Protein kinase C mediated anti-proliferative glucocorticoid-sphinganine synergism in cultured Pollard III prostate tumor cells.

PURPOSE: Experimental effort focused on the growth inhibition of an androgen-resistant prostatic carcinoma, using pharmacological inhibition of protein kinase C (PKC) as the therapeutic target. MATERIALS AND METHODS: Studies were performed in cell culture using the Pollard (PA) III androgen-insensitive spontaneous rat prostate tumor cells, and the human prostate tumor lines, PC-3 and LnCaP. Pharmacological agents included steroid hormones and PKC modulators; measured parameters of tumor growth/function included cell number, PKC activity and sphingolipid metabolism. RESULTS: Triamcinolone (TA) and sphinganine synergized to inhibit the proliferation rate of PA III prostate tumor cells by converging through separate mechanisms to inhibit protein kinase C. At five days of cell culture, 0.1 microM TA reduced both the soluble and particulate forms of PKC in association with a 35-40% reduction in cellular proliferation. Exogenous sphinganine, a competitive inhibitor at the regulatory domain of PKC had no anti-proliferative effect at 1 microM, but in combination with TA synergized to reduce proliferation 80-90%, three days in advance of any detectable inhibitory effect of TA alone on cell number. TA produced no discernable stimulation of endogenous free sphingosine production as evidenced by the lack of an effect on the activity of neutral membrane sphingomyelinase or in the turnover of total cellular sphingomyelin. Phorbol esters, but not cell permeable diglycerides, prevented the TA + sphinganine effect suggesting that a stable long term PKC activation was required for reversal. Steroid specificity studies of the synergistic response revealed that while other glucocorticoids mimicked TA, aldosterone was less active and representatives of the three major classes of sex steroids were inert. Tests of sphinganine specificity demonstrated that calphostin C, a chemically unrelated inhibitor of the regulatory site of PKC, also produced a supra-additive interaction with TA. Ceramides (C2 & C6), which were closely related chemically to sphinganine but lacked affinity for the regulatory subunit of PKC, were inactive in this system. Analyses of the cellular specificity of the TA-sphinganine synergism using the human prostate carcinoma cell lines PC-3 and LnCap revealed a true synergistic growth inhibition in the glucocorticoid receptor positive PC-3 line and no significant interaction in the glucocorticoid receptor negative LnCap cells. CONCLUSIONS: TA-induced reduction of PKC concentration coupled with sphinganine antagonism of PKC activation contributed to in a synergistic growth inhibition of an androgen resistant prostatic carcinoma.

Etiological considerations for the growth of stroma in benign prostatic hyperplasia.

The finding that human benign prostatic hyperplasia (BPH) consisted primarily of fibromuscular tissue has led to basic research into the hormonal control of the growth of male accessory sex organ smooth muscle. By using the separated epithelium and muscle layers of the guinea pig seminal vesicle, it was determined that the epithelium exhibited only reversible androgen-induced growths, whereas the muscle proved to be a target tissue for both androgen and estrogen, and exhibited irreversible growth responses. It was of particular interest that the normal androgen-dependent pubertal development of the muscle involved an approximate twofold increase in DNA, followed by the development of a complete and relatively selective androgenic insensitivity in this parameter. An understanding of the factors leading to this apparently normal androgen-dependent loss of the proliferative response in muscle may allow for the development of specific hypotheses for the reawakened stromal growth in BPH. Research on other organ systems focusing on the various factors and mechanisms involved in muscle growth regulation is briefly discussed.

Characterization and androgenic regulation of soluble protein kinases and protein phosphorylation in rat ventral prostate gland.

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.

Simplified non-enzymatic technique for isolation of epithelium and muscle from the dog prostate gland.

Epithelium and muscle from the normal dog prostate were isolated with a rapid, simple, non-surgical separation (NSS) technique, involving mechanical agitation of organ cubes in a high concentration (30 microM) of EDTA for tissue dispersion, and gentle pressure filtration for recovery of epithelium and muscle. Derivation of this NSS procedure utilized the guinea pig seminal vesicle, since it can be surgically separated (SS) into pure epithelium and muscle to serve as controls for the biochemical viability of NSS prepared tissues. The NSS procedure adopted for use yielded not only pure (greater than 95%) epithelium and muscle from intact seminal vesicle, but also activities of 5 alpha-reductase and concentrations of the cytosol estrophile in each tissue, which were not significantly different from the SS counterparts. In the dog prostate gland, the concentrations of 5 alpha-reductase and the cytosol estrophile in the NSS epithelium were 1.58 and 0.43 of the NSS muscle, respectively.

Androgen and estrogen effect on guinea pig seminal vesicle muscle: a combined stereological and biochemical study.

A combined electron microscopic stereological and biochemical study of the smooth muscle cells of guinea pig seminal vesicles was performed in intact, castrated, castrated and dihydrotestosterone- or estradiol-treated adult animals. Castration led to cell atrophy as determined stereologically by a decreased single cell volume and biochemically by no change in DNA content coupled with an increase in the DNA concentration. Treatment of castrates with dihydrotestosterone restored both the stereological and biochemical parameters of the cell size to slightly supranormal levels. The estrogen-induced increase in muscle weight and DNA content appeared to be due only to hyperplasia of muscle cells and not to a proliferation of fibroblasts or to infiltration by inflammatory cells. In all treatment groups, including the estrogen-treated castrates, more than 95% of the cells in the tissue were smooth muscle cells, and there was no evidence that polyploidy contributed to changes in DNA levels. In addition, in the estrogen-treated muscles, DNA concentration remained high, and the stereologically determined cell size remained low. Therefore, both morphological and biochemical evidence indicate that androgen induces hypertrophy, whereas estrogen induces hyperplasia of muscle cells. The correction of stereological and biochemical data validates the application of stereological cell size determination for smooth muscle cells in organs that hardly can be separated into stromal and epithelial components; eg, the prostate.

Androgens and estrogens in the plasma and prostatic tissue of normal dogs and dogs with benign prostatic hypertrophy.

The endogenous concentrations of certain androgens and estrogens have been quantified in the plasma and prostatic tissue from normal dogs and dogs with benign prostatic hypertrophy (BPH). In the tissue and plasma from both dog populations, the level of estrone was higher than that of estradiol. The concentration of testosterone in the tissue and plasma of both normal dogs and dogs exceeded the concentration of either estrogen. Prostatic levels of dihydrotestosterone greatly exceeded that of testosterone. A comparison of normal dogs and dogs with BPH revealed that in both the plasma and prostatic tissue the concentrations of estradiol and estrone were significantly elevated (P less tha 0.05) in the BPH dogs. Plasma and prostatic levels of testosterone did not differ between the two groups; however, the concentration of dihydrotestosterone in hyperplastic glands was approximately 4 times greater than normal.

Effect of prolactin on androgen metabolism by the guinea-pig sex accessory organs.

The in vitro actions of prolactin on the metabolism of testosterone by the guinea-pig liver and sex accessory organs have been investigated under a variety of conditions including various concentrations of ovine or bovine prolactin, addition of reduced nicotinamide adenine dinucleotide phosphatate (NADPH) or nicotinamide adenine dinucleotide (NAD) to incubation media and various lengths of incubations. Treatment of the epithelium and muscle of the guinea-pig seminal vesicle with NAD elicited a shift from normal reductive metabolism of testosterone toward oxidative metabolism. This shift toward oxidative metabolism of testosterone was particularly marked in the muscle of the guinea-pig seminal vesicle. Ovine prolactin was highly effective in inhibiting the reductive metabolism of testosterone by the sex accessory organs. This effect appeared primarily as a decrease in dihydrotestosterone formation and was noted in both the presence and absence of NADPH or NAD. Oxidized metabolites, androstenedione and androstandione, were apparently insensitive to prolactin treatment in vitro. Variations induced by prolactin in the concentrations of intracellular androgens may be important in either the normal or abnormal growth and function of male sex accessory organs.