Darrell K. Murray

Vitamin E and other antioxidants inhibit human prostate cancer cells through apoptosis.

Many human prostate cancer cells have escaped the apoptotic effects of natural regulators of cell growth such as transforming growth factor βl (TGF β‐1) and tumor necrosis factor (TNF).

Sphingolipids inhibit insulin and phorbol ester stimulated uptake of 2-deoxyglucose

Studies are presented demonstrating inhibition of both insulin and phorbol myristate acetate stimulated uptake of 2-deoxyglucose uptake by 3T3-L1 fibroblasts. Greatest inhibition of uptake was seen with sphinganine while sphingosine was also potent in this regard. Ceramide inhibited phorbol myristate acetate but not insulin stimulation of uptake. It is suggested that sphingolipid inhibition of glucose transport relates to the previously demonstrated effect of corticosteroids to increase membrane sphingomyelin and inhibit glucose transport.

Dexamethasone increases neutral sphingomyelinase activity and sphingosine levels in 3T3-L1 fibroblasts

The activity of neutral sphingomyelinase (EC 3.1.4.12) in a plasma membrane enriched fraction was found to be increased in dexamethasone treated cells. The elevation of sphingomyelinase activity was blocked by cycloheximide indicating that protein synthesis was required for the steroid action. Ceramidase (EC3.5.1.23) activity was unaffected by the dexamethasone treatment. Levels of sphingosine in 3T3-L1 Cells were also increased after treatment with 10(-7) M dexamethasone for 2 and 4 hours.

Inhibition of nuclear factor κB induces apoptosis following treatment with tumor necrosis factor α and an antioxidant in human prostate cancer cells

Abstract Transforming growth factor beta-1 (TGFβ-1) and tumor necrosis factor alpha (TNF-α), an activator of nuclear factor kappa B (NF-κB), modulate apoptosis and/or cell growth. This study was designed to investigate the activity of NF-κB and its regulation of inhibitor of apoptosis gene (c-IAP2) in two human prostate cancer cell lines, DU-145 (which is androgen unresponsive) and ALVA-101 (which is moderately androgen responsive). These cells were treated with and without various concentrations of a strong antioxidant, pyrrolidinedithiocarbamate (PDTC), and TNF-α at various time intervals. Following treatments, cell growth and apoptosis were determined by ELISA techniques. NF-κB activity was determined by electrophoretic mobility shift assay (EMSA), and c-IAP2 mRNA production was determined with Northern blot analysis. PDTC treatment significantly reduced cell growth up to 80% in both DU-145 and ALVA-101 cells. TNF-α and lower but not higher doses of PDTC combined demonstrated an additive inhibition of cell growth in both cell lines. Active NF-κB and c-IAP2 was blocked significantly following PDTC treatments, whereas treatments with TNF-α alone showed increased NF-κB activity and c-IAP2. However, when both PDTC and TNF-α were combined, nuclear presence of NF-κB and c-IAP2 were reduced significantly ( P

Oleic acid inhibits cholesteryl esterase and cholesterol utilization for testosterone synthesis in mouse Leydig cells.

We have observed that nonesterified fatty acids (NEFA) inhibit testosterone synthesis in response to luteinizing hormone (LH) in mouse Leydig cells, possibly by affecting cholesterol utilization or endogenous concentrations. We have now studied the influence of oleic acid (OA) on the cellular content of cholesterol, hydrolysis of cholesterol esters, and steroidogenesis in isolated mouse Leydig cells. OA (700 micromol/L added with fatty acid-free [FAF] albumin, 3 g/dL) significantly (P<.025) reduced testosterone production in response to LH (10 ng/mL), total cholesterol concentrations of Leydig cells and the culture medium, and cholesteryl esterase activity in the cytosol and mitochondria. We also studied the effects of OA on steroidogenesis and cellular cholesterol concentrations after treatments to increase cellular cholesterol. OA at lower concentrations (5 micromol/L with albumin, 0.1 g/dL) or low-density lipoprotein ([LDL] 4 micrograms protein/mL) increased cellular cholesterol (P<.01) without affecting basal steroidogenesis. These treatments failed to reverse the inhibitory (P<.05) effect of OA on testosterone synthesis following LH stimulation, but did significantly (P<.01) increase cellular cholesterol. In summary, OA appears to inhibit testosterone synthesis by inhibiting cholesteryl esterase activity.

Corticosteroid induced simultaneous changes in leukocyte phospholipids and superoxide anion production.

Abstract The effect of dexamethasone upon the phospholipid content and Superoxide anion production of human leukocytes has been investigated. Three mg dexamethasone was given by mouth to normal adult subjects and leukocytes isolated at 4, 8, 24, and 28 h following administration of the steroid. There was significant reduction in Superoxide anion production by leukocytes obtained 8 h and 24 h following administration of the steroid. There were also significant changes in the phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine content of the isolated leukocytes at 4 and 8 h following administration of the hormone. Exposure of isolated polymorphonuclear leukocytes to 8 × 10−7 M dexamethasone for 2 h also produced a significant decrease in phosphatidylcholine. An apparent increase in spingomyelin after both 2 and 4 h incubation was not statistically significant. These studies demonstrate an effect of corticosteroids to alter the phospholipid composition of leukocytes at the same time that Superoxide anion production by these cells is decreased. This finding is consistent with the concept that corticosteroid induced changes in phospholipid membranes may mediate some of the action of the hormone.

Heritability of the symptoms of benign prostatic hyperplasia and the roles of age and zonal prostate volumes in twins

OBJECTIVES Both benign prostatic hyperplasia and lower urinary tract symptoms (LUTS) have been shown to increase with age in men, but a causal relationship between prostate volume and symptoms has not been established. This study had two aims, to investigate the inter-relationships of age, symptoms, and various zonal measurements in the prostate and to assess the impact of heritable influences on symptom score. METHODS Eighty-three monozygotic twin pairs and 83 dizygotic twin pairs were studied to determine age and LUTS as assessed by the American Urological Association symptom score. Their prostate volumes (total, transition zone, and peripheral zone) were measured by transrectal ultrasound. RESULTS There was significant evidence of pairwise correlation between transition zone and symptom score (P = 0.04) and between age and symptom score (P = 0.03). Age also showed significant correlation with all volume measurements. Heritability appears to account for 82.6% of the variability in symptom score in men older than 50 years. CONCLUSIONS This study provides evidence that age and transition zone volume play a role in LUTS, but also that their influence is not strong. Estimates of heritability suggest that hereditary factors contribute substantially to LUTS.

Corticosteroids stimulate an increase in phospholipase A2 inhibitor in human serum.

A corticosteroid induced increase in a circulating inhibitor of serum phospholipase A2 activity is described. Inhibitor activity was found to be normally present in serum in agreement with the findings of other workers, and this activity was significantly increased by either acute or chronic administration of corticosteroids. The possible relation of this inhibitor to the known inhibitory effects of lipocortin and sphingomyelin on phospholipase A2 activity is briefly discussed.

Transforming growth factor beta-1 and beta-2 and type II receptor functional regulation of ALVA-101 human prostate cancer cells.

Transforming growth factor beta-1 (TGFbeta-1) causes apoptosis of many epithelial cells, including the prostate, but other secondary effects of TGFbeta-1 may be important in carcinogenesis. In a human prostate cancer cell line (ALVA-101), we determined the effects of TGFbeta-1 and TGFbeta type I and II receptor antibody on cell proliferation and TGFbeta-1 receptor binding. TGFbeta-1 and -2 and TGFbeta type II receptor mRNA expression levels were determined by polymerase chain reaction (PCR) and Northern blot analysis. A dose-responsive suppression (0.03 to 10 ng/mL) was observed for cells treated with TGFbeta-1 from 3 to 7 days (P < .01). Untreated cells had 1.1 x 10(3) (n = 3) TGFbeta receptors per cell, with a Kd of 0.20 nmol/L (n = 3) as determined by Scatchard analysis; treatment for 3 days with TGFbeta-1 (1 ng/mL) reduced the receptor number (0.9 x 10(3)) and the Kd (0.12 nmol/L). Antibodies to TGFbeta type I and II receptor stimulated proliferation with or without added TGFbeta-1 (50% +/- 5% above control, P < .01, n = 6). TGFbeta-1 and -2 and TGFbeta type II receptor mRNA expression was observed in untreated cells. In cells treated with TGFbeta-1, TGFbeta-1 mRNA was not affected by treatment, but expression levels of the TGFbeta type II receptor and TGFbeta-2 mRNA were moderately suppressed after 72 hours of treatment. Control cells actively produced TGFbeta-1 as measured by radioimmunoassay. The active and inactive forms of TGFbeta-1 were approximately equal, but TGFbeta-2 was secreted in smaller quantities than TGFbeta-1 and the inactive form of TGFbeta-2 predominated, with very small amounts of the active form. Our results suggest that the human prostate cancer cell line ALVA-101 retains negative control of proliferation in response to TGFbeta-1. Inhibition of endogenous TGFbeta action by antibodies to its receptor enhances the growth of ALVA-101 human prostate cancer cells, suggesting that endogenous TGFbeta exerts an inhibitory control on their growth and cellular function.

Testosterone is a potential augmentor of antioxidant-induced apoptosis in human prostate cancer cells

We have investigated the effect of antioxidant-induced apoptosis in human prostate cancer cell lines that is augmented by testosterone (T). In this study, DU-145 (androgen unresponsive), ALVA-101 (partially androgen responsive), and LNCaP (androgen responsive) were grown in tissue culture with RPMI 1640 medium, 5-10% fetal bovine serum (FBS), antibiotics and 5% CO2. Treatment with 2.5-20 microg/ml of PDTC significantly (P < 0.05, n = 6) lowered cell growth in all three cells 2-60% following treatment for 1-7 days. T (10(-12) M) alone enhances cell growth in androgen responsive cells. In contrast, the combination of PDTC and T significantly (P < 0.05, n = 6) augmented the PDTC induction of apoptosis in the androgen responsive cells, (ALVA-101 and LNCaP), but not in the androgen unresponsive cells (DU-145). PDTC reduced the nuclear NF-KB, as determined with an electrophoretic mobility shift assay (EMSA), to 50% of the control in LNCaP cells, 65% in ALVA-101 cells and 45% in DU-145 cells, but the combination of PDTC and T was not more potent than PDTC alone in any of the cell lines. PDTC suppressed both the AR mRNA and protein expression and reversed the stimulatory effect of T on androgen receptor (AR) protein synthesis in LNCaP and AVLA-101 cells. In conclusion, PDTC is a potent growth inhibitor and an inducer of apoptosis in human prostate cancer cells by reducing nuclear NF-kappaB and AR protein expression. PDTCs suppression of AR synthesis and nuclear NF-kappaB in response to T may contribute to its enhancement of apoptosis observed with T and PDTC compared to PDTC alone.