Phorbol Ester Inhibition Research Articles

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.

Phorbol ester inhibition of vasopressin-induced calcium efflux from cultured rat aortic myocytes

The effect of the protein kinase C activator TPA was investigated on AVP-induced 45Ca release from rat aortic myocytes. In the nanomolar range TPA, but not 4β-phorbol, reduced the brief 45Ca efflux produced by AVP in the presence or in the absence of extracellular calcium. The maximal effect of TPA was to abolish the response to a half maximally active concentration of AVP, and to reduce by 50% the maximal response to the hormone. These results suggest that protein kinase C activation can exert a negative control on the early AVP-induced calcium mobilization in vascular smooth muscle.

Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells.

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.

Modulation of phorbol ester receptors in mouse skin by application of quercetin.

The topical application of quercetin, an anti-tumor promoter, to mouse skin reduced the number of phorbol ester receptors, although quercetin did not inhibit specific 3H-12-O-tetradecanoylphorbol-13-acetate binding to a mouse skin particulate fraction. Quercetin, morin, kaempferol and luteolin inhibited activation of protein kinase C by teleocidin, and caused half-maximal activation at 25 microM. (+)-Catechin, which has been reported not to inhibit tumor-promoting activity, did not have any effect on these reactions. The modulation of phorbol ester receptors and inhibition of activation of protein kinase C are considered to be involved in the anti-tumor-promoting effect of quercetin in mouse skin. Diet containing 4% or 1% quercetin did not influence the action of teleocidin on mouse skin in a two-stage carcinogenesis experiment.

Kinetic evidence that 1,2-diolein inhibits phorbol ester binding to protein kinase C via a competitive mechanism

Diacylglycerols inhibit binding of [20- 3H]phorbol 12,13-dibutyrate ([ 3H]PDBu) to protein kinase C (the phorbol ester receptor). This inhibition could reflect competitive binding by the diglyceride. Alternatively, it might simply represent perturbation of the lipid environment required for binding activity. As predicted for a competitive mechanism, we report here that inhibitory concentrations of the diglyceride 1,2-diolein do not affect the off-rate of [ 3H]PDBu from its receptor. This behavior contrasts with that of arachidonic acid, which appears to interact via a mixed mechanism.

Phorbol ester inhibition of Na-H exchange in rabbit proximal colon.

In rabbit proximal colon, in vitro addition of phorbol 12,13-dibutyrate (PDB, 10(-7) M) to the serosal bathing medium inhibits mucosal (m)-to-serosal (s) unidirectional Na flux (JsmNa) without altering JsmNa or unidirectional Cl fluxes. Similar results were obtained when amiloride (2 X 10(-4) M) was added to the mucosal bathing medium. No additivity of effect was seen when tissues were exposed to both agents. Measurements with carboxyfluorescein reveal that the two agents cause equal decreases of intracellular pH (pHi), an effect that is dependent on the presence of extracellular Na (Na replacement also decreases pHi). No additivity of pHi effects is seen when both agents are added together. To determine the membrane site of this PDB-inhibitable Na-H exchange, Na influx across the luminal border of proximal colon was measured and was found to be inhibited equally by PDB and amiloride. We conclude that PDB, by activation of protein kinase C, inhibits electro-neutral amiloride-sensitive Na-H exchange in the luminal membrane of proximal colon.

Phorbol ester inhibition of hormonal induction of tyrosine aminotransferase

The liver specific enzyme, tyrosine aminotransferase, can be induced by glucocorticoids, cAMP analogs, or insulin. Each of these different inducing agents is believed to act through a separate pathway. The tumor promoting phorbol esters have been reported to stimulate phosphorylation of the insulin receptor and thereby decrease the ability of insulin to induce tyrosine aminotransferase. Our results demonstrate that TPA will not only inhibit the insulin stimulated increase in tyrosine aminotransferase, but will also inhibit induction of the enzyme by glucocorticoids or by cAMP.

Hydrocortisone inhibits phorbol ester stimulated release of histamine and arachidonic acid from rat mast cells

Purified rat peritoneal mast cells were incubated overnight with or without hydrocortisone (3 × 10 −6 M) and then stimulated with anti-IgE, somatostatin or a phorbol ester-ionophore combination, i.e. , 12-0-tetradecanoyl-phorbol-13-acetate and A23187. The release of both histamine and [1- 14C]arachidonic acid and its metabolites was determined. Hydrocortisone treatment markedly inhibited both anti-IgE and TPA-A23187 stimulated release, but not release stimulated by somatostatin. These results suggest that anti-inflammatory steroids may alter histamine release through an action involving the activation of the phosphatidylserine-calcium dependent protein kinase or its substrates.

Stoichiometric binding of diacylglycerol to the phorbol ester receptor.

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.

Effects of 6-thioguanine on communication competence and factors affecting reversibility of phorbol ester inhibition in V79 cells measured by the metabolic cooperation assay and by dye coupling.

One assay used to study the role of intracellular communication in tumor promotion examines chemically-induced inhibition of metabolic cooperation between 6-thioguanine (6-TG) sensitive (HGPRT+) V79 Chinese hamster cells in co-culture with 6-TG resistant (HGPRT-) cells. In the present study metabolic cooperation and Lucifer Yellow dye coupling were applied to detect intercellular communication. HGPRT+ cells were still communication competent when pre-incubated with 6-TG for up to 48 h before HGPRT- cells were plated. In both assays the phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) inhibited intercellular communication completely at 1 ng/ml. Using 6-TG metabolic cooperation, extensive washing of cells failed to reverse the inhibition caused by 100 ng TPA/ml after exposure for as little as 1 h. However, the effect of PDBu was completely eliminated if the drug was removed within 24 h. Complete blockade of dye coupling by 1, 10 or 100 ng TPA/ml was reversible after washing only at 1 ng/ml. Cells cultured in the presence of these TPA concentrations displayed significant reversal of dye coupling inhibition after 40 h. The results indicate that the onset of communication competence impairment between V79 cells by 6-TG is much slower than hitherto implied. Failure of intercellular communication to recover even from short exposure to 100 ng TPA/ml appears to be attributable to its lipophilic nature. Not enough TPA can be removed by washing to overcome its potent inhibitory action. These observations indicate that the physicochemical properties of a chemical must be taken into consideration before making judgements about the duration of inhibition.

Phorbol Ester Inhibition of Ovarian and Testicular Steroidogenesis in Vitro

Possible influences of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) upon gonadal steroidogenesis were investigated in vitro. Granulosa cells from hypophysectomized, estrogen-treated rats were cultured for 2 days in medium containing 0.1 microM androstenedione. Follicle-stimulating hormone (FSH) treatment increased estrogen, progesterone, and 20 alpha-hydroxypregn-4-en-3-one production. Concomitant TPA treatment inhibited FSH-stimulated estrogen production by up to 95% [concentration that induced 50% inhibition of steroid production (ED50), 1.1 ng/ml]. TPA also inhibited FSH-stimulated progesterone (ED50, approximately 0.6 ng/ml) and 20 alpha-hydroxypregn-4-en-3-one (ED50, approximately 1.1 ng/ml) production. N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate increased steroidogenesis; however, cotreatment with TPA blocked progestin but not estrogen production. The TPA inhibition of progestin biosynthesis was accompanied by decreases in FSH-stimulated pregnenolone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity without decreasing the activity of 20 alpha-hydroxysteroid dehydrogenase. In primary cultures of rat testicular cells, human chorionic gonadotropin treatment increased testosterone production 44-fold, whereas concomitant treatment with TPA inhibited testosterone production by up to 86% (ED50, 10 ng/ml). Cholera toxin and N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate also increased testosterone production, while the actions of these agents were decreased by TPA. The TPA suppression of testosterone production was associated with a decrease in accumulation of 17 alpha-hydroxyprogesterone and androstenedione and an increase in progesterone production, suggesting a specific inhibition of 17 alpha-hydroxylase and 17,20-lyase activities. These results demonstrate the inhibitory effects of a tumor promoter upon gonadotropin-stimulated steroidogenesis by cultured rat granulosa and Leydig cells through specific regulation of steroidogenic enzymes. Additional studies may assist in further elucidation of cellular mechanisms associated with carcinogenesis and steroidogenesis.

Mechanism of action of the phorbol ester tumor promoters: specific receptors for lipophilic ligands.

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.