cells exposed to AA and GLA were apparently able to move into the G2/M phase, the LiGLA exposed cells were mostly apoptotic and no cycling cells were present. The primary embryonic equine lung cells were fairly resistant to the cytotoxic effects of GLA and AA. From our studies we conclude that, although LiGLA was the most toxic to the cancer cells, it is apparently less selective, compared to AA and GLA, in the killing of cancer and normal cells. It would also appear that the lithium might have added to the cytotoxic effects of LiGLA. The mechanism needs to be clarified. INTRODUCTION
We and others have shown that gamma-linolenic acid (GLA) and arachidonic acid (AA), a metabolite of GLA, inhibit cell growth and induce apoptosis in transformed cells in vitro. 1-4 Non-malignant cells are also affected, but only at high doses (100 gg/ml), and then to a lesser extent than the effects seen in transformed cells. 4 Although we found AA to be slightly more toxic than GLA on various cancer cells, including SV40 large T antigen transformed cells and two lymphoblastoid lines, 4 others reported that the GLA toxicity seen in neuroblastoma and colon carcinoma cells could not be attributed to the formation of metabolites, and that the effects were only due to GLA.5 Recently it was shown that the lithium salt of GLA Received 4 March 1998 Accepted 11 March 1998 Correspondence to: Prof. J. C. Seegers, Department of Physiology, PO Box 2034, Pretoria, 0001, South Africa. Tel.: +27 12 319 2147; Fax: +27 12 319 2238
caused longer survival times in patients with inoperable pancreatic cancers. 6 It was also shown that lithium gamma-linolenate (LiGLA) is taken up b y cancer cells in vitro and in vivo, and that it is effectively metabolized to AA.7 We are, therefore, also interested in the effects of LiGLA on transformed and normal cells in vitro. The incidence of oesophageal cancer is very high in South Africafi The purpose of this study was to compare the effects of LiGLA, GLA and AA on cell cycle progression and apoptosis induction in transformed oesophageal cells, which were obtained from a squamous cell carcinoma. Since the effects of LiGLA have not previously been investigated on normal cells, the effects of the three compounds on primary embryonic equine lung cells were also investigated. We have previously shown that apoptosis induced after exposure to GLA and AA is p53 independent, and that the p53 status of the cells to be investigated is not relevant. 4 The effects of the compounds on the cell cycle and mitosis, as well as apoptosis induction, were studied with flow cytometry. 285
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MATERIALS AND METHODS
Onderstepoort, Pretoria, South Africa). The WHCO3 cells were grown in Eagle's BME with Earle's salts (Gibco BRL, Grand Island, NY) supplemented with 10% heat inactivated foetal bovine serum (Delta Bioproducts, Kempton Park, Pretoria, South Africa). The equine lung ceils were grown in Eagle's MEM with Earle's salts (Gibco BRL) supplemented with 10% foetal bovine serum. For the flow cytometric analysis, all cells were seeded at a concentration of 2 x 103 cells into 25 cm 2 flasks, and maintained at 37°C in a humidified incubator with 5% CO2. Near confluent WHCO3 and equine lung cells were exposed to HGLA, GLA and AA at doses of 50 btg/ml. The fatty acids were dissolved in ethanol. The concentration of ethanol never exceeded 0.05%. In all the experiments, control cultures treated with 0.05% ethanol were included. The WHCO3 cells were exposed for periods of 6 and 24 h, and the equine lung cells for 24 h. The cells
Materials
GLA, AA, RNAse and the DNA stains, propidium iodide and Hoechst 33342 were purchased from Sigma Chemical Co. (St Louis, MO, USA). LiGLA was supplied by Scotia Pharmaceuticals (Randburg, South Africa). All other chemicals used in this study were supplied by Sigma or J. T. Baker Chemical Co. (Phillipsberg, NJ, USA) Flow cytometry
The oesophageal cancer cells, line WHCO3, were obtained from Prof. Alan Thornley (Zoology Department, University of the Witwatersrand, Johannesburg, South Africa) and the primary embryonic equine lung cells from Prof. P. G. Howell (Department of Tropical Diseases,
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Fig. 1 DNA histograms of WILCO3 cells after 6 h exposure: (a) control cells treated with 0.05% ethanol, showing the normal cell cycle phases, a prominent Go/G1 peak (> 200 DNA content) is present; (b) AA treatment at 50 pg/ml doses, a broad band and peak of apoptotic cells below the Go/G1 (< 200 DNA), but no G0/G1 or GJM (> 400 DNA) peaks are present; (c) GLA treatment at 50 pg/ml caused a similar effect as that seen in (b); (d) LiGLA treatment at 50 pg/ml caused a very prominent apoptotic peak below the Go/G1 area, again no Go/G~ or GJM cells are seen.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
were trypsinized in equal volumes of trypsin (0.25%, Gibco BRL) and EDTA (1 riM, Gibco BIG). Cell pellets were made of the cells and the pellets washed with PBS. The cell pellets were fixed in 100% methanol and stored at -20°C for at least 24 h before the cells were stained with 1 ml of a phosphate buffered saline solution, containing 18 mg PI and 40 mg RNAse, for at least 20 rain at room temperature. The samples were analysed with a Coulter Epic-XS Flow Cytometer. At least 10 000 events were registered for each determination. The data was analysed with Multicycle AV software. Hoechst 33342 staining
To verify apoptosis induction by LiGLA, TK6 lymphoMastoid cells were exposed to GLA and LiGLA at doses of 50 gg/ml for 24 h. The technique for Hoechst staining of TK6 cells, as well as the cultivation of these cells were previously described. 4 RESULTS Cell cycle analysis of WILCO3 cells after 6 h
DNA histograms showing the effects of 50 ~tg/ml doses of AA, GLA and LiGLA on cell cycle patterns are shown in Figure 1. Marked increases in the area below the G0/G1 peak were seen in the flow cytometric analysis of AA, GLA and LiGLA exposed cells. A prominent apoptotic peak was seen in the cell cycle analysis of cells exposed to LiGLA. The DNA histograms of cells exposed to AA and GLA
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showed a broad band of cells below the G0/G1 peak. DNA histograms after 24 h are not shown; only the multicycle analysis of the data is shown in Figures 4 and 5. Quantification of the effects of AA, GLA and LiGLA after 6h
All three compounds markedly increased apoptosis in the WHCO3 cells (Fig. 2). LiGLA had the most prominent effect. LiGLA caused all of the remaining cycling cells to be blocked in the S-phase (Fig. 2). In the GLA and AA exposed cells, a small fraction of the cycling cells was in the G0/G 1 phase, and the rest blocked in the S-phase (Fig. 2). None of the treated cells moved to the Ga/M-phase
(Fig. 2). Cell cycle analysis of equine lung cells after 24 h
Only after 24 h could changes be seen. in the normal equine lung cells treated with either AA or GLA. Therefore only DNA histograms obtained after 24 h are shown (Fig. 3). The cells were m u c h more susceptible to the apoptosis induction effects of LiGLA than to that of AA or GLA (Fig. 3). Quantification of the effects of AA, GLA and LiGLA on WHCO3 and equine lung cells after 24 h
The effects of AA, GLA and LiGLA on apoptosis induction after 24 h exposure of malignant WHCO3 (DNA
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Fig. 2 Multicycle analysis of the raw data shown in Figure 1. After 6 h treatment, the effects of ethanol (vehicle), AA, GLA and LiGLA on apoptosis development, and on the various cell cycle phases in the WILCO3 cells, are shown. In the AA and GLA exposed cells, only a few G0/G1 cells are present; all three compounds caused marked increases in the S-phase (100% of cycling LiGLA treated cells are blocked in S-phase), but no GJM cells were present.
© 1998 Harcourt Brace & Co. Ltd
Prostaglandins, Leukotrienes and Essential FattyAcids (1998) 59(4), 285-291
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Fig. 3 DNA histograms of embryonic equine lung cells after 24 h exposure to 50 gg/ml of AA, GLA, and LiGLA: (a) control cells showing a small number of cells below the Go/G1 peak, a prominent Go/G1 peak, as well as a small peak of GJM cells; (b) and (c) after AA (b) and GLA (c) treatment similar patterns in the DNA histograms were seen, an increase above that of the control in apoptotic cells was present; (d) LiGLA treatment caused a prominent apoptotic peak and a smaller Go/G1 peak.
histograms not shown) and normal equine lung cells are compared in Figure 4. The most marked effect was seen after treatment with LiGLA in both cell types. The effect on the WHCO3 cells was much more pronounced than that seen in the normal cells (Fig. 4). More than 90% of the WHCO3 cells were apoptotic compared to only 43.8% of the equine lung cells (Fig. 4). AA and GLA also affected the malignant cells more than the normal cells (Fig. 4). The overall effects of AA and GILAon both cell types were much smaller than that of LiGLA (Fig. 4). The effects of AA, GLA and LiGLA on the remaining cycling equine lung cells after 24 h exposure are shown in Figure 5. No prominent block was seen in any of the cell cycle phases. A small increase in the G0/Gl-phase and a decrease in S-phase were present. The effects of AA and GLA on the remaining cycling cells are shown in Figure 6. Since more than 90% of the
LiGLA-exposed WCHO3 were apoptotic, too few cycling cells remained to be analysed. No effect of AA or GLA on any of the cell cycle phases was evident (Fig. 6). It would appear that cells exposed to these two fatty acids were able to move out of the S-phase block, which occurred after 6 h exposure (Fig. 2). It would also appear that the WHCO3 cells blocked in S-phase after 6 h exposure to LiGLA (Figs 1 and 2) eventually became apoptotic (Fig. 4). Morphological evidence of LiGLA-induced apoptosis
In a previous study4 we confirmed apoptosis induction observed with flow cytometry in lymphoblastoid TK6 cells exposed to AA and GLA, with Hoechst 33342 staining. In this study, the effects of GLA and LiGLA are compared in TK6 cells. Most of the TK6 cells exposed to 50 ~tg/ml LiGLA are apoptotic (Fig. 7C). This morphologic
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
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study using transformed lymphoblastoid cells also showed increased apoptosis development after treatment with LiGLA, compared with the effects seen after GLA treatment (Fig. 7b). This morphological study confirms that GLA causes marked apoptosis in transformed cells. DISCUSSION
The experiments described above again showed similar effects of AA and GLA on transformed cells (Figs 1, 2, 4 © 1998 Harcourt Brace & Co, Ltd
and 6). Both compounds inhibited mitosis and induced apoptosis in the oesophageal cancer cells, indicating that the two compounds probably activated similar cellular processes involved in cell growth and cell death. LiGLA was much m o r e to toxic to the oesophageal cells than the other two fatty acids (Figs 1, 2, 4 and 6). It would appear that the lithium either contributed to the cytotoxic effects, or that it enhanced the uptake of GLA into the cells. It was also interesting that LiGLA caused marked effects in the normal embryonic equine lung cells
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
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Fig. 7 Control TK6 cells are shown in (a). TK6 cells exposed to 50 #g/ml doses of GLA (b) and LiGLA (c) for 24 h show morphological lesions typical of apoptosis (arrows) confirming that LiGLA causes apoptosis (x400).
compared to AA and GLA (Fig. 4), which also indicates that the lithium per se m a y be toxic to the embryonic ceils. Because lithium enhanced the cytotoxic effects in both transformed and normal embryonic cells, the more pronounced selective toxicity for cancer ceils seen in AAand GLA-treated cells is diminished in LiGLA treatment. Further studies in which the effects of lithium alone on these cells should be determined are indicated. It is
known that lithium affects the phosphatidylinositol signal tranduction system. Lithium is an inhibitor of endogenous inositol recycling. 9 M t h o u g h it was reported that lithium stimulated cell proliferation in MCF-7 breast cancer cells, 1°,~1 cell proliferation was inhibited in mouse embryonic stem cells# The cytotoxic effects on the stem cells was not alleviated by the addition of exogenous inositol.gWelshons et al.lo reported that the enhanced cell
Prostaglandins! Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
p r o l i f e r a t i o n s e e n i n t h e MCF-7 cells was l i m i t e d b e c a u s e of t h e c y t o t o x i c effects of l i t h i u m . Doses i n t h e r a n g e of 0 . 6 - 1 . 2 m_M are r e g a r d e d as t h e r a p e u t i c a n d t h o s e i n t h e r a n g e of 1.5-5 m M a c t e d as m f f o g e n s i n MCF-7 cells; doses a b o v e 5 m M are toxic a n d are a s s o c i a t e d w i t h cell-kill. I° I n o u r studies, t h e l i t h i u m c o n c e n t r a t i o n n e v e r e x c e e d e d 0.186 raM. At t h e s e v e r y l o w levels, e m b r y o n i c e q u i n e l u n g cells were v e r y s u s c e p t i b l e to t h e c y t o t o x i c effects of l i f l l i u m g a m m a - l i n o l e n a t e (Figs 3, 4-5). It is u n c e r t a i n if t h e c y t o t o x i c effects are r e l a t e d to t h e i n h i b i t i o n of i n o s i t o l m e t a b o l i s m . It is also n o t clear if GLA a n d Li a c t e d s y n e r g i s t i c a l l y or i n d e p e n d e n t l y i n t h e a p o p t o s i s - i n d u c i n g effects. It is n o w k n o w n t h a t t h e r e are s e v e r n a p o p t o s i s - i n d u c i n g m e c h a n i s m s a v a i l a b l e i n cells. M o r e s t u d i e s are n e c e s s a r y to f u r t h e r e v a l u a t e t h e c y t o t o x i c effects of GLA, LiGLA a n d t h e v a r i o u s m e t a b o l i t e s of GLA o n cell g r o w t h a n d cell d e a t h i n v a r i o u s t y p e s of c a n c e r cells, i n o r d e r to clarify t h e different r e s p o n s e s r e p o r t e d i n t h e literature. 4'1z13
REFERENCES 1. Begin M. E., Ells G., Das U. N., Horrobin D. F. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986; 77: 1053-1062. 2. De Kock M., Lottering M.-L., Seegers J. C. Differential cytotoxic effects of gamma-linolelliC acid on MG-63 and HeLa cells. Prost~glandins Leukot Essent FatO¢Acids 1994; 51: 109-120. 3. De Kock M., Lottering M.-L., Grobler C. J. S., Viljoen T. C., Le Roux M., Seegers J. C. The induction of apoptosis in human cervical carcinoma (HeLa) cells by gamma-linolenic acid. Prostaglandins Leukat Essent Fatty Acids 1996; 55:403-411. 4. Seegers J. C., De Kock M., Lottering M.-L., Grobler C. J. S., Van
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Papendorp D. H., Shou Y., Habbersett R., Lehnert B. E. Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells. Prostaglandins Leukot Essent Fatty Acids 1997; 56: 271-280. 5. Hrelia S., BordonJ A., Biagi P., Rossi C..4., Bernarcli L., Horrobin D. F., Pession A. Gamma-linolenic acid supplementation can affect cancer cell proliferation via modification of fatty acid composition. Biochem Biophys Res Commun 1996; 225: 441-447.
6. Fearon K. C. H., Falconer J. S., Ross J. A., Carter D. C., Hunter J. O., Reynolds P. D., Tnffnell Q. An open-label phase I/II dose escalation study of the treatment of pancreatic cancer using lithium gamma-linolenate. Anticancer Res 1996; 16: 867-874. 7. De Antueno R., Elliot M., Ells G., Quiroga P., Jenkins K., Horrobin D. F. In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas. Bry Cancer 1997; 75: 1812-1818. 8. Van Rensburg S. J. Nutritional factors in human carcinogenesis. SA Cancer Bulletin; 26:153-159. 9. Duffy C., Kane M. T. Investigation of the role of inositol and phosphatidyl-inositol signal transduction system in mouse embryonic cells, f Reprod and FerEli~y 1966; 108: 87-93. 10. Welshons W. V., Engler K. S., Taylor J. A., Grady L. H., Curran E. M. Lffhium-stimulated proliferation and alteration of phosphoinositide metabolites in MCE-7 human breast cancer cells. J Cell Physio11995; 165: 134-144. 11. Taylor J. A., Grady L. H., Engler K. S., Welshons W. V. Relationship of growth stimulated by lithium, estradiol, and EGF to phospholipase C activity ill MCF-7 human breast cancer cells. Breast Caneer Res Treat 1995; 34: 265-277. 12. Grfffiths G., Jones H. E., Eaton C. L., Stobart A. K. Effect of n-6 polyunsaturated fatty acids on growth and lipid composition of neoplastic and non-neoplastic canine prostate epithelial cell cultures. Prostate 1997; 31: 29-36. 13. Sravan Kumar G., Das U. N. Cytotoxic action of alpha-linolenic and eicosapentanoic acids on myeloma cells in vitro. Prostaglandins Leukot Essent Fatty Acids 1997; 56: 285-293.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
C o m p a r a t i v e a n t i - m i t o t i c e f f e c t s of lithium g a m m a . l i n o l e n a t e , g a m m a linolenic acid and a r a c h i d o n i c acid, on t r a n s f o r m e d and e m b r y o n i c cells J. C. S e e g e r s , 1 M.-L. Lottering, 1 A. Panzer, 1 P. Bianchi, 2 J. H. S t a r k 2 ~Department of Physiology, University of Pretoda, Pretoria, South Africa 2Flow Cytometry Unit, Faculty of Life Sciences, University of the Witwatersrand, Johannesburg, South Africa
Summary The effects of gamma-linolenic acid (GLA), the lithium salt of gamma-linolenic acid (LiGLA) and arachidonic acid (AA) were compared at doses of 50 t~g/ml for periods of 6 and 24 h on cell cycle progression and apoptosis induction in transformed and in normal cells. In WILCO3 (oesophageal cancer) cells and on primary embryonic equine lung cells, we found LiGLA to be the most effective in apoptosis induction. After 24 h, 94% of the WHCO3 cancer cells and 44% of the primary embryonic equine lung cells exposed to LiGLA were apoptotic. The WILCO3 cancer cells were also very susceptible to the apoptosis-inducing effects of AA (56%) and GLA (44%), whereas the embryonic equine lung cells were much less affected by these two fatty acids. After 6 h exposure to all three compounds, most of the cycling WILCO3 cancer cells were blocked in S-phase. After 24 h treatment, some of the S-phase cells exposed to AA and GLA were apparently able to move into the G2/M phase, the LiGLA exposed cells were mostly apoptotic and no cycling cells were present. The primary embryonic equine lung cells were fairly resistant to the cytotoxic effects of GLA and AA. From our studies we conclude that, although LiGLA was the most toxic to the cancer cells, it is apparently less selective, compared to AA and GLA, in the killing of cancer and normal cells. It would also appear that the lithium might have added to the cytotoxic effects of LiGLA. The mechanism needs to be clarified. INTRODUCTION
We and others have shown that gamma-linolenic acid (GLA) and arachidonic acid (AA), a metabolite of GLA, inhibit cell growth and induce apoptosis in transformed cells in vitro. 1-4 Non-malignant cells are also affected, but only at high doses (100 gg/ml), and then to a lesser extent than the effects seen in transformed cells. 4 Although we found AA to be slightly more toxic than GLA on various cancer cells, including SV40 large T antigen transformed cells and two lymphoblastoid lines, 4 others reported that the GLA toxicity seen in neuroblastoma and colon carcinoma cells could not be attributed to the formation of metabolites, and that the effects were only due to GLA.5 Recently it was shown that the lithium salt of GLA Received 4 March 1998 Accepted 11 March 1998 Correspondence to: Prof. J. C. Seegers, Department of Physiology, PO Box 2034, Pretoria, 0001, South Africa. Tel.: +27 12 319 2147; Fax: +27 12 319 2238
caused longer survival times in patients with inoperable pancreatic cancers. 6 It was also shown that lithium gamma-linolenate (LiGLA) is taken up b y cancer cells in vitro and in vivo, and that it is effectively metabolized to AA.7 We are, therefore, also interested in the effects of LiGLA on transformed and normal cells in vitro. The incidence of oesophageal cancer is very high in South Africafi The purpose of this study was to compare the effects of LiGLA, GLA and AA on cell cycle progression and apoptosis induction in transformed oesophageal cells, which were obtained from a squamous cell carcinoma. Since the effects of LiGLA have not previously been investigated on normal cells, the effects of the three compounds on primary embryonic equine lung cells were also investigated. We have previously shown that apoptosis induced after exposure to GLA and AA is p53 independent, and that the p53 status of the cells to be investigated is not relevant. 4 The effects of the compounds on the cell cycle and mitosis, as well as apoptosis induction, were studied with flow cytometry. 285
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MATERIALS AND METHODS
Onderstepoort, Pretoria, South Africa). The WHCO3 cells were grown in Eagle's BME with Earle's salts (Gibco BRL, Grand Island, NY) supplemented with 10% heat inactivated foetal bovine serum (Delta Bioproducts, Kempton Park, Pretoria, South Africa). The equine lung ceils were grown in Eagle's MEM with Earle's salts (Gibco BRL) supplemented with 10% foetal bovine serum. For the flow cytometric analysis, all cells were seeded at a concentration of 2 x 103 cells into 25 cm 2 flasks, and maintained at 37°C in a humidified incubator with 5% CO2. Near confluent WHCO3 and equine lung cells were exposed to HGLA, GLA and AA at doses of 50 btg/ml. The fatty acids were dissolved in ethanol. The concentration of ethanol never exceeded 0.05%. In all the experiments, control cultures treated with 0.05% ethanol were included. The WHCO3 cells were exposed for periods of 6 and 24 h, and the equine lung cells for 24 h. The cells
Materials
GLA, AA, RNAse and the DNA stains, propidium iodide and Hoechst 33342 were purchased from Sigma Chemical Co. (St Louis, MO, USA). LiGLA was supplied by Scotia Pharmaceuticals (Randburg, South Africa). All other chemicals used in this study were supplied by Sigma or J. T. Baker Chemical Co. (Phillipsberg, NJ, USA) Flow cytometry
The oesophageal cancer cells, line WHCO3, were obtained from Prof. Alan Thornley (Zoology Department, University of the Witwatersrand, Johannesburg, South Africa) and the primary embryonic equine lung cells from Prof. P. G. Howell (Department of Tropical Diseases,
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Fig. 1 DNA histograms of WILCO3 cells after 6 h exposure: (a) control cells treated with 0.05% ethanol, showing the normal cell cycle phases, a prominent Go/G1 peak (> 200 DNA content) is present; (b) AA treatment at 50 pg/ml doses, a broad band and peak of apoptotic cells below the Go/G1 (< 200 DNA), but no G0/G1 or GJM (> 400 DNA) peaks are present; (c) GLA treatment at 50 pg/ml caused a similar effect as that seen in (b); (d) LiGLA treatment at 50 pg/ml caused a very prominent apoptotic peak below the Go/G1 area, again no Go/G~ or GJM cells are seen.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
were trypsinized in equal volumes of trypsin (0.25%, Gibco BRL) and EDTA (1 riM, Gibco BIG). Cell pellets were made of the cells and the pellets washed with PBS. The cell pellets were fixed in 100% methanol and stored at -20°C for at least 24 h before the cells were stained with 1 ml of a phosphate buffered saline solution, containing 18 mg PI and 40 mg RNAse, for at least 20 rain at room temperature. The samples were analysed with a Coulter Epic-XS Flow Cytometer. At least 10 000 events were registered for each determination. The data was analysed with Multicycle AV software. Hoechst 33342 staining
To verify apoptosis induction by LiGLA, TK6 lymphoMastoid cells were exposed to GLA and LiGLA at doses of 50 gg/ml for 24 h. The technique for Hoechst staining of TK6 cells, as well as the cultivation of these cells were previously described. 4 RESULTS Cell cycle analysis of WILCO3 cells after 6 h
DNA histograms showing the effects of 50 ~tg/ml doses of AA, GLA and LiGLA on cell cycle patterns are shown in Figure 1. Marked increases in the area below the G0/G1 peak were seen in the flow cytometric analysis of AA, GLA and LiGLA exposed cells. A prominent apoptotic peak was seen in the cell cycle analysis of cells exposed to LiGLA. The DNA histograms of cells exposed to AA and GLA
287
showed a broad band of cells below the G0/G1 peak. DNA histograms after 24 h are not shown; only the multicycle analysis of the data is shown in Figures 4 and 5. Quantification of the effects of AA, GLA and LiGLA after 6h
All three compounds markedly increased apoptosis in the WHCO3 cells (Fig. 2). LiGLA had the most prominent effect. LiGLA caused all of the remaining cycling cells to be blocked in the S-phase (Fig. 2). In the GLA and AA exposed cells, a small fraction of the cycling cells was in the G0/G 1 phase, and the rest blocked in the S-phase (Fig. 2). None of the treated cells moved to the Ga/M-phase
(Fig. 2). Cell cycle analysis of equine lung cells after 24 h
Only after 24 h could changes be seen. in the normal equine lung cells treated with either AA or GLA. Therefore only DNA histograms obtained after 24 h are shown (Fig. 3). The cells were m u c h more susceptible to the apoptosis induction effects of LiGLA than to that of AA or GLA (Fig. 3). Quantification of the effects of AA, GLA and LiGLA on WHCO3 and equine lung cells after 24 h
The effects of AA, GLA and LiGLA on apoptosis induction after 24 h exposure of malignant WHCO3 (DNA
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© 1998 Harcourt Brace & Co. Ltd
Prostaglandins, Leukotrienes and Essential FattyAcids (1998) 59(4), 285-291
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Fig. 3 DNA histograms of embryonic equine lung cells after 24 h exposure to 50 gg/ml of AA, GLA, and LiGLA: (a) control cells showing a small number of cells below the Go/G1 peak, a prominent Go/G1 peak, as well as a small peak of GJM cells; (b) and (c) after AA (b) and GLA (c) treatment similar patterns in the DNA histograms were seen, an increase above that of the control in apoptotic cells was present; (d) LiGLA treatment caused a prominent apoptotic peak and a smaller Go/G1 peak.
histograms not shown) and normal equine lung cells are compared in Figure 4. The most marked effect was seen after treatment with LiGLA in both cell types. The effect on the WHCO3 cells was much more pronounced than that seen in the normal cells (Fig. 4). More than 90% of the WHCO3 cells were apoptotic compared to only 43.8% of the equine lung cells (Fig. 4). AA and GLA also affected the malignant cells more than the normal cells (Fig. 4). The overall effects of AA and GILAon both cell types were much smaller than that of LiGLA (Fig. 4). The effects of AA, GLA and LiGLA on the remaining cycling equine lung cells after 24 h exposure are shown in Figure 5. No prominent block was seen in any of the cell cycle phases. A small increase in the G0/Gl-phase and a decrease in S-phase were present. The effects of AA and GLA on the remaining cycling cells are shown in Figure 6. Since more than 90% of the
LiGLA-exposed WCHO3 were apoptotic, too few cycling cells remained to be analysed. No effect of AA or GLA on any of the cell cycle phases was evident (Fig. 6). It would appear that cells exposed to these two fatty acids were able to move out of the S-phase block, which occurred after 6 h exposure (Fig. 2). It would also appear that the WHCO3 cells blocked in S-phase after 6 h exposure to LiGLA (Figs 1 and 2) eventually became apoptotic (Fig. 4). Morphological evidence of LiGLA-induced apoptosis
In a previous study4 we confirmed apoptosis induction observed with flow cytometry in lymphoblastoid TK6 cells exposed to AA and GLA, with Hoechst 33342 staining. In this study, the effects of GLA and LiGLA are compared in TK6 cells. Most of the TK6 cells exposed to 50 ~tg/ml LiGLA are apoptotic (Fig. 7C). This morphologic
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
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study using transformed lymphoblastoid cells also showed increased apoptosis development after treatment with LiGLA, compared with the effects seen after GLA treatment (Fig. 7b). This morphological study confirms that GLA causes marked apoptosis in transformed cells. DISCUSSION
The experiments described above again showed similar effects of AA and GLA on transformed cells (Figs 1, 2, 4 © 1998 Harcourt Brace & Co, Ltd
and 6). Both compounds inhibited mitosis and induced apoptosis in the oesophageal cancer cells, indicating that the two compounds probably activated similar cellular processes involved in cell growth and cell death. LiGLA was much m o r e to toxic to the oesophageal cells than the other two fatty acids (Figs 1, 2, 4 and 6). It would appear that the lithium either contributed to the cytotoxic effects, or that it enhanced the uptake of GLA into the cells. It was also interesting that LiGLA caused marked effects in the normal embryonic equine lung cells
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
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Fig. 7 Control TK6 cells are shown in (a). TK6 cells exposed to 50 #g/ml doses of GLA (b) and LiGLA (c) for 24 h show morphological lesions typical of apoptosis (arrows) confirming that LiGLA causes apoptosis (x400).
compared to AA and GLA (Fig. 4), which also indicates that the lithium per se m a y be toxic to the embryonic ceils. Because lithium enhanced the cytotoxic effects in both transformed and normal embryonic cells, the more pronounced selective toxicity for cancer ceils seen in AAand GLA-treated cells is diminished in LiGLA treatment. Further studies in which the effects of lithium alone on these cells should be determined are indicated. It is
known that lithium affects the phosphatidylinositol signal tranduction system. Lithium is an inhibitor of endogenous inositol recycling. 9 M t h o u g h it was reported that lithium stimulated cell proliferation in MCF-7 breast cancer cells, 1°,~1 cell proliferation was inhibited in mouse embryonic stem cells# The cytotoxic effects on the stem cells was not alleviated by the addition of exogenous inositol.gWelshons et al.lo reported that the enhanced cell
Prostaglandins! Leukotrienes and Essential Fatty Acids (1998) 59(4), 285-291
© 1998 Harcourt Brace & Co. Ltd
Anti-mitotic effects on transformed and embryonic cells
p r o l i f e r a t i o n s e e n i n t h e MCF-7 cells was l i m i t e d b e c a u s e of t h e c y t o t o x i c effects of l i t h i u m . Doses i n t h e r a n g e of 0 . 6 - 1 . 2 m_M are r e g a r d e d as t h e r a p e u t i c a n d t h o s e i n t h e r a n g e of 1.5-5 m M a c t e d as m f f o g e n s i n MCF-7 cells; doses a b o v e 5 m M are toxic a n d are a s s o c i a t e d w i t h cell-kill. I° I n o u r studies, t h e l i t h i u m c o n c e n t r a t i o n n e v e r e x c e e d e d 0.186 raM. At t h e s e v e r y l o w levels, e m b r y o n i c e q u i n e l u n g cells were v e r y s u s c e p t i b l e to t h e c y t o t o x i c effects of l i f l l i u m g a m m a - l i n o l e n a t e (Figs 3, 4-5). It is u n c e r t a i n if t h e c y t o t o x i c effects are r e l a t e d to t h e i n h i b i t i o n of i n o s i t o l m e t a b o l i s m . It is also n o t clear if GLA a n d Li a c t e d s y n e r g i s t i c a l l y or i n d e p e n d e n t l y i n t h e a p o p t o s i s - i n d u c i n g effects. It is n o w k n o w n t h a t t h e r e are s e v e r n a p o p t o s i s - i n d u c i n g m e c h a n i s m s a v a i l a b l e i n cells. M o r e s t u d i e s are n e c e s s a r y to f u r t h e r e v a l u a t e t h e c y t o t o x i c effects of GLA, LiGLA a n d t h e v a r i o u s m e t a b o l i t e s of GLA o n cell g r o w t h a n d cell d e a t h i n v a r i o u s t y p e s of c a n c e r cells, i n o r d e r to clarify t h e different r e s p o n s e s r e p o r t e d i n t h e literature. 4'1z13
REFERENCES 1. Begin M. E., Ells G., Das U. N., Horrobin D. F. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986; 77: 1053-1062. 2. De Kock M., Lottering M.-L., Seegers J. C. Differential cytotoxic effects of gamma-linolelliC acid on MG-63 and HeLa cells. Prost~glandins Leukot Essent FatO¢Acids 1994; 51: 109-120. 3. De Kock M., Lottering M.-L., Grobler C. J. S., Viljoen T. C., Le Roux M., Seegers J. C. The induction of apoptosis in human cervical carcinoma (HeLa) cells by gamma-linolenic acid. Prostaglandins Leukat Essent Fatty Acids 1996; 55:403-411. 4. Seegers J. C., De Kock M., Lottering M.-L., Grobler C. J. S., Van
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Papendorp D. H., Shou Y., Habbersett R., Lehnert B. E. Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells. Prostaglandins Leukot Essent Fatty Acids 1997; 56: 271-280. 5. Hrelia S., BordonJ A., Biagi P., Rossi C..4., Bernarcli L., Horrobin D. F., Pession A. Gamma-linolenic acid supplementation can affect cancer cell proliferation via modification of fatty acid composition. Biochem Biophys Res Commun 1996; 225: 441-447.
6. Fearon K. C. H., Falconer J. S., Ross J. A., Carter D. C., Hunter J. O., Reynolds P. D., Tnffnell Q. An open-label phase I/II dose escalation study of the treatment of pancreatic cancer using lithium gamma-linolenate. Anticancer Res 1996; 16: 867-874. 7. De Antueno R., Elliot M., Ells G., Quiroga P., Jenkins K., Horrobin D. F. In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas. Bry Cancer 1997; 75: 1812-1818. 8. Van Rensburg S. J. Nutritional factors in human carcinogenesis. SA Cancer Bulletin; 26:153-159. 9. Duffy C., Kane M. T. Investigation of the role of inositol and phosphatidyl-inositol signal transduction system in mouse embryonic cells, f Reprod and FerEli~y 1966; 108: 87-93. 10. Welshons W. V., Engler K. S., Taylor J. A., Grady L. H., Curran E. M. Lffhium-stimulated proliferation and alteration of phosphoinositide metabolites in MCE-7 human breast cancer cells. J Cell Physio11995; 165: 134-144. 11. Taylor J. A., Grady L. H., Engler K. S., Welshons W. V. Relationship of growth stimulated by lithium, estradiol, and EGF to phospholipase C activity ill MCF-7 human breast cancer cells. Breast Caneer Res Treat 1995; 34: 265-277. 12. Grfffiths G., Jones H. E., Eaton C. L., Stobart A. K. Effect of n-6 polyunsaturated fatty acids on growth and lipid composition of neoplastic and non-neoplastic canine prostate epithelial cell cultures. Prostate 1997; 31: 29-36. 13. Sravan Kumar G., Das U. N. Cytotoxic action of alpha-linolenic and eicosapentanoic acids on myeloma cells in vitro. Prostaglandins Leukot Essent Fatty Acids 1997; 56: 285-293.
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