Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Research Articles

Possible anti-tumour-promoting activity of components in Japanese soybean fermented food, Natto: effect on gap junctional intercellular communication.

In order to detect any protective agent against tumor formation, we examined the anti-tumor-promoting effect of a Japanese traditional soybean fermented food, Natto. Dye transfer was employed as an assay method. When fluorescent dye was microinjected into cultured BALB/3T3 cells, the dye was transformed into the neighboring cells through the gap junction. This dye transfer was blocked by the treatment with the tumor promoters 12-O-tetra-decanoylphorbol-13-acetate (TPA), a high concentration of NaCl and lithocholic acid (LCA). This reduction of the dye transfer by TPA treatment was not observed when the cells were pretreated with retinoic acid, an anti-tumor promoter. Thus, the recovery of the dye transfer in TPA-treated BALB/3T3 cells was proven to ge a good indicator for detecting some possible anti-tumor promoters. After extraction and fractionation of Natto, we obtained an active fraction (H1) which showed recovery of the dye transfer in TPA-treated cells. The fraction contained straight-chain saturated hydrocarbons. A comparison of the fraction and the authentic samples by GC analysis suggests that the H1 fraction contained straight-chain saturated hydrocarbons from around C30 to C32. Among these hydrocarbons, hentriacontane (C31) was found at the highest concentrations, and was shown to have the highest activity. Hentriacontane at a very low concentration of 0.65 ng/ml was shown to recover the dye transfer inhibited by the treatment with TPA as well as with NaCl and LCA.

Initiating activity of quinones in the two-stage transformation of BALB/3T3 cells.

2-tert-Butyl-1,4-benzoquinone (BQ), a metabolite of the rodent carcinogen 3-tert-butyl-4-hydroxyanisole (3-BHA), has been shown previously to have initiating activity for cell transformation. In this paper, we examined the initiating activity of quinones in a two-stage transformation assay using BALB/3T3 cells. Cells were treated first with a quinone and then with the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA). The quinones tested were 1,4-benzoquinone (pQ), phenyl-1,4-benzoquinone (PhQ), menadione and 2,5-di-tert-butyl-1,4-benzoquinone (DBQ) in addition to BQ. pQ is a metabolite of benzene and phenacetin, and PhQ is a metabolite of o-phenylphenol (OPP) and sodium o-phenylphenate (OPP-Na). All of the tested quinones induced transformation in the presence of TPA but not in its absence. The extent of transformation caused by quinones followed by TPA was weak but statistically significant. Thus these quinones were shown to act as initiators in the transformation of BALB/3T3 cells. This result suggests that BQ, pQ and PhQ may be involved in carcinogenesis by 3-BHA, benzene, phenacetin, OPP and OPP-Na in vivo. Menadione has been reported to cause cytotoxic effects and mutations through active oxygen generation from semiquinone radicals. DBQ has two bulky substitutes which interfere with covalent bonds with DNA. Menadione and DBQ exhibited initiating activity in the present study. This result suggests that active oxygens generated from semiquinone radicals may play a role in the initiation of cell transformation.

Inhibition of the tumor promoting action of 12-O-tetradecanoylphorbol-13-acetate by tubeimoside III isolated from Bolbostemma paniculatum.

As tubeimoside I isolated from Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae) has been shown to suppress tumor promoter effects, tubeimoside III from the same plant was tested in vitro and in vivo against the action of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tubeimoside III, the natural analog of tubeimoside I, also had an anti-inflammatory effect on mouse ear edema induced by arachidonic acid and TPA and a potent anti-tumor promoting effect on two-stage carcinogenesis of mouse skin after topical application. However, the important difference in bioactivities between tubeimosides I and III is the non-activity of tubeimoside III as an inhibitor of tumor promotion if administered orally. Differences in metabolism connected with different routes of the compound may be one of a number of explanations of the important difference.

Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumor. II.

To search for possible anti-tumor-promoters, we carried out a primary screening of twenty-four 29-nor-cucurbitacin glucosides isolated from the roots of Cayaponia tayuya (Cucurbitaceae) using an in vitro synergistic assay system. Of these glucosides, cayaponosides B (5), B3 (7), D (8), D3b (22) and C2 (23) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, 5 and 23 exhibited remarkable anti-tumor-promoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Spectroscopic characterization of specific phorbol ester binding to PKC-receptor sites in membranes in situ.

Electron paramagnetic resonance (EPR) spectra were measured for the spin labelled phorbol-12,13-diester [5,6]PA bound to membranes of the particulate fraction of mouse brain tissue rich in PKC receptors. [5,6]PA is a bioactive derivative of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), carrying the spin label in a doxyl group in position 7' of the 12-O-tetradecanoyl residue. A mathematical model based on special algorithms (Griffith, O.H. and Jost, P.C. Spin labeling: theory and applications, 1976) allows a satisfactory reconstruction of the experimental spectrum. It reveals that in the experimental spectrum the signal from the [5,6]PA molecules bound non-specifically to the lipid bilayer of the membranes is superimposed by the signal of [5,6]PA molecules bound specifically, i.e. to the active site of PKC (approximately 10% of total EPR signal intensity). Moreover, interpretation of spectral parameters indicates that in [5,6]PA molecules bound specifically the tetradecanoyl chain exhibits a larger motional freedom compared to that in [5,6]PA bound non-specifically. These new findings are in accordance with views featured independently by two recent molecular models of interaction of PKC with cellular membranes (8,9).

Molecular Cloning of TPAR1, a Gene Whose Expression Is Repressed by the Tumor Promoter 12-O-Tetradecanoylphorbol 13-Acetate (TPA)

We previously isolated a partial cDNA sequence, termed TPAR1 (TPA repressed gene 1), from a cDNA library constructed from C3H10T1/2 mouse embryo fibroblasts treated with TPA, using a differential screening procedure. (M.D. Johnson et al. Mol. Cell. Biol. 7, 2821-2829, 1987). In the present study, we have cloned two corresponding full-length 1.9- and 3.4-kb cDNAs of TPAR1 from murine cDNA libraries. Sequence analysis of these TPAR1 cDNAs revealed that they encode 89 and 93 amino acid polypeptides, respectively, with a putative leader sequence and show significant homology with the human cytokine interleukin-8 (IL-8) and its superfamily. Genomic DNA isolation and structural characterization provide evidence that the TPAR1 mRNAs are transcribed from a single gene with alternative splicing. TPAR1 mRNAs are expressed ubiquitously among adult mouse tissues as three major transcripts, 1.9, 3.4, and 6.5 kb, whose expression depends on the tissue type. The levels of TPAR1 mRNAs were markedly decreased in fibroblasts following TPA treatment and also in serum-deprived quiescent fibroblasts stimulated by serum. The levels of TPAR1 mRNAs were dramatically down-regulated in regenerating rat liver when compared to normal adult liver. In addition, there was no detectable expression of TPAR1 in three rat hepatoma cell lines and several transformed fibroblast cell lines. Thus, the TPAR1 gene is a new member of the cytokine IL-8 superfamily, whose expression is down-regulated in rapidly dividing cells. Further studies are required to determine whether it plays a negative role in controlling cell proliferation and tumorigenesis.

Ornithine decarboxylase gene expression is aberrantly regulated via the cAMP signal transduction pathway in malignant H-ras transformed cell lines.

We have tested the hypothesis that H-ras transformed cells contain alterations in signal pathways important in controlling the expression of ornithine decarboxylase (ODC), the highly regulated rate-limiting activity in the biosynthesis of polyamines. Mouse 10T1/2 fibroblasts and a series of 10T1/2 H-ras transformed cell lines were treated with stimulators of cAMP synthesis (forskolin and cholera toxin), a biologically stable analogue of cAMP (8-bromo-cAMP), and an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine). Elevations in ODC gene expression were noted in H-ras transformed cells that were not observed in parental 10T1/2 fibroblasts. The forskolin-mediated effects were not detected with 1,9-dideoxyforskolin, a compound structurally related to forskolin, which does not activate adenyl cyclase. The effects observed with cholera toxin were not detected when cells were treated with the purified subunits of this compound, indicating that the toxin-induced effects were cAMP-specific. Actinomycin D treatment prior to forskolin exposure reduced the elevation observed in ODC gene expression indicating the involvement of the transcriptional process. Furthermore, we observed that cycloheximide treatment of malignant but not benign H-ras transformed cells significantly elevated ODC message level. Treatment of malignant cells with both cycloheximide and forskolin together resulted in a further additive elevation in ODC message, but a similar treatment of benign tumor cells reduced the forskolin-mediated increase in ODC message. In addition, treatment of H-ras transformed cells with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10T1/2 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin‐1α mediates phorbol ester‐induced inflammation and epidermal hyperplasia

The topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to murine skin produces acute inflammatory and hyperplastic responses that have been associated with the promotion stage of skin carcinogenesis. It has been shown in a previous study that TPA induces the expression of the highly inflammatory cytokine, interleukin (IL) -1 alpha, in the epidermis of SENCAR mice. The goal of this study was to investigate the role of IL-1 alpha in several TPA-induced responses in skin. Topical application of TPA (1 microgram) enhanced the production of immunoreactive IL-1 alpha protein, primarily associated with the suprabasal keratinocytes. IL-1 alpha intradermally injected in the dorsal surface significantly increased (P < 0.001) vascular permeability at low concentrations (1-1000 microU) and increased (P < 0.001) inflammatory cell infiltration and epidermal hyperplasia at higher concentrations (10(3) U). TPA produced fourfold increases in vascular permeability as measured by Evans blue dye leakage; this effect was prevented by intradermal injection of anti-IL-1 alpha antibody (25-75 micrograms). Furthermore, injected anti-IL-1 alpha antibody significantly reduced (P < 0.001) TPA-induced inflammatory cell infiltration and epidermal hyperplasia. This study suggests that IL-1 alpha directly or indirectly mediates the inflammatory and hyperplastic responses elicited by topical treatment with TPA.

Gene activation studied by immunological methods.

Gene activation can be studied at several levels: transcription (mRNA), translation (proteins), or phenotypical alterations (functional activity or morphology). These levels can be studied in situ or biochemically by the use of specific probes for normal or altered DNA, mRNA, or proteins. Immunological probes are potent tools for studies of alterations induced by xenobiotics in target organs. When the effects of xenobiotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ and biochemical techniques are necessary. Antibodies to normal or altered cellular constituents are used for identification, quantitation, and cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-acetylaminofluorene (AAF) forms DNA adducts, which can be identified immunologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentrations in proliferating cells and preneoplastic foci in the livers of AAF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to phenotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for studies of gene activation. In mouse skin, the keratins K1 and K10 are only expressed in suprabasal, differentiating cells. BrdU pulse chase experiments combined with double immunofluorescence have revealed that K1 and K10 are sequentially turned on 18 to 24 hr after DNA synthesis and are followed by suprabasal migration. After a single application of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of cantharidin or 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis induces a cell population shift that causes altered keratin distribution

Abstract The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) causes changes in epidermal protein expression, especially in the major differentiation products keratins K1 and K10. These keratins and filaggrin were studied in a pulse-labelled cell cohort in hairless mouse epidermis stimulated to proliferate by TPA or the hyperplasiogen cantharidin. Cells in DNA synthesis were pulse-labelled by 5-bromo-2-deoxyuridine (BrdU) 16 h after topical application of cantharidin or TPA. The BrdU-labelled cell cohort, the two keratins, and filaggrin were spatially mapped by paired immunofluorescence staining. Both cantharidin- and TPA-treated epidermis displayed altered distributions of K1 and K10 with expression only in the outermost cell layers, but the start of their postreplicative expression parallelled that in normal epidermis (18 h for K1 and 24 h for K10 after the last round of DNA synthesis). Cantharidin- and TPA-induced epidermal hyperplasia showed increased basal cell proliferation, accelerated suprabasal migration, and shortened transit time. Thus, the newly formed hyperplastic epidermis was composed of keratinocytes with a lower mean cellular age than that seen in unperturbed epidermis, which caused altered distribution of K1 and K10. Both hyperplastic and normal epidermis showed filaggrin expression in stratum granulosum; this started earlier in treated (30–36 h) than in untreated (96 h) skin. We concluded that the postmitotic onset of K1 and K10 expression was unaltered in regenerative epidermis, whereas filaggrin expression was considerably accelerated and thus influenced by the cell kinetic changes.

Phorbol ester modulation of a novel cytoplasmic protein binding activity at the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA and role in message stability.

The R2 gene of ribonucleotide reductase is elevated in BALB/c 3T3 fibroblasts treated with the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment increased the half-life of the R2 message by 3-fold, showing that TPA regulates R2 gene expression by a post-transcriptional mechanism(s). A 20-nucleotide (nt) TPA-responsive region was found within the R2 mRNA 3'-untranslated region (3'UTR). Ultraviolet cross-linking detected a novel 45-kDa protein-R2 mRNA complex migration band that bound selectively to the 20-nt fragment and did not bind to the 5'UTR or the coding region of the R2 message, or to the 3'UTRs of mRNA from several other genes, or to the homopolymer poly(A) sequence. The-45 kDa protein-R2 mRNA binding activity observed in unstimulated cells was markedly down-regulated after TPA treatment. Deletion of a 201-nt region, containing the 20-nt sequence, from the 3'UTR caused stabilization of hybrid chloramphenicol acetyltransferase mRNA in the absence of TPA treatment. Furthermore, in vitro decay reaction mixtures supplemented with the 20-nt sense RNA transcript resulted in stabilization of R2 message. A model is presented of R2 message regulation in which a cis-element within the 20-nt sequence of the 3'UTR interacts with a cytosolic protein to form a 45-kDa protein-mRNA binding complex. The TPA-induced alteration of R2 message stability is at least in part due to the down-regulation of the 45-kDa protein-mRNA binding activity which is linked to a reduction in the rate of R2 mRNA degradation.

Growth inhibition of human melanoma-derived cells by 12-O-tetradecanoyl phorbol 13-acetate.

In vitro growth of 6 human melanoma-derived cell lines was inhibited markedly by the phorbol-ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of several isoforms of protein kinase C (PKC). Utilizing PKC isoform-specific antibodies in immunoblotting experiments, we found that the PKC alpha and PKC epsilon isoforms were expressed in all of the 6 melanoma cell lines tested, whereas the PKC beta isoform was expressed at detectable levels in only 2 of the 6 cell lines. The SK-Mel-173 melanoma cell line, which had relatively high levels of PKC beta mRNA and protein expression, and which was also the most sensitive to cell growth inhibition by TPA, was used to isolate clones whose growth was less inhibited by TPA. Immunoblotting experiments revealed that in parental SK-Mel 173 cells PKC beta was rapidly down-regulated to below detectable levels after treatment for 48 hr with TPA, but that in TPA-resistant variant clones there was negligible down-regulation of PKC beta by TPA. On the other hand, treatment of parental and TPA-resistant SK-Mel 173 cells with TPA led to partial down-regulation of PKC alpha in both cell lines. Total PKC enzyme activity was also greater in TPA-resistant cells than in parental SK-Mel 173 cells. Our results show that TPA might inhibit the growth of melanoma cells by causing down-regulation of specific isoforms of PKC that are required to maintain the growth of these cells.

Two inhibitors of gap junctional intercellular communication, TPA and endosulfan: different effects on phosphorylation of connexin 43 in the rat liver epithelial cell line, IAR 20.

The skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the chlorinated insecticide, endosulfan, are two potent inhibitors of gap junctional intercellular communication. In the present study the effects of TPA and endosulfan on cell communication have been investigated in IAR 20 rat liver epithelial cells, as well as the effects of these compounds on connexin 43 (cx43), the main gap junction protein in this cell line. The results clearly demonstrate that at non-toxic doses both compounds inhibited the cell communication by at least 90% within 5 min. The communication was partially restored after 4 h of TPA exposure and almost fully restored by 24 h, whereas in endosulfan-exposed cells the communication was completely down-regulated for the whole exposure-period of 24 h. Immunoblots of IAR 20 cell extracts indicated that TPA initially caused an increased phosphorylation of cx43. A normal phosphorylation pattern was observed after 4 h when the cell communication was restored. Immunoblot analysis after endosulfan-exposure showed a slightly increased phosphorylation of cx43 after 10 min treatment, gradually followed by dephosphorylation during the rest of the 24 h treatment period. Immunostaining of IAR 20 cells showed that both compounds caused a rapid disappearance of cx43 from the cell membrane. After 4 h of exposure immunofluorescent cx43-plaques started to reappear in the cell membrane, although less pronounced in endosulfan-treated cells. However, after 24 h of endosulfan-exposure a high number of cx43-spots was demonstrated. These results indicate that different mechanisms are responsible for the inhibition of gap junctional intercellular communication induced by TPA and by endosulfan, at least during the later part of the 24 h exposure-period. TPA causes a marked hyperphosphorylation of cx43, whereas endosulfan increases phosphorylation initially only slightly but longer exposure-periods lead to hypophosphorylation. Thus, phosphorylation as well as dephosphorylation seem to be important factors involved in the regulation of the function of cx43 in this cell line.

HL-60 cell differentiation induced by phorbol- and 12-deoxyphorbol-esters.

The human promyelocytic leukaemia cell (HL-60) undergoes differentiation into a macrophage-like form when exposed to both tumour promoting- and non-promoting phorbol esters. We have investigated the effect of the two non-promoting phorbol esters, 12-deoxyphorbol-13-O-phenylacetate (Dopp) and 12-deoxyphorbol-13-O-phenylacetate-20-acetate (Doppa) on HL-60 cultures, and compared them with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All phorbol esters tested were found to be able to stop HL-60 proliferation and induce cell adherence and morphological changes characteristic of differentiation. TPA, fully differentiating at 1 nM, was more potent than Dopp and Doppa, which required 100 nM for full differentiation effects within the 4 day study. Doppa initially appeared weaker than Dopp at inhibiting incorporation of thymidine, the earliest effect studied, but we were able to detect rapid C-20 deacylation of Doppa, converting it to Dopp, using an HPLC protocol presented here. A detailed study of this thymidine incorporation inhibition showed that both TPA (10 nM or greater) and Dopp (500 nM or greater) have very similar time courses, with 50% inhibition occurring at approximately 12 h, in contrast to Doppa which had a significantly delayed time course at all doses tested. Exposure tests indicated that Dopp and Doppa could be washed from the cells much more easily than TPA. The data presented here strongly support the notion that the metabolic conversion of Doppa to Dopp by HL-60 cells was necessary to mediate its differentiating effects. Since protein kinase C (PKC)-beta 1, present in HL-60 cells, has been found to be the only PKC isotype activated so far in vitro by Doppa, our results suggest that activation of this isotype is not sufficient to drive HL-60 differentiation in vivo.

Effects of increased expression of protein kinase C on radiation-induced cell transformation.

The in vitro oncogenic transformation of C3H 10T1/2 cells by ionizing radiation is known to be enhanced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). It is also known that the activation of protein kinase C (PKC) by TPA is an important step in its tumor-promoting effect. In the present study, we examined the effects of overexpression of a specific isoform of PKC, PKC beta 1 on gamma-ray-induced transformation of 10T1/2 cells. In addition, the effects of overexpression of PKC beta 1 on the malignant phenotype of a previously transformed 10T1/2 cell line were also evaluated. Derivatives of 10T1/2 cells that stably overexpress PKC beta 1 were obtained by transduction with the retroviral expression vector pMV7 carrying the rat PKC beta 1 cDNA sequence. We found that the parental 10T1/2 cells and a control cell line 10T1/2 MV7, which carried only the pMV7 vector without the cDNA insert, expressed dose-dependent transformation frequencies when exposed to gamma-rays. On the other hand, concurrently treated PKC-overexpressing cells that had an 11-fold increase in enzyme activity (PKC-4 cells) failed to yield any morphologically identifiable foci. Cell lines that expressed lower levels of PKC beta 1 were partially resistant to transformation by gamma-rays. Clonogenic survival data indicated that this observation was not due to radioresistance per se. Thus, overexpression of PKC beta 1 did not appear to function as an endogenous substitute for TPA in promoting radiation-induced transformation. Furthermore, overexpression of PKC did not reverse the transformation phenotypes in tumorigenic 10T1/2 cells once it was established. These findings are discussed with respect to the specific roles of individual isoforms of PKC in growth control.

Phospholipid peroxidation in tumor promoter-exposed mouse skin.

We have investigated lipid peroxidation in the skin of CD1 mice following single or repeated topical applications of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). A substantial accumulation of hydroxyphospholipids, to levels 3-5 times control values, followed exposure to two or more TPA treatments (24-72 h intervals), whereas single applications were ineffective. Sodium borohydride reduction increased the yield of product by approximately 50%, suggesting the additional presence of phospholipid hydroperoxides in the oxidized lipids. Straight phase HPLC analysis of the constituent hydroxy fatty acids, followed by gas chromatography/mass spectrometry, revealed that oxidized derivatives of linoleic acid, including 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODE), were the primary products. Stereochemical analysis showed ratios of S to R stereoisomers of 1.3 for 13-HODE and 1.27 for 9-HODE, which implied that TPA-induced peroxidation was primarily due to free radical oxidation, although a partial contribution of enzyme (lipoxygenase) activity is possible. The TPA-induced peroxidation was greater in the epidermis than in the dermis. Pre-exposure of mouse skin to the anti-inflammatory agent fluocinolone acetonide, antioxidants and enzyme (phospholipase A2 and lipoxygenase) inhibitors lowered the peroxidation response to subsequent exposure to TPA. Phospholipid peroxidation products may be useful markers of oxygen radical production in TPA-exposed mouse skin with possible relevance to tumor promotion.

Enhancement of human papillomavirus type 18 gene expression in HeLa cells by 12-O-tetradecanoylphorbol-13-acetate, 3 beta,5 alpha-dihydroxycholestan-6-one, and cholesterol.

Human papillomavirus type 18 (HPV18) is involved in the genesis of cervical cancer through expression of its viral oncoprotein in infected cells. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was found to increase the level of HPV18 transcripts in an HPV18-harboring cervical cancer cell line, HeLa. A similar increase in HPV18 expression was also observed on treatment of the cells with an oxygenated cholesterol, 3 beta,5 alpha-dihydroxycholestan-6-one (yakkasterone). The effects on HPV18 expression elicited by TPA and yakkasterone were repressed by a protein kinase inhibitor, staurosporine. Treatment of the cells with cholesterol under serum-free conditions also resulted in an apparent increase of HPV18 expression.

Inhibition of two-stage skin carcinogenesis as well as complete skin carcinogenesis by oral administration of TMK688, a potent lipoxygenase inhibitor.

1-([5'-(3''-methoxy-4''-ethoxycarbonyloxyphenyl)-2',4'- pentadienoyl]aminoethyl)-4-diphenylmethoxypiperidine (TMK688) is a potent and orally active 5-lipoxygenase inhibitor having anti-histamine activity in its moiety. Recently, we have found that TMK688 also inhibits epidermal cyclooxygenase activity with a potency similar to its inhibiting 5-lipoxygenase. Oral administration of 30 mg/kg TMK688, a dose which markedly inhibits tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated LTB4 formation in mouse skin, markedly inhibited both TPA-promoted and a non-TPA-type tumor promoter anthralin-promoted skin tumor formation in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice. The inhibitory effect of TMK688 was not due to any damage inflicted on the initiated cells but due to its antitumor-promoting activity. TMK688 not only inhibited two-stage skin carcinogenesis but also inhibited benzo[a]pyrene-caused complete skin carcinogenesis. Throughout the tumorigenesis experiment, the survival rate of animals was 100% and the TMK688-treated mice looked healthy. The body weight gain of TMK688-treated mice was not significantly different from that of non-treated mice. Both TMK688 and 1-([5'-(3''-methoxy-4''-hydroxyphenyl)-2',4'-pentadienoyl]amino eth yl]-4-diphenylmethoxypiperidine (TMK777), an active metabolite of TMK688 having more potent 5-lipoxygenase inhibitory activity and less potent cyclooxygenase inhibitory activity than TMK688, inhibited epidermal 8-lipoxygenase activity induced by a topical application of TPA to mouse skin. The 8-lipoxygenase inhibitory activity of TMK777 was approximately 5 times more potent than that of TMK688. Indomethacin, a typical cylcloxygenase inhibitor, in topical doses which almost completely inhibit epidermal PGE2 formation, failed to inhibit or only slightly inhibited DMBA-initiated and TPA-promoted skin tumor formation. These results suggest that the cyclooxygenase inhibitory effect of TMK688 is not essential for its anti-tumor promoting activity. Although at present a possible contribution of anti-histamine activity cannot be ruled out completely, the anti-tumor promoting action of TMK688 may most probably be related to its anti-lipoxygenase activity. TMK688 seems to be a promising agent for the prevention of skin carcinogenesis.

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

In mouse skin in vivo the irritant and hyperplasiogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) strongly increased the epidermal content of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, but not of leukotriene LTB4. This effect was completely suppressed by the selective leukotriene biosynthesis inhibitor MK-886. Intragastric administration of MK-886 prevented phorbol ester-induced ear edema, but not epidermal hyperproliferation and tumor promotion. These data indicate that leukotrienes are involved in the pro-inflammatory effects of the phorbol ester, whereas its hyperproliferative and tumor-promoting activities do not depend on 5-lipoxygenase-catalyzed leukotriene formation. This action differs from several non-selective inhibitors of lipoxygenases that were found to inhibit tumor promotion in initiated mouse skin.

Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors.

To search for possible anti-tumor-promoters, we carried out a primary screening of 21 cucurbitane triterpenoids using an in vitro assay system. Of these triterpenoids, scandenoside R6 (6), 23,24-dihydrocucurbitacin F (14), 25-acetyl-23,24-dihydrocucurbitacin F (15), 2-O-beta-D-glucopyranosyl-23,24-dihydrocucurbitacin F (17) and cucurbitacin F (18) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Further, compounds 14 and 17 exhibited remarkable anti-tumor-promotion effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.