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.

m-Calpain activation/depletion is associated with androgen-induced reduction of protein kinase C and proliferation of male accessory sex organ smooth muscle cells.

PURPOSE: In the guinea pig seminal vesicle smooth muscle (SVM), androgen-dependent proliferation and terminal differentiation appear to be coupled to protein kinase C (PKC). This is based on the observations that both the soluble (cytosolic) enzyme and the Triton X-100 solubilizable form of the particulate enzyme were reduced during proliferation but were androgen-resistant in the amitotic state of adults. The purpose of the present investigation was to determine if the reduction in PKC activity was linked to the translocation of the activated enzyme to acceptor sites in the Triton X-100 insoluble fraction of the cell or reflected enzyme depletion due to proteolysis by androgen-dependent activation of the u- and/or m-calpains. MATERIALS AND METHODS: SVM was harvested from treated animals, homogenized and separated into soluble and particulate components. The particulate material was further fractionated into Triton X-100 soluble and insoluble fractions. PKC activity was determined in all fractions using radioactive ATP. Cultures of pure smooth muscle cells from both human prostate and SVM were also employed to assess the role of calpain in smooth muscle growth. RESULTS: During androgen-induced proliferation, instead of a translocation of PKC activity to the Triton X-100 insoluble particulate fraction of the cell, PKC activity in this fraction was significantly reduced. The m-calpain was the only isoform detected in SVM. At the peak of androgen-induced DNA synthesis in pre-pubertal castrate animals, m-calpain decreased 45% whereas in proliferative resistant SVM of adult castrates the protease was not significantly affected by androgen treatment. In pure smooth muscle cultures from the SVM as well as human prostate glands calpeptin the cell permeable inhibitor of calpains produced a concentration-dependent inhibition (IC50 approximately equal to 35 microM) of cellular proliferation. CONCLUSIONS: Given that biochemical assays of calpain quantify the residual proenzyme and that upon activation calpain is rapidly degraded, our findings indicate that m-calpain activation occurs in association with androgen-induced degradation of PKC and SMC proliferation. Thus in vitro inhibition of m-calpain activation is antiproliferative. The relative resistance of m-calpain in adult SVM may be an important component of the terminal differentiation process in normal smooth muscle.