title: Protein kinase C mediated anti-proliferative glucocorticoid-sphinganine synergism in cultured Pollard III prostate tumor cells.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Urology, West Virginia University, Robert C. Byrd Health Sciences Center, Morgantown 26506, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sosnowski
authors.forename: C
authors.initials: C
authors.lastname: Stetter-Neel
authors.forename: D
authors.initials: D
authors.lastname: Cole
authors.forename: J P
authors.initials: JP
authors.lastname: Durham
authors.forename: M G
authors.initials: MG
authors.lastname: Mawhinney
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D008297:Male; D011471:Prostatic Neoplasms / Q000473:pathology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014221:Triamcinolone / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D005938:Glucocorticoids; D014221:Triamcinolone; D011493:Protein Kinase C; D013110:Sphingosine; C005682:safingol