Regulation of ovarian UT-OC-2 carcinoma cells by cytokines: effects on cell proliferation, activation of transcription factors and apoptosis.

BACKGROUND: It is known that immunologic factors are involved in the regulation of the growth of ovarian carcinoma and that granulocytes are often found on the site of ovarian cancer. Therefore, we chose to investigate the effects of cytokines on UT-OC-2 ovarian endometrioid adenocarcinoma cells in vitro. In order to investigate the molecular mechanisms involved, the activation of two key DNA-binding proteins, AP-1 and transcription factor NF-kappaB (NF-kappaB), was studied. Since DNA extracted from the UT-OC-2 cells showed fragmentation typical of apoptosis, we also studied the effects of cytokines on this event. METHODS: The effects of the studied cytokines on the proliferation of UT-OC-2 cells were investigated by (125)I-deoxyuridine incorporation. The activation of DNA-binding proteins was studied by electrophoretic mobility shift assay. Statistical analyses were performed by Student's t-test. RESULTS: Interferon alpha (IFN-alpha), transforming growth factor beta(1) (TGF-beta(1)), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) all had a significant inhibitory effect on cell proliferation. Granulocyte colony stimulating factor (GM-CSF) did not alter cell proliferation significantly. Transcription factors AP-1 and NF-kappaB were both found to be constitutively active in UT-OC-2 ovarian carcinoma cells. We were able to show that IFN-gamma, TGF-beta(1) and TNF-alpha all increased the binding activity of transcription factor AP-1 (AP-1). The binding activity of transcription factor NF-kappaB was not altered by any of the cytokines studied, with the exception of IFN-gamma. IFN-gamma had also a clear inhibitory effect on the apparent magnitude of apoptosis, whereas TNF-alpha and TGF-beta(1) showed no effect. CONCLUSION: The results of this study show that TNF-alpha, TGF-beta(1), and IFN-gamma are able to inhibit the proliferation of UT-OC-2 ovarian carcinoma cells. Activation of AP-1 seems to be involved in the growth-regulating processes induced by IFN-gamma, TGF-beta(1) and TNF-alpha; IFN-gamma is also able to increase the binding activity of NF-kappaB and inhibited apoptosis in ovarian cancer cells.

Effect of the cessation of long-term hormone replacement therapy on plasma plasminogen activator inhibitor-1 and fibrinogen.

OBJECTIVE: Hormone replacement therapy (HRT) has generally been documented to reduce plasminogen activator inhibitor-1 (PAI-1) and fibrinogen levels in plasma of postmenopausal women. We used a wash out protocol to study whether stopping long-term HRT with estrogen alone or a combination of estrogen-progestin have different effects on these markers of hemostasis. STUDY DESIGN: Thirty healthy postmenopausal women on HRT participated. Fifteen had estradiol valerate, and 15 had estradiol valerate and levonorgestrel. Each was studied after long-term HRT (period 1), four weeks after cessation of the treatment (period 2, wash out), and three weeks after reintroducing the therapy (period 3). RESULTS: In the estrogen group, PAI-1 increased by 18% during the wash out period (P=0.013) and decreased by 22% after reintroduction of therapy (P=0.001). In the combined therapy group, there was a trend of PAI-1 to increase by 18% when therapy was discontinued (P=0.17), and it decreased by 25% after reintroduction of hormone replacement therapy (P=0.036). Fibrinogen was initially lower in the estrogen group compared with the combined therapy group (p=0.014), and did not change during wash out. CONCLUSION: This wash out study shows that cessation of long-term HRT unfavorably increases PAI-1, but appears to have no adverse effect on fibrinogen.

Hepatic lipase C-480T genotype-dependent benefit from long-term hormone replacement therapy for atherosclerosis progression in postmenopausal women.

Hepatic lipase (HL) is a lipolytic enzyme that hydrolyzes triglycerides and phospholipids in almost all major classes of lipoproteins. The HL gene has a functional promoter polymorphism at position -480, which affects transcription and leads to CC, CT, and TT genotypes. We investigated the effect of long-term hormone replacement therapy (HRT) on the progression of atherosclerosis in a 5-yr follow-up observational study of 88 postmenopausal women with different HL genotypes (CC, n = 49; CT, n = 34; TT, n = 5). These women, aged 45-71 yr, were divided into three groups based on the use of HRT. The HRT-EVP group (n = 26) used sequential estradiol valerate (EV) plus progestin (levonorgestrel), the HRT-EV group used EV alone (n = 32), and the control group (n = 30) used no HRT. The HRT-EV and HRT-EVP groups started estrogen at menopause for estrogen-deficiency symptoms, whereas the control group took no estrogen due to either the absence of such symptoms or a dislike of estrogen therapy. In addition to serum lipid concentration and HL genotype, the atherosclerosis severity score (ASC) for the abdominal aorta and carotid arteries was determined by ultrasonography. There was a significant interaction between HRT therapy and HL genotypes on the increase in ASC (P = 0.046) after adjustment for age, body mass index, changes in high-density lipoprotein cholesterol and baseline ASC. In subjects with the T allele, the progression of ASC was significantly faster in the control group than the HRT group (P = 0.0006), whereas in the CC genotype, there were no significant differences in ASC progression between the control and HRT groups. Our results suggest that the beneficial effect of HRT on atherosclerosis progression was restricted to women with the T allele, in whom the progression of ASC was slower by half. These results may help us understand in greater detail the benefits and possible risks associated with HRT in atherosclerotic diseases.

Sequentially combined estradiol valerate plus levonorgestrel therapy decreases 18:1 trans-fatty acid content of plasma lipids in healthy postmenopausal women.

Trans-fatty acids (TFA) have been classified as atherogenic dietary constituents but the effect of hormone replacement therapy (HRT) on their concentrations is not known. We used a washout protocol to study the effect of long-term estrogen and combined estrogen-progestin HRT on plasma elaidate (18:1t), which is the trans isomer of oleate and the major TFA in the diet. The study group comprised 15 women receiving estradiol valerate HRT and 15 women receiving combined HRT with estradiol valerate and levonorgestrel. The concentrations of elaidate in plasma phospholipids, cholesteryl esters and triglycerides were determined by gas chromatography. At baseline, the total plasma elaidate concentration was lower in the combined HRT group than in the estradiol valerate HRT group (p < 0.01). In the combined HRT group, the concentration of elaidate increased significantly after withdrawal of HRT (p < 0.001) and decreased again to the baseline level after restart of therapy (p < 0.001). These changes were due to decreases in the concentrations of phospholipids and triglycerides; in phospholipids there was also a proportional decrease of elaidate. There were no changes in elaidate in women receiving estradiol valerate alone. Our results suggest that long-term combined HRT treatment decreases plasma TFA, which is not achieved by estrogen alone.

Impact of long-term hormone replacement therapy on in vivo and in vitro markers of lipid oxidation.

Postmenopausal hormone replacement therapy (HRT) with estrogen has been suggested to inhibit oxidation of low-density lipoprotein (LDL) in vitro, but progestins may oppose this effect. We studied whether estrogen HRT and combined HRT with estrogen and progestin differ in their ability to resist in vivo and in vitro oxidation of lipids. Study group included 15 women on oestradiol valerate (mean age 56 years, treatment duration 10.5 years) and 15 women on combined HRT with oestradiol valerate and levonorgestrel (mean age 58 years, treatment duration 11.3 years). In addition to lipid and apolipoprotein concentrations, the lagtime of LDL to oxidation, the rate of the propagation phase and the maximum concentration of conjugated dienes were recorded as indices of LDL susceptibility to copper-induced oxidation in vitro. As an in vivo marker of oxidative stress we measured 24-h excretion of urinary 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha). All measurements were done after long-term HRT (baseline), after 4 weeks pause and again 3 weeks after reintroduction of HRT. High-density lipoprotein (HDL) cholesterol and apolipoprotein AI concentrations were significantly higher and LDL to HDL ratio significantly lower after long-term oestradiol valerate therapy than after combined therapy. Simultaneously, the triglyceride and lipoprotein (a) levels were higher in the estrogen group. Susceptibility of LDL to oxidation and the level of 8-iso-PGF2alpha were similar in both groups at all measurement points, and treatment group was not a statistically significant determinant of these markers at baseline. According to these results, estrogen and combined HRT do not differ in their abilities to oppose LDL oxidation in vitro or systemic oxidative stress in vivo, but have differential effects on blood lipids.

Fatty acid and cholesterol composition of the uterine artery intima in relation to menopausal status, age, and serum cholesterol.

OBJECTIVES: Estrogens modulate lipid metabolism and the increased risk of atherosclerosis in postmenopausal women is at least partly due to the reduction of estrogen production after menopause. We studied the effect of menopause on the contents of long-chain fatty acids, free cholesterol (FC) and cholesterol ester (CE) in uterine artery wall. METHODS: The uterine artery intima samples were obtained in connection with surgery of 21 postmenopausal and 51 premenopausal women. The amount of FC, CE and phospholipid fatty acids were measured by gas chromatography after extraction and fractionation and these lipid values were related to menopausal status, age and serum total and low-density lipoprotein (LDL) cholesterol levels. RESULTS: Premenopausal females had significantly less intimal FC (161 +/- 50 vs. 407 +/- 276 microg/100 mg wet weight, P = 0.003) and CE (19 +/- 34 vs. 305 +/- 348 microg/100 mg wet weight, P = 0.050) and smaller proportion of linoleic acid out of all phospholipid fatty acids (4.2 vs. 7.2%, P = 0.002) than postmenopausal women after adjustment with age. The content of CE (r = 0.34, P = 0.025) and the FC-to-CE ratio (r = -0.45, P = 0.002) correlated with age in premenopausal but not in postmenopausal women. Moreover, the intimal content of CE correlated with the percentage of intimal phospholipid linoleic acid in postmenopausal women (r = 0.79, P = 0.020). The same was true for FC (r = 0.73, P < 0.001). CONCLUSIONS: These results indicate that CE and FC accumulation into the wall of uterine artery depends on menopausal status, independently of age, and that the phospholipid long-chain fatty acid composition differs significantly between premenopausal and postmenopausal women. This suggests that estrogens may be involved in the regulation of artery wall lipid composition.

Effects of long-term estrogen replacement therapy versus combined hormone replacement therapy on nitric oxide-dependent vasomotor function.

Postmenopausal hormone replacement therapy (HRT) with estrogen may increase production of the predominant endothelium-derived vasodilator nitric oxide (NO) and consequently improve vascular reactivity. In contrast, concurrent progestin therapy may oppose this beneficial effect. We studied the effect of long-term estrogen HRT and combined HRT on vasomotor function and on plasma nitrate, which reflects the amount of NO in the circulation. As lipid peroxidation affects NO production and impairs endothelial function, we also measured the amount of the in vivo lipid peroxidation marker urinary 8-iso-prostaglandin F(2 alpha). The study group comprised 15 women receiving estradiol valerate HRT (mean age, 56 yr; treatment duration, 10.5 yr) and 15 women receiving combined HRT with estradiol valerate and levonorgestrel (mean age, 58 yr; treatment duration, 11.3 yr). The peak flow velocity (PFV) and pulsatility index of the common carotid and internal carotid artery and the abdominal aorta were measured by ultrasonography after long-term HRT (baseline), after a 4-wk pause and again 3 wk after reintroducing HRT. A statistically significant interaction between the groups and time points was observed in the PFV of the internal carotid artery (P = 0.011). In women taking estradiol valerate, the PFV values decreased significantly after withdrawal of HRT (P = 0.007) and increased again to the baseline level after reintroduction of therapy (P < 0.001). In women receiving combined HRT, the PFV remained stable over all study periods. At baseline, the PFV of women taking estradiol valerate correlated with the plasma nitrate concentration in the common carotid artery (r = 0.646; P = 0.009) and in the abdominal aorta (r = 0.579; P = 0.024). For pulsatility index and urinary 8-iso-prostaglandin F(2 alpha) excretion, there were no significant differences between the groups. Our results suggest that the favorable effects of long-term estrogen treatment on blood flow are at least partly mediated through NO. The addition of levonorgestrel to the treatment regimen appears to abolish this effect.

Effect of long-term hormone replacement therapy on atherosclerosis progression in postmenopausal women relates to myeloperoxidase promoter polymorphism.

Myeloperoxidase (MPO) is an oxidative enzyme present in phagocytes and atherosclerotic lesions. The MPO gene has a promoter polymorphism -463G/A, which leads to high (GG) and low expression (AG, AA) genotypes. We investigated the effect of long-term hormone replacement therapy (HRT) on the progression of atherosclerosis in a 5-yr follow-up study of postmenopausal women with different MPO genotypes. Eighty-seven nonsmoking postmenopausal women, aged 45-71 yr, were divided into three groups based on the use of HRT. The HRT-EVP group (n = 25) used sequential estradiol valerate plus progestin, the HRT-EV group used estradiol valerate alone (n = 32), and the control group (n = 30) used no HRT. The atherosclerosis severity score (ASC) for abdominal aorta and carotid arteries was determined by ultrasonography, and the MPO genotype was analyzed. In subjects with the GG genotype, the progression of ASC was significantly faster in the control group than in the HRT group (genotype by time interaction, P = 0.042), whereas in A allele carriers there were no significant differences in ASC progression between control and HRT. The effects of HRT on atherosclerosis progression in subjects with the GG genotype seem to be especially beneficial compared with controls with the same genotype but without HRT. These results may help us understand in greater detail the benefit and possible risk of HRT in atherosclerotic diseases.

The effect of hormone replacement therapy on atherosclerotic severity in relation to ESR1 genotype in postmenopausal women.

OBJECTIVE: The atheroprotective action of estrogen is mediated by estrogen receptors (ESR) 1 and 2, expressed in atherosclerotic lesions. The effects of hormone replacement therapy (HRT) and ESR1 PvuII genotypes on atherosclerosis have not previously been studied prospectively in postmenopausal women. METHODS: We investigated the effect of HRT on the progression of atherosclerosis in a 5-year follow-up study of 88 postmenopausal women aged 45-71 years at baseline allocated into three groups based on the use of HRT. The HRT-EVP group (n=26) used sequential estradiol valerate (EV) plus progestin (P), the HRT-EV group EV alone (n=32), and a control group (n=30) was without HRT. The atherosclerosis severity score (AS) of the abdominal aorta and carotid arteries were determined by sonography and the ESR1 PvuII genotypes (P/P, P/p and p/p) by PCR. RESULTS: HRT, time and ESR1 PvuII genotype had a statistically significant or borderline significant main effect on AS during 5-year follow-up (P=0.004, P<0.001 and P=0.090, respectively), when analyzed by repeated measures analysis of variance. There was a significant genotype-by-treatment (HRT-EVP and control groups) interaction for AS (P=0.034). In response to HRT-EVP, subjects with P/P, compared with those with P/p and p/p genotypes, had a less increase in AS (1.61+/-1.14 vs. 1.71+/-1.27 vs. 2.43+/-1.27). Baseline AS as covariate in similar model does not change the significant interaction effect between HRT-EVP and control groups (P=0.036). But this effect was not found between HRT-EV and control groups. CONCLUSIONS: Our results suggest that the effect of HRT-EVP in postmenopausal women on progression of AS may be determined in part by the genotype of ESR1 PvuII.

Effect of long-term hormone replacement therapy on atherosclerosis progression in postmenopausal women relates to functional apolipoprotein e genotype.

Apolipoprotein (apo)E gene epsilon4 allele carrier status modulates the responses of lipoprotein metabolism to hormone-replacement therapy (HRT). We investigated the effect of long-term HRT on the progression of atherosclerosis in postmenopausal women with or without apoE epsilon4 allele. One hundred forty-one nonsmoking postmenopausal women, 45-71 yr old, were divided into 3 groups based on the use of HRT. The HRT-EVP group (n = 61) used sequential estradiol valerate (EV) plus progestin (P), the HRT-EV group used EV alone (n = 40), and a control group had no HRT. Of these 141 women, 93 participated in a 5-yr follow-up study in 1998. In addition to serum lipid concentration and apoE genotype, the atherosclerosis severity score of the abdominal aorta and carotid arteries was determined by sonography. In apoE4-negative subjects, the progression of atherosclerosis severity score was significantly faster in control than in the HRT groups (genotype-by-time interaction P = 0.0026); whereas in apoE4-positive subjects, there were no significant differences in atherosclerosis severity score progression between the control and HRT groups. The effects of HRT on atherosclerosis progression in subjects with no apo epsilon4-allele seems to be especially beneficial, compared with controls with same phenotype status but without HRT. These results may help us to understand, in more detail, the benefit and possible risk of HRT on atherosclerotic diseases.