Artificial Analogs of Naturally Occurring Tumor Promoters as Biochemical Tools and Therapeutic Leads

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.     

Structural basis of protein kinase C activation by diacylglycerols and tumor promoters.

Protein kinase C is a ubiquitous and important regulatory enzyme. The enzyme is physiologically activated in a temporary manner by (S)-diacylglycerols (DAGs), which are themselves generated by the phospholipase C mediated hydrolysis of polyphosphoinositides. The (S)-DAGs specifically bind to the regulatory domain of PKC and cause the activation of the PKC toward substrate. Minor modifications in the DAG result in inactive molecules. On the other hand, the structurally diverse, polycyclic tumor promoters also specifically activate PKC by binding to the same effector site as do the DAGs. The object of this paper is to present a discrete structural model that accounts for the activation of PKC by both the tumor promoters and the DAGs. The unique model presented is based on experimentation rather than on computer-driven hypotheses which, experience has shown, generally produce incorrect structural models when applied to PKC. The model described here begins with a structural analysis of the tumor-promoting debromoaplysiatoxins (DATs). DAT is an ideal starting molecule, because it is conformationally rigid with a known relative and absolute configuration, and it is synthetically manipulable. The pharmacophore of DAT was experimentally determined, and this pharmacophore serves as a template for further analyses. This template is used to predict the active conformer of the acylic DAGs; this conformer is then used to reveal the pharmacophore of various families of tumor promoters. The overall model presented is consistent with published structure-activity studies on the tumor promoters and makes testable predictions that have proven to be correct thus far.     

Regulation of T-cell-derived protein kinase C activity by vitamin A derivatives

Protein kinase C (PKC) is a ubiquitous enzyme linked to transmembrane signal transduction. It regulates agonist-mediated activation of intracellular events that result in growth and differentiation in a variety of cells and tissues. PKC is the cellular receptor for phorbol ester tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), that bind to, and directly activate, this enzyme. Vitamin A analogs (retinoids) have been known to antagonize biologic effects of phorbol esters, e.g., promotion of skin tumor formation; however, the extract mechanism(s) of this action is not clear. To analyze the effects of retinoids on T-cell-derived PKC, we partially purified the enzyme from human leukemic T cells (Jurkat) and examined the effects of different vitamin A analogs on its activity. Furthermore, the regulatory effects of retinoids on PKC activity were compared with those of common membrane phospholipids. Retinal inhibited PKC activation induced by TPA, as well as by diacylglycerol, the physiologic activator of PKC. The observed inhibition resulted from competition with phospholipid (phosphatidylserine) and was selective for the phospholipid-dependent C kinase; cAMP-dependent protein kinase, which is phospholipid-independent, was not affected by retinal. The inhibitory effect of retinal on PKC activity was similar to that of phosphatidylcholine. Retinoic acid, in contrast to retinal, induced a Ca2+-dependent activation of PKC, thus substituting for phosphatidylserine. Furthermore, PKC activation by retinoic acid was similar to that by phosphatidylserine, the natural phospholipid cofactor, in that both could be inhibited by phosphatidylcholine and augmented by phosphatidylinositol. The inhibition or activation of PKC by retinal or retinoic acid, respectively, was independent of whether the terminal aldehyde (retinal) or carboxyl (retinoic acid) groups were in the trans or cis configuration. Other vitamin A analogs tested did not affect PKC activity. The results demonstrate that different retinoids and phospholipids may have positive or negative cooperativity in PKC activation, thereby regulating its enzymatic activity and affecting the resulting intracellular activation events. These findings suggest that at least part of the biologic effects of retinoids in general, and their modulation of T-cell function in particular, may be mediated via the influence of their intracellular metabolites on PKC, and that this mechanism may account for some of the antagonistic effects of retinoids on TPA-mediated responses in cells.     

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

Background  

Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.     

Contribution of individual PKC isoforms to breast cancer progression

The protein kinase C (PKC) family of serine/threonine kinases has been intensively studied in cancer since their discovery as major receptors for the tumor-promoting phorbol esters. The contribution of each individual PKC isozyme to malignant transformation is only partially understood, but it is clear that each PKC plays different role in cancer progression. PKC deregulation is a common phenomenon observed in breast cancer, and PKC expression and localization are usually dynamically regulated during mammary gland differentiation and involution. In fact, the overexpression of several PKCs has been reported in malignant human breast tissue and breast cancer cell lines. In this review, we summarize the knowledge available on the specific roles of PKC isoforms in the development, progression, and metastatic dissemination of mammary cancer. We also discuss the role of PKC isoforms as therapeutic targets, and their potential as markers for prognosis or treatment response.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

Phorbol esters can persistently replace interleukin-2 (IL-2) for the growth of a human IL-2-dependent T-cell line

A selected clone from an IL-2-dependent human T-cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutylate (PDBu). Thus, a TPA(PDBu)-dependent T-cell line, designated TPA-Mat, was established from IL-2-dependent T cells. The TPA-dependency of TPA-Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA-Mat cells still showed IL-2-dependent growth. However, the TPA (PDBu)-dependent growth of TPA-Mat did not seem to be mediated by an autocrine mechanism of IL-2 or by any other growth factor production, because these factors were not detected in TPA-Mat cell supernatants. Therefore, the phorbol esters substituted for IL-2 and may be directly involved in transduction of growth signals in TPA-Mat cells. Although activity of PKC was down-regulated, messenger ribonucleic acid (mRNA) of the PKC beta-gene was detected in TPA-Mat cells cultured with PDBu. Furthermore, the growth of TPA-Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL-2-dependent TPA-Mat cells.     

New insights into the regulation of protein kinase C and novel phorbol ester receptors.

Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways.     

Induction of tumor cell resistance to macrophage-mediated lysis by phorbol esters: a postbinding event

Activated macrophages can recognize, bind to, and lyse tumor cells in an antibody-independent manner. We have found that tumor cells pretreated with phorbol esters are markedly less susceptible to macrophage-mediated cytolysis, although the initial binding step is unaffected. Tumor cells preincubated with tumor-promoting phorbol esters (10(-8)-10(-6) M) were rendered resistant to macrophage kill whereas non-tumor-promoting derivatives were inactive in protecting tumor cells against cytolysis. Inhibition of [3H]phorbol-12,13-dibutyrate binding by other phorbol esters correlated with their potency as tumor promoters and their ability to render tumor cells resistant to macrophage killing. The role of protein kinase C as the receptor to phorbol esters was implicated by inhibition of PDBu binding by phenothiazine derivatives. This suggests a possible mechanism for the resistance of phorbol ester-treated tumor cells to macrophage-mediated cytolysis.     

Human T lymphocyte activation by tumor promoters: role of protein kinase C

Protein kinase C (PKC) has a major role in a ligand-receptor-mediated signal transduction system in a variety of cell types including T lymphocytes. One of the early phenotypic changes associated with T cell activation is the expression of cell surface receptors for interleukin 2 (IL 2). To test the role of PKC in regulation of IL 2 receptor (IL 2-R) expression and T cell activation in general, we used tumor promoters (TP) as modulators of PKC and compared their effects on intact human T cells and on the enzymatic activity of T cell-derived PKC in a cellfree system. In T cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced IL 2-R expression and proliferation associated with cytosol-to-membrane PKC translocation. A dose of TPA (1 to 4 ng/ml) that induced about 50% of the maximal activation of PKC in the enzymatic assay also induced half-maximal effects on cell proliferation, IL 2-R expression, and PKC redistribution in intact T cells. Structure-function studies with several phorbol ester analogs and non-phorbol ester TP directly correlated tumor promotion activity with the ability to activate PKC and induce IL 2-R. An inhibitor of PKC, chlorpromazine, was found to suppress TPA-mediated proliferation and IL 2-R expression, and inhibited T cell-derived PKC by competing with the phospholipid. Ca2+ ionophore, which synergizes with TPA in induction of T cell proliferation, facilitated the TPA-induced PKC translocation to the membrane. The results thus demonstrate a direct correlation between the effects of various chemicals on: subcellular redistribution of PKC in T cells; induction of T cell proliferation and IL 2-R expression; and activation of T cell-derived PKC in vitro. These data provide further support for the role of PKC in transduction of activation signals in T cells and in regulation of IL 2-R expression.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.     

Synthesis and biological evaluation of 2-alkoxycarbonylallyl esters as potential anticancer agents

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin.     

Further characterization of tumor-promoter-mediated activation of protein kinase C

Tumor promoting phorbol esters are able to activate Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C) in a reconstituted system. Indol alkaloid teleocidin, a tumor promoter, has been found to be as potent as tumor promoters from the series of phorbol esters and mezerein in activating the mouse brain enzyme. Chemically unrelated tumor promoters such as tetrachlorodibenzo-p-dioxin, anthralin and phenobarbital are devoid of effect. Diacylglycerol 1,2 diolein strongly activated the enzyme whereas 1,3 diolein like 1,2 distearin were poor activators and 1,3 distearin was inactive. Although tumor-promoter-or diacylglycerol-mediated activation of protein kinase C was observed in the presence of 0.5mM EGTA, the reaction requires traces of Ca2+. Tumor promoters did not prevent inhibitory action of antipsychotic phenothiazines and local anesthetics but appear to increase IC50 of these drugs.     

Chemical modifications of resveratrol for improved protein kinase C alpha activity

Resveratrol (1) is a naturally occurring phytoalexin that affects a variety of human disease models, including cardio- and neuroprotection, immune regulation, and cancer chemoprevention. One of the possible mechanisms by which resveratrol affects these disease states is by affecting the cellular signaling network involving protein kinase C alpha (PKCα). PKCα is a member of the family of serine/threonine kinases, whose activity is inhibited by resveratrol. To study the structure-activity relationship, several monoalkoxy, dialkoxy and hydroxy analogs of resveratrol have been synthesized, tested for their cytotoxic effects on HEK293 cells, measured their effects on the membrane translocation properties of PKCα in the presence and absence of the PKC activator TPA, and studied their binding with the activator binding domain of PKCα. The analogs showed less cytotoxic effects on HEK293 cells and caused higher membrane translocation (activation) than that of resveratrol. Among all the analogs, 3, 16 and 25 showed significantly higher activation than resveratrol. Resveratrol analogs, however, inhibited phorbol ester-induced membrane translocation, and the inhibition was less than that of resveratrol. Binding studies using steady state fluorescence spectroscopy indicated that resveratrol and the analogs bind to the second cysteine-rich domain of PKCα. The molecular docking studies indicated that resveratrol and the analogs interact with the protein by forming hydrogen bonds through its hydroxyl groups. These results signify that molecules developed on a resveratrol scaffold can attenuate PKCα activity and this strategy can be used to regulate various disease states involving PKCα.