Of phorbol

Conventional and novel PKC isozymes are potently activated by phorbol esters, heterocyclic compounds found in the milky sap exuded by plants of the Euphorbiaccae family. This sap was used medicinally as a counterirritant and cathartic agent over the millennia we now know that the active ingredients, phorbol esters, specifically bind to the Cl domain, the diacylglycerol sensor described above. In fact, their ability to recruit PKC to membranes is so effective that phorbol esters cause maximal activation of conventional PKCs, bypassing the requirement for Ca2+. This module is found in a number of other proteins in addition to PKC, so the profound effects of phorbol esters on cells are mediated by other proteins in addition to PKC. 

Experiments were conducted in which purified trichloroethylene (1 mg in acetone) was applied to the shaved backs of female ICR/Ha Swiss mice (Van Duuren et al. 1979). In an initiation-promotion study, a single application of trichloroethylene was followed by repeated application of phorbol myristate acetate (PMA) promoter. In a second study, mice were treated with trichloroethylene three times per week without a promoter. No significant tumor incidences were observed in these studies. Doses used in these studies were well below the maximum tolerated dose, which is often not reached in dermal studies. 

ROY s, SEN c K, KOBUCHi H and PACKER L (1998) Antioxidant regnlation of phorbol ester-indnced adhesion of hiunan Jurkat T-cells to endothehal cells Free Radical Biology and Medicine 25, 229—41. 

KATiYAR s K and MUKHTAR H (1997) Inhibition of phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate-caused inflammatory responses in SENCAR mouse skin by black tea polyphenols . Carcinogenesis, 18 1911-16. 

The Cu semicorrin complex (68a) has been successfully used as the catalyst for cyclization of alkenyl diazoketones, though the reactions of some substrates showed modest enantioselectivity (Scheme 74).276 Shibasaki et al. have successfully used the cyclization of diazoketone with Cu bis(oxazoline) (101) for the construction of the CD ring skeleton of phorbol.277... 

Activation of PI-PLC-linked receptors, such as the mAChR, results in increased PKC activity. Since the addition of phorbol esters, which are PKC agonists (see Ch. 20), results in phosphorylation of Raf, this mechanism may provide an explanation for the ability of PI-PLC-coupled receptors to activate MAPK. A recently discovered protein tyrosine kinase PYK2, which is enriched in the CNS, is also activated by PKC. Like PTK-X, PYK2 phosphorylates SHC and recruits the Grb2-SOS complex, which results in activation of the MAPK cascade. PYK2 is also activated by... 

In animal studies, mirex (a nonmutagenic hepatocarcinogen) promoted mouse skin squamous carcinomas and papillomas after initiation with 7,12-dimethyl-benz[a]anthracene (DMBA) for 1 week. Mirex, also, potentiated the promotional potency of the phorbol ester tumor promoter, 12-0 -tetradecanoylphorbol-13-acetate (TPA). There was a 90% incidence (activation) of the c-Ha-ras tumor gene in these co-promoted tumors. When both mirex and TPA gave a similar tumor yield, only the TPA response was associated with biochemical markers of enhanced cell proliferation, induction of epidermal ornithine decarboxylase activity and increased DNA synthesis, and hyperplasia. Thus, there is evidence for a dual effect of mirex during co-promotion first, as an independent tumor promoter with a mechanism different than that of phorbol esters and second, as a compound that also potentiates skin tumor promotion by TPA (Meyer et al. 1993, 1994 Moser et al. 1992, 1993). 

As indicated above in the section on "Genotoxic Effects", it is likely that mirex and chlordecone are tumor promoters and not tumor initiators. Initiators irreversibly alter DNA by a mutation, chromosomal aberration, or other alteration. Promoters act by facilitating the proliferation of previously initiated preneoplastic cells. One of the mechanisms for promotion is believed to involve suppression of inhibitory proliferative control through inhibition of gap-junctional-mediated intercellular communication as well as enzyme induction (Trosko et al. 1983). The results of studies to evaluate the promotional activity potential of mirex in mice indicate that mirex is a mouse skin cancer promoter but exerts this toxicity through a hitherto unknown mechanism that is different from that of phorbol esters, such as TPA (Meyer et al. 1993, 1994 Moser et al. 1992, 1993). Unlike initiation, promotion is a reversible process to a point. This implies, at least in theory, that there may be justification for setting NOAELs for promoters. 

Kiss, Z., 1990, Effects of phorbol ester on phosphohpid metabolism. Prog. Lipid Res. 29 141-166... 

Kiss, Z., and Deh, E., 1995, Preferenhal inhibition of phorbol ester-induced hydrolysis of phosphatidylethanolamine by N-acetylsphingosine in NIH 3T3 fibroblasts. FEES Lett. 

Long-chain ester derivatives of phorbol, a tetracyclic diterpene from the seed oil of Croton tiglium L., including its most abundant representative, 12-0-tetradecanoylphorbol-13-acetate (65), are potent activators of protein kinase G (PKG) and are used as standard tumor promoters for the study of experimental carcinogenesis in animal models." ... 

As mentioned in chapter 2.3.1, in many cases it is difficult to distinguish whether the effects of phorbol esters are due to activation or inactivation of... 

Results Indicating no Influence of Phorbol Esters in MDR Modulation... 

Ahn CH, Kong JY, Choi WC, Wang MS (1996) Selective inhibition of the effects of phorbol ester on doxorubicin resistance and P-glycoprotein by the protein kinase C inhibitor l-(5-isoquinolinesulfonyl)-2-methylpipera2inc (H7) in multidrug-resistant MCF-7/Dox human breast carcinoma cells. Biochem Pharmacol 52 393-399... 

O Driscoll KR, Teng KK, Fabbro D, Greene LA, Weinstein IB (1995) Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis. Mol Biol Cell 6 449-458 Ohmi Y, Ohta A, Sasakura Y, Sato N, Yahata T, Santa K, Habu S, Nishimura T (1997) The role of phorbol ester-sensitive protein kinase C isoforms in lymphokine-activated killer cell-mediated cytotoxicity dissociation between perforin-dependent and Fas-dependent c otoxicity. Biochem Biophys Res Commun 235 461-464... 

Ward NE, O Brian CA (1991) Distinct patterns of phorbol ester-induced downr -lation of protein kinase C activity in adriamycin-selected multidrug resistant and parental murine fibrosarcoma cells. Cancer Lett 58 189-193 Warenius HM, Seabra LA, Maw P (1996) Sensitivity to cis-diamminedichloro-platinum in human cancer cells is related to ejq>ression of cyclin D1 but not c-raf-1 protein. Int J Cancer 67 224-231... 

Shibasaki and co-workers used an intramolecular nitrile oxide cycloaddition to prepare the skeleton of phorbol (272) (Scheme 6.99), a tumor promoter that activates protein kinase C (PKC) (333). Nitroalkene 268 was elaborated in several steps from (+)-3-carene (267) and was subjected to cycloaddition by means of -chlorophenyl isocyanate-triethylamine to give cycloadduct 269 in 88% yield. Reductive hydrolysis employing Raney Ni and boric acid afforded hydroxyketone 270, that was subsequently used for the construction of the optically active derivative 271, which contains the phorbol skeleton (333). 

Evidence accumulating from various laboratories points to a role for PKC in mediating the action of lithium in a number of cell systems and the brain (Manji and Lenox 1994, in press]. Currently available data suggest that short-term lithium exposure facilitates a number of PKC-mediated responses, whereas longer-term exposure results in an attenuation of phorbol ester-mediated responses, which may be accompanied by a downregulation of PKC (S. M. P. Anderson et al. 1988 J. A. Bitran et al. 1990 M. S. Evans et... 

Anderson SMP, Godfrey PP, Grahame-Smith DG The effects of phorbol esters and lithium on 5-HT release in rat hippocampal slices. Br J Pharmacol 93 96P, 1988 Andrade C, Gangadhar BN, Swaminath G, et al Predicting the outcome of endogenous depression following electroconvulsive therapy. Convulsive Therapy 4 169-174, 1988... 

TUmor promotion has been demonstrated in a number of experimental models, but the mechanism of promotion has been studied most extensively in mouse skin, rat liver, and cell culture. In skin, the most potent promoters are fatty add esters of phorbol, a plant-derived ditei>... 

Armuth, V. (1976). Leukaemogenic action of phorbol in intact and thymec-tomized mice of different strains, Br. J. Cancer 34,516. 

Baird, W.M. and Boutwell, R.K. (1974). "nunor-fHamoting activity of phorbol and four diesters of phorbol in mouse skin, Cancer Res. 31,1074. 

Obviously, there is some kind of cross-talk between PKC and cAMP in the modulation of intercellular coupling, since cAMP can inhibit the uncoupling effect of phorbol esters if cells are exposed to both agents from the start of the experiment [Kanno et al., 1984], but this protective effect can be abolished by the protein synthesis inhibitor cycloheximide [Enomoto et al., 1984] in Balb/ cells. 

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