Effect of diterpene esters on actin cytoskeleton of SV40-transformed keratinocytes is not reproduced by diacylglycerols

The tumor promoter 12-O-tetradecanoylpborbol-13-acetate (TPA)induces a disorganization of the actin cytoskeleton and itsredistribution at the periphery of the cells in SV40-transformedhuman keratinocytes. This phenomenon is induced by other diterpeneesters, such as 4-O-methyl TPA, 12-O-ethacrynylphorbol-13-acetate(EPA), 12-O-retinoylphorbol-13-acetate (RPA) and mezerein, whichexert either convertogenic or promoting effects in skin tumordevelopment in vivo. Two diacylglycerols: oleyl-acetyl glycerol(OAG) and dioctanoylglycerol (DOG) do not induce the disorganizationof actin. Thus, the effects of these compounds, as they arefound in SV40-transformed human keratinocytes, do not exhibitclearcut correlations to either their protein kinase C activatingabilities, or their effects on different stages of multistagecarcinogenesis in mouse skin in vivo.     

Effects of complete and incomplete tumor promoters on hair growth, angiogenesis, and tenascin expression in the skin of NMRI mice

Although the phorbol esters 12-O-tetradecanoylphorbol- 13-acetate(TPA) and 12-0-retinoylphorbol-13-acetate (RPA) are almost equipotentinducers of an Inflammatory and hyperplastic response in NMRImouse skin, TPA acts as a converting or complete and RPA asa non-converting or incomplete skin tumor promoter. Bearingin mind the converting effect of skin wounding and TGFfß,we have addressed the question of a relationship between conversionand stromal events. For this purpose we have compared the effectsof TPA and RPA on dermal angiogenesis, hair regrowth and theexpression of tenascin in NMRI mouse skin in vivo. In the reticularpart of the dermis, shaving alone induced angiogenesis peakingat day 14, concomitant with hair regrowth. While TPA exhibiteda stimulatory effect on both parameters, RPA suppressed shavinginducedangiogenesis and delayed the onset of hair regrowth. Whetherthis difference in the phorbol ester effects being consistentwith the working hypothesis is critical for conversion, remainsto be shown. In the papillary dermis, however, RPA was morepotent than TPA in inducing vascularization and tenascin expression.The kinetics of both responses corresponded to the time courseof hyperproliferation induced by the phorbol esters in the overlayingepidermis, i.e. may be related to promotion proper, rather thanconversion.     

Effect of mouse skin tumor promoters upon [3H]uridine exchange and focus formation in cultures of C3H/10T1/2 mouse fibroblasts

The abilities of 4-O-methyl-12–O-tetradecanoylphorbol-13-acetate(4–O-methyl-TPA) and mezerein to promote the process oftransformation were evaluated in cultures of C3H/ 10T1/2 mouseembryo fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine.Mezerein was found to be as potent as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate(TPA) for the promotion of focus formation, eliciting a promotionresponse at concentrations that ranged from 100 to 2500 ng/ml.4-O-Methyl-TPA (25–2500 ng/ml) did not promote focus formation,but was mitogenic for confluent cultures. The effects of promotingand non-promoting compounds upon intercellular communicationwere then evaluated to determine if a rapid assay for the inhibitionof communication might serve as a surrogate for the relativelylong term, labor-intensive cell transformation assay. Inhibitedintercellular communication, as measured by inhibition of [3H]uridineexchange between cells, appeared to correlate with the abilityof phorbol related compounds to promote transformation. However,the potent promoter 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin didnot inhibit [³H]uridine transfer. Inhibition of intercellularcommunication may thus be diagnostic of the promoting potentialof phorbol-related compounds in C3H/10T1/2 cultures, but maynot be an appropriate end point for the study of carcinogenicdioxins.     

Enhancement of collagen-degrading enzymes in the dermis after one topical application of tumor-promoting phorbol esters

The effect of tumor-promoting and non-promoting skin mitogenson the induction of matrix degradation in the dermis of mouseskin has been examined. A stimulation of active collagenolyticand proteolytic enzyme levels was observed after applicationof the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate(TPA) and 12-O-retinoylphorbol-13-acetate (RPA) as well as thenon-promoting skin irritant Caionophore A 23187, but not withthe non-irritant mitogen 4-O-methyl-TPA. It therefore appearsthat the enhancement of collagenolytic and proteolytic enzymesafter tumor promoter treatment is mainly due to the inflammationthat is always caused by the promoter. However, a subfractionof collagenolytic enzymes that is not extracted from the dermiswith 0.5 M Nacl but only with 5 M urea is specifically increasedafter treatment with TPA and RPA. This fraction is absent inA 23187-or-4-O-methyl-TPA-treated dermis. This indicates thatpart from inflammation-induced matrix degradation there is alsostimulation of enzymes which are directly related to tumor promotion.     

SENCAR mouse skin tumors produced by promotion alone have A to G mutations in codon 61 of the c-rasHa gene

SENCAR mice, developed by selective breeding for high susceptibilityto skin carcinogenesis by initiation with 7, 12-dimethylbenz[a]anthraceneand promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA),form squamous papillomas in     

Dehydroepiandrosterone and two structural analogs inhibit 12-O-tetradecanoylphorbol-13-acetate stimulation of prostaglandin E2 content in mouse skin

Dehydroepiandrosterone, a naturally occurring adrenal steroid,is a highly effective tumor chemopreventive agent in laboratorymice and rats, inhibiting spontaneous breast cancer and chemicallyinduced tumors of the lung, colon, skin, liver and thyroid.Dehydroepiandrosterone blocks three processes that have beenimplicated in experimental tumorigenesis: (i) carcinogen activationthrough the mixed-function oxidases, (ii) 12-O-tetradecanoylphorbol-13-acetatestimulation of superoxide anion production in neutrophils, and(iii) 12-O-tetradecanoylphorbol-13-acetate stimulation of [³H]thymidineincorporation in mouse epidermis. All of these effects of dehydroepiandrosteronevery likely result from glucose-6-phosphate dehydrogenase inhibitionand a lowering of the NADPH cellular pool. It is now reportedthat oral administration of dehydroepiandrosterone (0.2% inthe diet) for two weeks inhibits the stimulation in prostaglandinE₂ content in mouse epidermis produced by topical applicationof 12-O-tetradecanoylphorbol-13-acetate. Two synthetic steroids,16-fluoro-5-androsten-17-one and 16-fluoro-5-androstan-17-one,which are more potent inhibitors of the above three processesin tumorigenesis and are also more effective than dehydroepiandrosteronein inhibiting skin papilloma development in the mouse, are moreactive in suppressing prostaglandin E₂ induction by 12-O-tetradecanoylphorbol-13-acetate.These two structural analogs, which also lack specific side-effectsassociated with dehydroepiandroster one treatment, may findapplication as cancer chemopreventive drugs in humans.     

Inhibitation of pro-inflammatory cytokine gene expression and papilloma growth during murine multistage carcinogenesis by pentoxifylline

Topical application of 12-O-tetradecanoylphorbol-13-acetate(TPA) to the dorsal epidermis of Sencar mice induces synthesisof pro-inflammatory cytokines, including interleukin-1     

Tumor-promoting phorbol esters cause a stable reduction of dermal collagen in mouse skin

Chronic treatment with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) causes permanently increased levels of active collagenolytic enzymes in the dermis and leads to a stable reduction of dermal collagen content. Non-promoting skin mitogens like the Ca-ionophore A 23187 or the 4-O-methylether of TPA, while being active stimulators of collagenolytic enzymes, do not support chronic collagen degradation throughout the experimental period.On the other hand, TPA-induced collagen degradation is not necessarily influenced by inhibition of tumor promotion. Fluocinolone acetonid (FA), an inhibitor preventing not only tumor development but also chronic inflammation and the establishment of a stationary hyperplasia, has been compared with retinoic acid (RA) which has no influence on either the inflammatory reaction or hyperplasia. While FA inhibited the dermal effects of TPA almost completely, RA at a dose that prevented tumor development by 80% had no effect whatsoever in this respect.Therefore, we conclude that both epidermal proliferation and inflammation are accompanied by collagenolytic reactions in the dermis. During chronic treatment sustained collagenolysis correlates with inflammation and/or the establishment of a stationary hyperplasia. Like these it can be regarded as a necessary but insufficient condition of tumor promotion (second stage).     

Spin-labeled phorbol esters and their interactions with cellular membranes. III. Skin irritant and tumor-promoting activities of spin-labeled phorbol-12,13-diesters and relationships to their particular structures

Sixteen ‘doxy’ spin-labeled 12, 13-(acetate, acylates)of phoebol were assayed in NMRI mice for irritant and for tumor-promotingactivity. The spin-labeled positionally isomeric (ⁿ, ^m)PA-andAP(ⁿ, ^m)-type esters carry straight aliphatic acyl chains ofdifferent overall lengths ^N(number of C atoms). Within the chainsthe ‘doxy’ label is located in different positions(nm). The potency of some of the esters as irritants and aspromoters is comparable to or even higher than that of the prototypediterpene ester promoter 12-^O-tetradecanoylphorbol-13-acetateand its positional isomer 12-O-acetylphorbol-13-tetradecanoate.Their irritancies on the ear and their promoting activitieson the back skin depend strongly on the structural featuresof the acyl chain carrying the spin label. Based upon the bioactivitiesof individual esters biologically meaningful probes were definedfor investigations of the molecular interaction of phorbol-ester-typepromoters with cellular targets for electron paramagenetic resonance.     

Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-n.m.r. and GC-MS spectroscopy

After a single in vivo application of 20 nmol of the ‘complete’tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) orof the ‘incomplete’ promoter 12-O-retinoylphorbol-13-acetate(RPA) to the back skin of mice, a major arachidonic acid metabolite(AAMX) was found in cell-free epidermal preparations after additionof arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenaseinhibitors indicated that this metabolite is a lipoxygenaseproduct. The metabolite has been purified by sequential useof t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z, 9E, 11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and ¹H-n.m.r.spectroscopy. To assist in the characterization of AAMX, a referencemixture of 8-HETE and 9-HETE was used for which a complete structuralanalysis could be performed using one- and twodimensional ¹H-n.m.r.at 500 MHz. The production of 8-HETE has been shown to startwith a lag phase of 3 h after TPA treatment and to reach a maximumafter 24–48 h. Nonpromoting phorbol esters are 10-fold,the Ca²⁺-ionophore A 23187 100-fold, less efficient in inducingin vivo the lipoxygenase responsible for 8-HETE production.3-Day-old neonatal mice cannot be induced to generate 8-HETEin response to TPA, whereas 8-day-old mice show an extremelystrong response. Neither primary basal keratinocytes, isolatedfrom TPA-treated mouse epidermis nor TPA-treated epidermal celllines generate 8-HETE, indicating that the metabolite may notoriginate from these epithelial cells.     

Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects

The effect of the convertogenic (‘first-stage’)tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) andthe nonconvertogenic (‘second-stage’) tumor promoterRPA (12-O-retinoylphorbol-13-acetate) on chromosomes was investigatedin HeLa cells which have previously been shown to exhibit aradiomimetic response to TPA. As in the case of mouse keratinocytes,only TPA had a significant clastogenic activity at non-cytotoxicconcentrations ranging from 10^–8 to 10^–6 M measuredafter 24 and 48 h. The values observed with RPA did not differsignificantly from values observed in the presence of the solvent(acetone, 0.2%). The response to TPA was saturable with respectto the dose of TPA. The chromosomal aberrations (mostly gapsand breaks) were predominantly of the chromatid type. Isochromatidaberrations were caused by a 24 or 48 h treatment with 10^–6M TPA. The aberrations appear as early as 6–8 h afterTPA application, i.e. as soon as the cells have recovered fromTPA-induced inhibition in G₂-phase. Even a 30 min exposure to10^–7 M TPA gives the same yield of aberrations as longertreatment, i.e. the response to TPA is ‘saturable’with respect to time. Both TPA and the non-clastogenic RPA causea temporal G₂-delay thus indicating that the G₂-inhibition isnot related to induction of chromosomal aberrations by TPA.The data are consistent with the hypothesis that TPA induceschromosomal aberrations via a receptor-mediated pathway.     

Inhibitory effect of topical application of polymerized ferulic acid, a synthetic lignin, on tumor promotion in mouse skin two-stage tumorigenesis

A dehydrogenation polymer of ferulic acid (DHP-FA), a syntheticlignin, was evaluated for its ability to inhibit tumor promotionby 12-O-tetradecanoylphorbol-13-acetate (TPA) in the 7,12-dimethylbenz[a]anthracene-treatedskin of female ICR mice. Topical application of DHP-FA inhibitsTPA-induced tumor promotion, whereas a monomeric ferulic aciddoes not show the inhibitory effect.     

Chemopreventive effect of 4'-demethyl epipodophyllotoxin on DMBA/TPA-induced mouse skin carcinogenesis

The chemopreventive effect of topical application of 4'-demethylepipodophyllotoxin (DMEP), an antimitotic agent, on a two-stageskin carcinogenesis model in Swiss Albino mice induced by 9,10-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated.Two topical applications with 0.24% DMBA over 1 week, followedlater by 5 nmol of TPA twice weekly produced 100% incidenceof tumors in these animals by 18 weeks. Treatment of animalswith DMEP (until the end of the experiment), 30 min before TPAtreatment, significantly reduced the tumor incidence, tumorvolume and the conversion efficiency of papillomas to squamouscell carcinomas. The tumor formation and growth was also delayedby DMEP pre-treatment. Application of DMEP protected againstthe losses provoked in levels of glutathione and activity ofcatalase and superoxide dismutase in skin and liver of animalsby the application of DMBA/TPA. Thus, DMEP might possibly beexerting its chemopreventive activity by acting as an antioxidant. Abbreviations: CAT, catalase; DMBA, 9,10-dimethylbenz[a]anthracene; DMEP, 4'-demethyl epipodophyllotoxin; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase; TPA, 12-O-tetradecanoylphorbol-13-acetate. ² To whom correspondence should be addressed Email: neeta{at}medinst.ernet.in     

Epidermal growth factor induces cytogenetic damage in mammalian cells

It has been previously shown that potent tumor promoters, suchas the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA),can induce both chromosomal aberrations and sister chromatidexchanges (SCE) in mammalian cells. We show here that epidermalgrowth factor (EGF) is also capable of inducing SCE. EGF andTPA did not enhance the frequency of SCE in an additive fashion,suggesting these agents do not act by completely independentmechanisms.     

Effects of five phorbal esters on gap junctional lintercellular communication, morphological transformation and epidermal growth factor binding in Syrian hamster embryo cells

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), 12-deoxyphorbol-13-phenylacetate(DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPP A),sapintoxin D (SAP D) and sapintoxin A (SAP A) on the decreasein [¹²⁵I]epidermal growth factor (EGF) binding (indicating proteinkinase C activation), suppression of gap junctional intercellularcommunication (GJIC) and induction of morphological cell transformation(MCT) in Syrian hamster embryo (SHE) cells were investigated.All five phorbol esters were found to reduce [¹²⁵I]EGF bindingin early passage SHE cells at comparable concentrations. DOPPA was     

Cytogenetic effects caused by phorbol ester tumor promoters in primary mouse keratinocyte cultures: correlation with the convertogenic activity of TPA in multistage skin carcinogenesis

The effects of the convertogenic (‘first-stage’)tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), thenon-convertogenic (‘second stage’) tumor promoter12-O-retinoylphorbol-13-acetate (RPA) and the non-promotingphorbol esters 4-O-MeTPA and 4--PDD on the chromosomes of mousekeratinocytes in primary cultures were investigated. In thesetarget cells of tumor promotion TPA caused severe numericaland structural chromosomal aberrations, which were evident aftertwo cell cycles and accumulated after multiple applications.Numerical aberrations were visible as hypo- and hyperdiploidy,with non-random loss or gain of specific chromosomes. The clastogeniceffects were evident as simple alterations such as gaps andbreaks, but more severe alterations such as tri- and quadriradialchromatid interchanges and ring chromosomes, as well as translocationscould be observed. The structural aberrations were nonrandomlydistributed in the genome and chromosomes no. 1, 2, 5, 6 and18 were preferentially involved in rearrangements. In additionto the aneuploidogenic, clastogenic and recombinogenic effectsinduced by TPA, short treatment with this tumor promoter wasefficient in producing cytogenetic equivalents of gene amplification,i.e. double minute chromosomes. The cytogenetic effects werenot merely due to cytotoxicity since they occurred after a lowTPA dose (10^–8 M) and did not considerably increase witha higher dose (10^–6 M). Moreover, at both dose levelscell cycle traverse of mouse keratinocytes was not drasticallyaltered. In contrast, the non-convertogenic tumor promoter RPAand the non-promoting phorbol esters 4--PDD and 4-O-MeTPA (atthe same dose level) did not cause any substantial chromosomalalterations. This discrepancy between the action of TPA andRPA suggests that effects which result in chromosomal alterationsin the target cells may be critical for the conversion stageof skin tumor promotion. This conclusion is supported by experimentswith substances such as antipain and eicosa-5,8,ll,14-tetraynoicacid which inhibit both tumor induction in initiated skin andthe cytogenetic alterations induced by TPA in cultured keratinocytes.These studies provide for the first time the possibility ofdifferentiating between convertogenic and non-convertogenictumor promoters in an in vitro assay using the target cellsof mouse skin carcinogenesis.     

Inhibition of 7, 12-dimethylbenz[{alpha}]anthracene-induced lung tumorigenesis in A/J mice by food restriction is reversed by adrenalectomy

Prior work has demonstrated that food restriction of mice markedlysuppresses12-O-tetradecanoylphorbol-13-acetate (TPA) promotionof skin papillomas and adrenalectomy prior to initiating foodrestriction completely reverses the tumor inhibitory effectof underfeeding. In the present experiment the effect of foodrestriction, with or without prior adrenalectomy, on 7,12-dimethylbenz[     

Inhibition of tumor promotion in benzo[a]pyrene-initiated CD-1 mouse skin by crocetin

The effects of topical application of crocetin on 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced promotion of skin tumors, hyperplasia, hydrogenperoxide, ornithine decarboxylase (ODC) and inflammation wereevaluated in female CD-1 mice. Topical application of crocetin(0.2 or 1.0 µmol) with TPA (15 nmol) twice weekly for20 weeks to mice previously initiated with benzo[     

Phorbol ester-induced leukotriene biosynthesis and tumor promotion in mouse epidermis

In mouse skin in vivo the irritant and hyperplasiogenic tumorpromoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stronglyincreased the epidermal content of the cysteinyl leukotrienesLTC₄, LTD₄ and LTE₄, but not of leukotriene LTB₄. This effectwas completely suppressed by the selective leukotriene biosynthesisinhibitor MK-886. Intragastric administration of MK-886 preventedphorbol ester-induced ear edema, but not epidermal hyperproliferationand tumor promotion. These data indicate that leukotrienes areinvolved in the pro-inflammatory effects of the phorbol ester,whereas its hyperproliferative and tumor-promoting activitiesdo not depend on 5-lipoxygenase-catalyzed leukotriene formation.This action differs from several non-selective inhibitors oflipoxygenases that were found to inhibit tumor promotion hiinitiated mouse skin.