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Cervix: II linoleic acid conversion during cervical carcinogenesis
Poster Presentations Wednesday 23 July
EFA & Eicosanoids 1997
-
Edinburgh
249
P145
P146
Apoptosis induction, angiogenesis and growth inhibition of Colon-26 adenocarcinomas with local delivery of diclofenac in hyaluronan.
Cervix: I Comparison of total fatty acid compositions in intraepithelial and infiltrating lesions of the cervix
Brown J, Freemanfle C, Colville-Nash P, Somerville K, Seed M, Willoughby D. Experimental Pathology, William Harvey Research Institute, London EC1M 6BQ UK.
L Louw and A-M Engelbrecht, Department of Anatomy & Cell Morphology, University of the OFS, Bloemfontein, 9300, South Africa.
Aim: NSAIDs reduce clinical colonic adenocarcinoma growth and topical diclofenac (Dc) in 2.5% hyaluronan (HA) inhibits basal cell carcinoma, and actinic keratosis. This could be due to effects on tumour growth, angiogenesis or apoptosis. Methods:Colon-26 cells viability (MTT), growth (thymidine), and apoptosis (acridine orange/ethidium bromide) was studied with and without diclofenac in vitro. In vivo sub-cutaneous tumour growth, angiogenesis (carmin/gelatin vascular casts), proliferation (mitotic figures) and apoptotic (Apoptag) indices were assessed. Results: Dc reduced proliferation and viability in vitro at 104 and 3x103M respectively, and stimulated apoptosis. In vivo, colon-26 tumour growth was preceded by angiogenesis and reduced apoptotic (AI, from 0.32_+0.05 day 7, to 0.04_+0.02% day 14 *** and mitotic indices (MI, from 5.59_+0.40 day 7 to 3=49+0.35% day 14). Topical De/HA inhibited tumour prostaglandin synthesis, b) retarded tumour a) to growth ('[/C ratio 0.174; fig a, topical 120 T aqueous cream 8 control O, topical E Dc/HA []) and "~ 6 g 8o angiogenesis (fig b). MI remained unaltered in vivo by Dc, whilst AI was 40 increased from 0.05 2 + 0.01 to 0.15 _+ 0.01% *~* atday 14 0 I , i i ] ~ ~ and from 0.05 + 0.02 2 6 10 14 0 2 6 tO 14 to 0.32 + 0.04% *~* at day 20 vs. HA Days alone. Tumour Days necrosis was also increased. Topical HA had slight antitumour and anti-angiogenesis activity. Conclusion: Dc inhibits colon-26 proliferation and viability, whilst inducing apoptosis in vitro. Anti-tumour activity in vivo is accompanied by angiogenesis inhibition and apoptosis induction. The substantial quantities of Dc delivered locally in HA may exhibit antitumour activity in similar fashion to those seen in vitro and explain its clinical efficacy.
In this study, the possible role of fatty acids and their metabolites during cervical carcinogenesis, was investigated. Since membrane lipids play a key role in cell proliferation and differentiation, disturbances in the fatty acid compositions of cell membranes and the modulation of membrane fatty acid compositions received attention in several in v i t r o studies. There are, however no reported studies where the actual total fatty acid compositions bare been determined during the different stages of cervical carcinogenesis. I n t h i s e x v i v o study, the fatty acid compositions of normal tissue, intraepithelial neoplasia and infiltrating lesions of the cervix were compared. The fatty acid profiles that were determined, make it possible to speculate about the metabolic pathways and to correlate these with possible therapeutical implications. Lipids were extracted from biopsies of normal tissue (n=36), cervical intraepithelial neoplasia (CIN) (n=47), and infiltrating lesions (n=74). Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by GLC. Essential fatty acid deficiency (EFAD) in the intraepithelial lesions, compared with normal tissue (linoleic acid, p < 0.01), and in infiltrating lesions, compared with intraepitbelial lesions (linoleic acid and arachidonie acid, p < 0.01) were observed. High levels of oleic acid were also observed when infiltrating lesions were compared with normal tissue (p < 0.01). There are many factors which may contribute to EFAD in cancer cells, but the hltman papilloma virus, prolonged cytokine-mediated reactions, free radical reactions and lipid peroxidation must be considered major role players in cervical carcinogenesis. From a therapeutic viewpoint, substantial changes in the fatty acid composition of the membranes can be produced in cancer cells by selective fatty acid supplementation strategies. At present, modifications of the fatty acid compositions of cell membranes represent an experimental model that has promoted increased understanding of lipid transportation, membrane remodelling, and the relationship between membrane lipids and membrane function. The clinical feasibility of using modification of fatty acids in cancer by diet or perfusion as an adjunct to standard therapies, should be tested.
P147
P148
Cervix: H Linoleie acid conversion during cervical carcinogenesis.
Cervix II1: Free fatty acid profiles in cervical carcinogenesis.
Louw L, Engelbrecht A-M. Department of Anatomy and Cell Morphology, Faculty of Medicine, University of the Free State, Bloemfontein, 9300, South Africa.
L o u w L, Engelbrecht A-M. Anatomy and Cell Morphology, Faculty of Medicine, University of the Free State, Bloemfontein, 9300, South Africa.
It is well established that there are significant alterations in the metabolic pathways during carcinogenesis. This study indicates and discusses the possibilities of linoleic acid conversion during stages of cervix cancer. Patient biopsies of normal tissue, cervical intraepithelial neoplasia and infiltrating lesions of the cervix (n=20 of each) were obtained. Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by GLC. A progressive decline in the relative percentages of linoleic acid (18: 2n-6; LA) could be observed in squamous epithelial cells of the cervix, during their transformation from normal tissue (N) to intraepithelial neoplasia (CIN) and infiltrating lesions (Ca) of the cervix. LA was significantly lower in Ca, compared with normal tissue and CIN (p < 0.01) in both cases; 95% CI for median of differences [- 9.28%; -4.55%] and [-5.87%; -2.28%]. Also, arachidonic acid (20: 4n-6; ARA) was significantly lower in Ca, compared with CIN (p < 0.01: 95% CI for median of differences - 5.02%; -1.47%). The significant decrease in LA is in accordance with a tendency observed by us amongst tumoar entities progressing from a benign toward a malignant state, and even between severe and less severe malignancies. The decrease in linoleic acid levels may be indicative of increased metabolism, since increased levels of DGLA, the precursor of the l-series prostaglandins, occurred in cancer cells of the cervix. The high concentrations of DGLA and C20:2n-6 in cervical cancer cells may indicate that delta-8-desaturase is very active in cancer cells. Alternative pathways (e.g. elongation and delta-8-desaturation) may enable the desuturation of LA when delta-6-desuturase is not active. The conversion of LA to I3-HOD in cervical carcinogenesis is currently under investigation. There is ample evidence that human viral infections are associated with reduced levels of linoleic acid and thus participate in the depletion of essential fatty acids in cancer cells. Another important risk factor in the development of cervical cancer seems to be related to an exposure of the cervix to injuries, leading to inflammatory reactions involving excessive superoxide generation. This excessive oxygen radicals stimulate lipid peroxidation which then results in the depletion of essential fatty acids.
Membrane lipids and free fatty acids play a key role in cell proliferation and differentiation. The free fatty acid concentration in normal cells is well regulated through active incorporation into phospholipids, tdglycerides and cholesterol esters. Upon stimulation the released (free) fatty acids (FFAs) may act as regulators or can be metabdiised to biological active compounds. FFAs may reflect active cell metabolism, since they act directly as messengers for protein kinase C, which induce signal transduction and cell regulation. The purpose of this study is to evaluate the role of individual FFAs during the stages of cervical cancer. Punch biopsies of normal tissue (N), and intraepithelial lesions (CIL) and infiltrating lesions (Ca) of the cervix were obtained from women referred for hysterectomy, colposeopy or staging of infiltrating cancer. FFAs were determined according to the method of Pace-Asciak and fatty acid methyl esters were analysed by GLC. The results can be summadsed as follow: 16:1n-7 was detected only in Ca; 18:0 was significantly lower in Ca compared with normal tissue and CIN (p < 0,01 in both cases); 18:1n-7 was detected only in CIN and Ca, where Ca was significantly higher compared with normal tissue and CIN; 20:4n-6 was significantly lower in Ca compared with normal tissue and CIN (p < 0.01 in both cases); 20:3n-6 and 22:4n-6 and 2 2 : 6 n - 3 were detected only in Ca. Essential fatty acid deficiency is prevalent in cervical carcinogenesis. Free fatty ac;ds serve as substrates for the generation of free radicals. Excessive generation of the hydroxyl radical close to cellular membranes enables it to attack free fatty acids, as well as the fatty acids side chains of the phospholipids, producing hydroperexides. Accumulation of these agents in a membrane inflicts oxidative damage, thus reducing its fluidity and permeability, then disrupting its function and causing it to collapse. In addition, lipid hydroperoxides can decompose to yield a range of highly cytotoxic products such as aldehydes, which can further perpetuate the tissue damage. This damage may be an essential prerequisite to enable carcinogens to initiate the neoplastic process. Free radical determination is currently under investigation in our laboratory.
EFA & Eicosanoids 1997
-
Edinburgh
249
P145
P146
Apoptosis induction, angiogenesis and growth inhibition of Colon-26 adenocarcinomas with local delivery of diclofenac in hyaluronan.
Cervix: I Comparison of total fatty acid compositions in intraepithelial and infiltrating lesions of the cervix
Brown J, Freemanfle C, Colville-Nash P, Somerville K, Seed M, Willoughby D. Experimental Pathology, William Harvey Research Institute, London EC1M 6BQ UK.
L Louw and A-M Engelbrecht, Department of Anatomy & Cell Morphology, University of the OFS, Bloemfontein, 9300, South Africa.
Aim: NSAIDs reduce clinical colonic adenocarcinoma growth and topical diclofenac (Dc) in 2.5% hyaluronan (HA) inhibits basal cell carcinoma, and actinic keratosis. This could be due to effects on tumour growth, angiogenesis or apoptosis. Methods:Colon-26 cells viability (MTT), growth (thymidine), and apoptosis (acridine orange/ethidium bromide) was studied with and without diclofenac in vitro. In vivo sub-cutaneous tumour growth, angiogenesis (carmin/gelatin vascular casts), proliferation (mitotic figures) and apoptotic (Apoptag) indices were assessed. Results: Dc reduced proliferation and viability in vitro at 104 and 3x103M respectively, and stimulated apoptosis. In vivo, colon-26 tumour growth was preceded by angiogenesis and reduced apoptotic (AI, from 0.32_+0.05 day 7, to 0.04_+0.02% day 14 *** and mitotic indices (MI, from 5.59_+0.40 day 7 to 3=49+0.35% day 14). Topical De/HA inhibited tumour prostaglandin synthesis, b) retarded tumour a) to growth ('[/C ratio 0.174; fig a, topical 120 T aqueous cream 8 control O, topical E Dc/HA []) and "~ 6 g 8o angiogenesis (fig b). MI remained unaltered in vivo by Dc, whilst AI was 40 increased from 0.05 2 + 0.01 to 0.15 _+ 0.01% *~* atday 14 0 I , i i ] ~ ~ and from 0.05 + 0.02 2 6 10 14 0 2 6 tO 14 to 0.32 + 0.04% *~* at day 20 vs. HA Days alone. Tumour Days necrosis was also increased. Topical HA had slight antitumour and anti-angiogenesis activity. Conclusion: Dc inhibits colon-26 proliferation and viability, whilst inducing apoptosis in vitro. Anti-tumour activity in vivo is accompanied by angiogenesis inhibition and apoptosis induction. The substantial quantities of Dc delivered locally in HA may exhibit antitumour activity in similar fashion to those seen in vitro and explain its clinical efficacy.
In this study, the possible role of fatty acids and their metabolites during cervical carcinogenesis, was investigated. Since membrane lipids play a key role in cell proliferation and differentiation, disturbances in the fatty acid compositions of cell membranes and the modulation of membrane fatty acid compositions received attention in several in v i t r o studies. There are, however no reported studies where the actual total fatty acid compositions bare been determined during the different stages of cervical carcinogenesis. I n t h i s e x v i v o study, the fatty acid compositions of normal tissue, intraepithelial neoplasia and infiltrating lesions of the cervix were compared. The fatty acid profiles that were determined, make it possible to speculate about the metabolic pathways and to correlate these with possible therapeutical implications. Lipids were extracted from biopsies of normal tissue (n=36), cervical intraepithelial neoplasia (CIN) (n=47), and infiltrating lesions (n=74). Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by GLC. Essential fatty acid deficiency (EFAD) in the intraepithelial lesions, compared with normal tissue (linoleic acid, p < 0.01), and in infiltrating lesions, compared with intraepitbelial lesions (linoleic acid and arachidonie acid, p < 0.01) were observed. High levels of oleic acid were also observed when infiltrating lesions were compared with normal tissue (p < 0.01). There are many factors which may contribute to EFAD in cancer cells, but the hltman papilloma virus, prolonged cytokine-mediated reactions, free radical reactions and lipid peroxidation must be considered major role players in cervical carcinogenesis. From a therapeutic viewpoint, substantial changes in the fatty acid composition of the membranes can be produced in cancer cells by selective fatty acid supplementation strategies. At present, modifications of the fatty acid compositions of cell membranes represent an experimental model that has promoted increased understanding of lipid transportation, membrane remodelling, and the relationship between membrane lipids and membrane function. The clinical feasibility of using modification of fatty acids in cancer by diet or perfusion as an adjunct to standard therapies, should be tested.
P147
P148
Cervix: H Linoleie acid conversion during cervical carcinogenesis.
Cervix II1: Free fatty acid profiles in cervical carcinogenesis.
Louw L, Engelbrecht A-M. Department of Anatomy and Cell Morphology, Faculty of Medicine, University of the Free State, Bloemfontein, 9300, South Africa.
L o u w L, Engelbrecht A-M. Anatomy and Cell Morphology, Faculty of Medicine, University of the Free State, Bloemfontein, 9300, South Africa.
It is well established that there are significant alterations in the metabolic pathways during carcinogenesis. This study indicates and discusses the possibilities of linoleic acid conversion during stages of cervix cancer. Patient biopsies of normal tissue, cervical intraepithelial neoplasia and infiltrating lesions of the cervix (n=20 of each) were obtained. Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by GLC. A progressive decline in the relative percentages of linoleic acid (18: 2n-6; LA) could be observed in squamous epithelial cells of the cervix, during their transformation from normal tissue (N) to intraepithelial neoplasia (CIN) and infiltrating lesions (Ca) of the cervix. LA was significantly lower in Ca, compared with normal tissue and CIN (p < 0.01) in both cases; 95% CI for median of differences [- 9.28%; -4.55%] and [-5.87%; -2.28%]. Also, arachidonic acid (20: 4n-6; ARA) was significantly lower in Ca, compared with CIN (p < 0.01: 95% CI for median of differences - 5.02%; -1.47%). The significant decrease in LA is in accordance with a tendency observed by us amongst tumoar entities progressing from a benign toward a malignant state, and even between severe and less severe malignancies. The decrease in linoleic acid levels may be indicative of increased metabolism, since increased levels of DGLA, the precursor of the l-series prostaglandins, occurred in cancer cells of the cervix. The high concentrations of DGLA and C20:2n-6 in cervical cancer cells may indicate that delta-8-desaturase is very active in cancer cells. Alternative pathways (e.g. elongation and delta-8-desaturation) may enable the desuturation of LA when delta-6-desuturase is not active. The conversion of LA to I3-HOD in cervical carcinogenesis is currently under investigation. There is ample evidence that human viral infections are associated with reduced levels of linoleic acid and thus participate in the depletion of essential fatty acids in cancer cells. Another important risk factor in the development of cervical cancer seems to be related to an exposure of the cervix to injuries, leading to inflammatory reactions involving excessive superoxide generation. This excessive oxygen radicals stimulate lipid peroxidation which then results in the depletion of essential fatty acids.
Membrane lipids and free fatty acids play a key role in cell proliferation and differentiation. The free fatty acid concentration in normal cells is well regulated through active incorporation into phospholipids, tdglycerides and cholesterol esters. Upon stimulation the released (free) fatty acids (FFAs) may act as regulators or can be metabdiised to biological active compounds. FFAs may reflect active cell metabolism, since they act directly as messengers for protein kinase C, which induce signal transduction and cell regulation. The purpose of this study is to evaluate the role of individual FFAs during the stages of cervical cancer. Punch biopsies of normal tissue (N), and intraepithelial lesions (CIL) and infiltrating lesions (Ca) of the cervix were obtained from women referred for hysterectomy, colposeopy or staging of infiltrating cancer. FFAs were determined according to the method of Pace-Asciak and fatty acid methyl esters were analysed by GLC. The results can be summadsed as follow: 16:1n-7 was detected only in Ca; 18:0 was significantly lower in Ca compared with normal tissue and CIN (p < 0,01 in both cases); 18:1n-7 was detected only in CIN and Ca, where Ca was significantly higher compared with normal tissue and CIN; 20:4n-6 was significantly lower in Ca compared with normal tissue and CIN (p < 0.01 in both cases); 20:3n-6 and 22:4n-6 and 2 2 : 6 n - 3 were detected only in Ca. Essential fatty acid deficiency is prevalent in cervical carcinogenesis. Free fatty ac;ds serve as substrates for the generation of free radicals. Excessive generation of the hydroxyl radical close to cellular membranes enables it to attack free fatty acids, as well as the fatty acids side chains of the phospholipids, producing hydroperexides. Accumulation of these agents in a membrane inflicts oxidative damage, thus reducing its fluidity and permeability, then disrupting its function and causing it to collapse. In addition, lipid hydroperoxides can decompose to yield a range of highly cytotoxic products such as aldehydes, which can further perpetuate the tissue damage. This damage may be an essential prerequisite to enable carcinogens to initiate the neoplastic process. Free radical determination is currently under investigation in our laboratory.