Effect of fatty acids on estradiol and testosterone binding to whole DU-145 prostate cells.

Cancer of the prostate is one of the leading causes of cancer related deaths in men. An important role in the development of prostate cancer is played by androgens and androgen ablation is therefore currently used in cancer treatment. In the past, estrogens were widely used in treatment of prostate cancer, but there are indications that estrogens could also be involved in carcinogenesis. Lately, much research has been done on the modulation of the binding of steroid hormones to their receptors by polyunsaturated fatty acids (PUFAs), which could interfere with the steroid hormone's message. Therefore, the aim of this study was to determine in whole DU-145 human prostate cells the effect of EFAs and their metabolites on the binding and affinity of the estrogen receptor (ER) and androgen receptor (AR) to estradiol (E(2)) and testosterone (T), respectively. Fatty acids were dissolved in ethanol and added to the cell culture in a final ethanol concentration of 0.2% on the fourth day of incubation. The results showed that the PUFAs under investigation inhibited the AR's capacity, in contrast to the ER's capacity which was stimulated. However, the dissociation constants (K(d)) of the AR and ER complexes in the presence of the PUFAs, were as follows. Except for eicosapentaenoic acid (EPA) which decreased the AR dissociation constant and EPA and alpha-linolenic acid (ALA) which increased the ER dissociation constant, the remaining FAs had no significant effect on the K(d) values of both the AR and ER complexes. According to these priliminary results it is postulated that men should benefit with a diet rich in certain essential polyunsaturated fatty acids although its function remains to be clarified.

Cytotoxicity of combined essential fatty acids on a human prostate cancer cell line.

In this study the effect of single and concomitantly added n-6 or n-3 polyunsaturated fatty acids (PUFAs) was investigated on human prostate cells. Data obtained from the single fatty acids (FAs) experiments showed that except for oleic acid (OA), arachidonic (AA) and linoleic acid (LA), which had very little (less than 10% cells dead) effect on the cells, an increase in dead cells was observed at physiological concentrations of, eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA) and alpha-linolenic acid (ALA). However, this was not the case when combining these acids at physiological concentrations. A slight increase in cell death was only obtained with three combinations of ALA, namely with AA, OA, or GLA. Other combinations with ALA, such as with LA or EPA, had respectively no effect on cell number or increased the cell number by causing less cells to die. Other PUFAs combinations tested, did not show the three groups mentioned with ALA, but only the last two types, namely, no effect, or a decrease in the amount of cell death. The latter might mean that the FA combination had stimulated the cells, since a decrease in the amount of dead cells was observed. Therefore, it is concluded that the characteristics of combined FAs may differ from single FAs, which may explain some controversies in the literature and in response to treatments.

Cytokine levels affected by gamma-linolenic acid.

This study was undertaken to assess whether gamma-linolenic acid (GLA) in the form of evening primrose oil (EPO) could affect rat serum cytokines, interferon-gamma (IFN-gamma), monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha). The following diets were administered: control, glucan, Freund's adjuvant and glucan plus Freund's adjuvant with and without GLA. In the presence of GLA, the IFN-gamma and MCP-1 levels were significantly decreased in contrast to the control group of TNF-alpha, which was significantly stimulated. On account of interaction between diets and GLA, the remaining diet groups of TNF-alpha were either not affected or were inhibited in the presence of GLA. The observations indicate that GLA may modulate the level of serum IFN-gamma, MCP-1 and TNF-alpha, which may be a worthwhile line of treatment in certain human diseases.

Does urokinase play a role in renal stone formation?

Renal stone formation is a complex multifactorial disease, and it is believed that the initial step in the pathogenesis of urolithiasis must be the precipitation of an organic matrix of mucoproteins followed by precipitation of minerals onto this matrix. An important factor in this process may be the activity and/or concentration of the urinary enzyme, urokinase, which would affect the level of urinary mucoproteins such as uromucoid. In support of this hypothesis, ELISA studies were conducted to investigate the urokinase concentrations in urine obtained from males (22-60 years) with and without renal stones. These results showed a significant decrease in urinary urokinase concentration of renal stone patients which, once again, underlines the possible involvement of urokinase in renal stone formation. Therefore, it seems logical to conclude that urokinase may play an integral role in this multifactorial disease.

A possible role for enzymes in tumour-cell invasion.

The complex molecular and cellular processes of metastatic invasion as well as the anti-invasion possibilities are summarized. Invasion by neoplastic cells is a major obstacle to successful cancer therapy. Enzymes such as hyaluronidase, sialyltransferase, urokinase-type plasminogen activator, plasmin, matrix metalloproteinases, and others, play central roles in the catabolism of extracellular matrix macromolecules. However, this process can be opposed by inhibitors of these enzymes. Both invasion (promoters) and anti-invasion factors (suppressors) need further investigation, to clarify the role of these factors in the aetiology and possibly in the treatment and prognosis of metastatic cancer.

The effect of essential fatty acids on growth and urokinase-type plasminogen activator production in human prostate DU-145 cells.

Urokinase-type plasminogen activator (uPA) is an important protease enzyme in carcinogenesis, and is involved in both invasion and metastasis of cancer. Increased uPA activity and decreased essential fatty acid (EFA) levels have been reported in cancer. This phenomenon may be explained by the fact that certain EFAs, such as gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA), inhibit uPA activity. The effect of EFA on human prostate DU-145 cell growth and uPA production is still unknown and was investigated in this study. Data obtained from the different unsaturated fatty acids showed that oleic acid (OA) and EPA enhanced DU-145 cell proliferation at 0.004 and 0.04 mM for up to 4 days. However, alpha-linolenic acid (ALA), linoleic acid (LA), GLA and arachidonic acid (AA) suppressed cell proliferation under the same conditions, possibly as a result of inhibition of DNA and protein synthesis as measured using labelled thymidine and glycine incorporation. In contrast to the cell proliferation, uPA production was inhibited by all the unsaturated fatty acids under investigation. Therefore, the absence of EFAs, as reported, may affect invasion and metastasis of cancer.

Influence of acetylsalicylic acid and metabolites on DU-145 prostatic cancer cell proliferation.

Conflicting reports have been published on the anti-tumour activities of acetylsalicylic acid in various cancers. Therefore, the effect of acetylsalicylic acid and its major metabolites has been studied on human prostatic carcinoma DU-145 cells. Investigations concentrated on the influence of acetylsalicylic acid, salicylic acid and salicyluric acid, on cell proliferation, DNA- and protein synthesis of DU-145 cells. DNA and protein synthesis determinations were done in vitro by [3H]thymidine and [3H]glycine incorporation, respectively. No effect on cell plating efficiency was observed, however proliferation studies showed that acetylsalicylic acid and salicylic acid inhibited cell growth (10 mM, 100% inhibition). No significant effect on cell proliferation was ascertained with salicyluric acid. Both DNA and protein synthesis were 40% inhibited by 0.1 mM acetylsalicylic acid. This study demonstrates that acetylsalicylic acid exhibits a significant influence on cell growth of prostatic DU-145 cells. These preliminary results may contribute to a better understanding of the anti-tumour capabilities of acetylsalicylic acid.

Binding of estradiol to whole prostatic DU-145 cells in the presence and absence of tamoxifen and acetylsalicylic acid.

Conflicting results have been obtained with regard to the estradiol receptor (ER) capacity of human prostatic tissue. Human prostatic DU-145 cells have been found to be ER-negative with immunohistochemical assays. The object of this investigation was to determine if whole DU-145 cells, which had been grown in monolayer culture, have ER and, if so, to confirm the finding with antiestrogens. After cells had been lysed, a Bmax of 44.7 +/- 4.0 fmol/mg (Kd = 0.6 +/- 0.6 nM) was obtained. Subcellular localization studies showed that the estrogen receptor level in the cytoplasmic fraction was approximately 10 times higher than in the nuclear fraction. Competitive binding studies showed that tamoxifen, DES, and acetylsalicylic acid decreased estradiol binding. The dissociation constants and relative affinities for tamoxifen, DES, and acetylsalicylic acid were 0.2 nM (281.7%), 0.2 nM (224.0%), and 0.8 nM (78.43%), respectively. However, 5 alpha-dihydrotestosterone and metabolites of acetylsalicylic acid had no effect in competitive binding studies. These results may contribute to a better understanding of prostatic carcinogenesis, which may in turn lead to more effective treatment.

In vivo effects of urease-producing bacteria involved with the pathogenesis of infection-induced urolithiasis on renal urokinase and sialidase activity.

Many hypotheses have been proposed for renal stone formation. It has been argued that with infection-induced renal stones the hydrolysis of urea by bacterial urease increases urinary pH, with consequent stone formation. Unfortunately, this theory is not applicable to the micro-organisms that do not produce urease (e.g. Escherichia coli). It has been recently reported that E. coli reduces the urinary urokinase activity of male rats, but does not influence the urinary sialidase activity. This study has now been expanded to the urease-producing bacteria Proteus mirabilis, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Micrococcus luteus. Subcutaneous injections with these bacteria were found to significantly (P < 0.003) reduce the UK activity of extrarenally obstructed kidneys. The urease-producing mammalian skin bacterium, M. luteus, was, however, the exception (P = 0.1079). In contrast to S. epidermidis, P. aeruginosa and M. luteus (P < 0.0213), P. mirabilis and S. aureus had no effect on renal sialidase activity (P < 0.4047). These results may explain why Proteus species are predominant in infection-induced renal stones. According to the urokinase-sialidase hypothesis, a decrease in urinary urokinase activity should increase the uromucoid levels, whilst no effect on the urinary sialidase activity should favour conversion of urinary uromucoid to mineralizable matrix. These conditions may lead to renal stone formation. An increase in urinary pH resulting from urease-producing micro-organisms will increase salt precipitation on the uromucoid. It is thus concluded that urease-producing bacteria may play a double role in renal stone formation.

Can linoleic acid and gamma-linolenic acid be important in cancer treatment?

This hypothesis proposes that the essential fatty acids (EFAs), linoleic acid (LA) and gamma-linolenic acid (GLA), play important roles in cancer treatment. Oxidation of LA by lipoxidase especially increases tumour cell death, whilst GLA inhibits urokinase-type plasminogen activator (uPA) activity. Increased uPA activity is: firstly, responsible for cancer invasion and metastasis and secondly, responsible for proteolysis of lipoxidase which favours a decrease in cancer cell death. Addition of LA and GLA to available therapeutic regimens may be worth considering in cancer treatment.

Modulation of lipoxidase activity by urokinase-type plasminogen activator.

Malignant cells show increased urokinase (UK) activity and decreased peroxidation of essential fatty acids (EFA). In order to explore this phenomenon the effect of UK on the lipoxidase activity was spectrophotometrically investigated. Decreased lipoxidase activity was obtained with increased UK concentrations (r = -1.000, p < 0.0001). This proteolytic effect of UK on lipoxidase was eliminated with the addition of the UK inhibitor leupeptin. These results suggest that the increase in UK activity in malignant cells may decrease the lipoxidase activity and thus peroxidation of EFA. The effectiveness of a given EFA in killing cancer cells would therefore depend on the modulation of the lipoxidase activity by the UK-type plasminogen activator.

The effect of gamma-linolenic acid and eicosapentaenoic acid on urokinase activity.

Urokinase (UK) is an important protease enzyme in carcinogenesis, and is involved in the invasion and metastasis of cancer. Thus, regulation of UK activity is likely to be important in healthy cell metabolism. As it has been reported that a decrease in delta 6-desaturated essential fatty acid (EFA) metabolites occurs in malignant cells and that gamma-linolenic acid (GLA) and eicosapentaenoic acid (EPA) exert antimutagenic effects, the effects of GLA and EPA on UK activity have been investigated in this study. Both GLA (n-6) and EPA (n-3) acted as competitive inhibitors of UK with Ki values of 120 and 96 microM respectively. No modification of plasmin activity occurred with either 1.4 x 10(-4) M GLA or EPA. These results could explain why malignant cells with decreased EFA concentrations show increased UK activity. The addition of EFAs to available therapeutic regimens may be worth considering in the treatment of cancer.

Pyelonephritis: renal urokinase and sialidase (neuraminidase) activity in rats fed a standard laboratory diet.

Renal stone formation can be caused by many different and varied disturbances, some of which are poorly understood. The relationship between urinary infection and renal stone formation has not been completely clarified. It is argued that renal stones form primarily as a consequence of the hydrolysis of urea by the bacterial enzyme urease. However, no explanation is given for microorganisms that produce urease only occasionally or not at all. The question arises as to whether the infection-induced microorganisms might not be playing a double role in renal stone formation by not only producing urease, but also by affecting in vivo urokinase (UK) and sialidase (SA) activity. With this in mind, the effect of Escherichia coli on renal UK and SA activity has been studied in male rats with a normal diet. The renal UK (P = 0.208) and SA (P = 0.2135) activities did not differ significantly between the two kidneys of the same rat. In contrast, when drainage from one kidney of a rat was externally obstructed, the UK and SA activities differed significantly between kidneys (P < 0.015). An increase in UK (r = 0.6456, P < 0.0001) and SA (r = 0.7507, P < 0.0001) activity was observed over time in the obstructed kidney. Subcutaneous injections with E coli reduced the UK activity of the obstructed kidney significantly (p = 0.017). However, the SA activity remained the same (P = 0.3929).(ABSTRACT TRUNCATED AT 250 WORDS)

Pyelonephritis: renal urokinase activity in rats on essential fatty acid diets.

This study was undertaken to assess whether additions of different oils to the diets of male rats would affect the renal urokinase (UK) activity of healthy and pyelonephritic kidneys. Four groups of fatty acid diets were studied: fat-free, coconut oil, fish oil and evening primrose oil (EPO). Pyelonephritis was obtained by unilateral extrarenal urinary obstruction and subcutaneous injection of Escherichia coli. The UK activity of the non-obstructed kidneys did not differ statistically between rats infected and not infected with bacteria (P > 0.056), except within the coconut oil group. A statistically decreased UK activity was obtained with bacteria injected animals on a coconut oil diet (P < 0.0001). This phenomenon, namely a decrease in UK activity, was also seen with pyelonephritic kidneys of rats on fat-free, coconut and fish oil diets (P < 0.0065). However, the UK activity of the obstructed kidneys with and without infection in the EPO group remained similar (P = 0.8477). These results suggest that the UK activity in infection-induced renal stones may be restored by EPO containing diets and may be of high relevance in the prevention and treatment of infection-induced renal stones. This revelation now needs to be more fully investigated.

Sialic acid concentration and sperm motility.

Sialic acid (SA) forms an integral part of the sperm membrane, and the masking properties of SA are well documented. Masking of estrogen and melatonin receptors on the sperm midpiece by SA has been demonstrated. In this paper we report on the correlation of sperm bound SA concentration and motility. We found a negative correlation between the spermbound SA concentration and both the quantitative and qualitative motility. The spermbound SA correlated positively although not significantly with the spermatozoal ATP concentration. SA may mask the receptors on the sperm membrane, affecting sperm metabolism. This will inhibit the breakdown of ATP to its lower nucleotides resulting in poor motility. This may be another reason to believe that SA may play a key role in sperm motility.

The detachment cascade during metastasis.

The cause of detachment of tumour cells during metastasis is still one of the most intriguing questions of tumour propagation. A hypothesis is suggested herein for lysis of extracellular matrix that could ultimately lead to the detachment and spreading of malignant cells. According to this theory a certain optimal estrogen level initiates a series of enzymatic activations that culminate in detachment and spreading of tumour cells.

Putative melatonin receptor in human spermatozoa.

Melatonin is present in human semen, and may affect sperm motility. The presence of melatonin receptors on spermatozoa has not yet been reported. Detection of melatonin-binding sites may be limited because of the masking of such sites by sialic acid. Spermatozoa were obtained from eligible human donors, incubated with neuraminidase to remove sialic acid residues, and saturation binding assays were carried out using 2-125I-melatonin as a receptor probe. Consistent 125I-melatonin binding could only be obtained after spermatozoa were treated with neuraminidase. Scatchard analysis revealed a low-affinity binding site (ML-2) with a Kd value of 127 +/- 6 nM and a Bmax of 25 +/- 4.5 fmol/mg protein. These results present evidence of melatonin-binding sites in spermatozoa. Sialic acid possibly regulates the binding of melatonin to these sites.

The effect of calcium and magnesium ions on urinary urokinase and sialidase activity.

The effect of a promoter (calcium) and an inhibitor (magnesium) of urolithiasis was spectrophotometrically studied on urokinase (0.45 IU) and sialidase (5 mM). Although these mineral did not affect the sialidase activity, total inhibition of urokinase activity was observed with either 0.05 M calcium chloride or 0.1 M magnesium chloride. This observation might explain why calcium and magnesium respectively function as a promoter and an inhibitor of stone formation.