Cytotoxic Tigliane Diterpenoids from Croton damayeshu.

Chemical investigation of an EtOH extract of the twigs and leaves of Croton damayeshu afforded 10 new tigliane diterpenoids, crodamoids A-J (1-10), along with five known compounds. Their structures were elucidated by physical data analysis. Compounds 8, 9, and 15 displayed cytotoxic effects against two human tumor cell lines, A549 and HL-60 (IC50: 0.9-2.4 µM).

A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis.

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.

Development of a sensitive in vitro method for identifying tumor promoters.

The identification and characterization of nongenotoxic carcinogens represents a significant challenge to toxicologists. In vitro methods for identifying tumor promoters with suitable sensitivity and specificity have been particularly elusive. Experiments are described which suggest that the human promyelocytic leukemia cell line HL-60 provides a sensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture which exhibits high phagocytic activity. Fluorescent latex particles were used as sensors to highlight the phagocytic phenotype and permitted the use of flow cytometry to automatically quantitate particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tumor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo promoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 pg of TPA per ml caused statistically significant increases in phagocytic activity. The experiments described herein suggest that in vitro differentiation of HL-60 cells may reflect the promoter-dependent modifications to gene expression that are observed in vivo during the promotion phase of carcinogenesis. The described method may represent a sensitive promoter screening assay which is both rapid and economical.

Phorbol ester-induced ventricular fibrillation in the Langendorff-perfused rabbit heart: antagonism by staurosporine and glibenclamide.

Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4 beta-phorbol,12,13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, 1-oleoyl-2-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4 alpha-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu+staurosporine treated hearts (P < 0.05 vs control). To further study the profibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induced activation of PKC induces redistribution of PKC activity and is associated with the development of VF.

Phorbol myristate acetate lung injury and airway responsiveness to aerosol histamine in awake sheep.

To test the hypothesis that phorbol myristate acetate (PMA), a potent granulocyte activator, would increase airway responsiveness to aerosol histamine, we quantitated airway responsiveness to aerosol histamine in nine awake sheep before and 4 1/2 hours after intravenous PMA, 5 micrograms/kg. A dose-response curve to aerosol histamine was constructed by administering histamine aerosols of 0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 mg/ml sequentially until dynamic lung compliance (Cdyn) decreased to less than or equal to 65% baseline. The dose of histamine that caused a reduction of Cdyn to 65% of baseline (ED65Cdyn) was determined by linear interpolation. We then administered PMA, defined a new baseline Cdyn 4 1/2 hours later, and administered aerosol histamine as before. Control experiments were performed on another day in eight of the nine sheep by administering aerosol histamine 4 1/2 hours apart without infusing PMA. Histamine responsiveness did not change significantly during 4 1/2 hours in the control experiments. Control (preaerosol histamine) Cdyn decreased from 0.090 +/- 0.010 L/cm H2O before PMA to 0.048 +/- 0.004 L/cm H2 4 1/2 hours after PMA. The mean pre-PMA ED65Cdyn for the nine sheep was 3.80 +/- 0.87 mg/ml, and the mean post-PMA ED65Cdyn was 2.13 +/- 0.98 mg/ml (p less than 0.05). When individual dose-response curves are examined, the small statistical increase in aerosol histamine responsiveness does not appear important, especially when the increase is compared to the changes previously reported after endotoxemia in unanesthetized sheep.

Antipromotional activity of dietary N-(4-hydroxyphenyl)retinamide in two-stage skin tumorigenesis in CD-1 and SENCAR mice.

The activity of the synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), as a promoter and as an inhibitor of tumor promotion in mouse skin was investigated using CD-1 and SENCAR mice. Dietary administration of 4-HPR inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both mouse strains, although protective activity was observed only at high TPA doses. Dietary 4-HPR had no promoting activity in mice receiving initiation and no TPA promotion. These data suggest that retinoid promotion of skin tumorigenesis may be specific to retinoic acid, and is not necessarily characteristic of the entire chemical class.

Ionizing radiation as an initiator in the mouse two-stage model of skin tumor formation.

The initiating potential of a range (7.5 to 22.5 Gy) of 4 MeV X rays was studied using the mouse skin two-stage model of carcinogenesis. A single dose of radiation was followed by 60 weeks of promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA) (8 nmol, two times per week). Since the proliferative state of a target cell population is known to influence carcinogen-induced tumorigenesis, we also investigated the effect of TPA on tumor incidence when applied as a single dose (17 nmol) 24 h prior to irradiation. Evidence presented here indicates that ionizing radiation can act as an initiator in this model system. All groups of animals that were promoted with TPA developed papillomas regardless of radiation treatment; however, only those groups of animals that received irradiation followed by TPA promotion developed squamous cell carcinomas. The incidence of nonepidermal tumors was similar between all radiation dose groups and was independent of TPA promotion. Our results also indicate that TPA pretreatment prior to irradiation results in an overall increase in the total tumor incidence, including both epidermal and nonepidermal tumors.

Increased persistence of induced mutants of Chinese hamster cells by 12-O-tetradecanoylphorbol-13-acetate.

Hypoxanthine phosphoribosyltransferase deficient mutants of Chinese hamster ovary cells induced by ethyl methanesulfonate usually do not maintain their phenotype during growth in non-selective medium immediately following the induction. This phenomenon, called poor "persistence" of the induced mutation, is in most cases unrelated to growth rate but results from establishment of contact with wild type cells (Bradley, W. E. C. Exp. Cell Res., 129: 251, 1980). We report here that 12-O-tetradecanoylphorbol-13-acetate, a strong tumor promoter, increases the persistence of these mutants.

The role of prostaglandin E1 in ornithine decarboxylase induction by tumor promoters.

The effect of topical application of PGE on induction of ODC in mouse epidermis was measured. When direct induction of ODC by TPA was blocked by also applying indomethacin, maximum ODC activity occurred only when PGE was applied simultaneously with TPA 4 1/2 hr before killing of the mice. If either TPA or PGE was applied at other times, ODC activity decreased substantially. Induction of ODC by mezerein was blocked by indomethacin but restored by PGE, as was observed with TPA, but induction by ethyl phenylpropiolate was not affected by indomethacin or PGE. DMBA did not cause a consistent increase in ODC activity, nor was its inductive action affected by indomethacin or PGE. However, another weak inducer, acetic acid, exhibited elevated ODC activity when PGE was also applied. Inhibition by topical retinoic acid of ODC induction by TPA was partially overcome in a dose-response fashion by PGE. The results indicate that at least 2 events, elevation of PGE and another independent event, are required for induction of ODC activity. It appears that TPA causes at least 4 independent events essential for tumor promotion. A model for the events in the 2-stage tumor promotion model is proposed.

The skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate induces transcription of the c-fos proto-oncogene in human bladder epithelial cells.

The effect of a single treatment with the skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the cellular proto-oncogenes, c-myc, c-rasHa, c-rasKi and c-fos was examined in the non-tumorigenic human bladder epithelial cell line HCV 29. TPA (1 microgram/ml) increased the transcription of the c-fos gene of HCV 29 at least 50-fold, and this stimulation was observed within minutes. The response was transient, and was accompanied by a rapid and transient change in cell morphology. The expression of c-myc, c-rasHa and c-rasKi were not enhanced by the TPA treatment. These results show that human bladder epithelial cells respond to a known skin tumor-promoter, TPA, by altering the transcription of a specific proto-oncogene in these cells.

Inhibitory effect of toluene on tumor promotion in mouse skin.

In the two-stage mouse model for skin tumorigenesis with phorbol-12-myristate-13-acetate (PMA) as promoter, topical application of 40 microliters of toluene 2X/week at the initiation/promotion site (the back) reduced the average number of tumors/mouse (ANT/M) to approximately one-fourth that of controls. Control procedure involved initiation of C3H mice with benzo[a]pyrene (BaP) and CD-1 mice with 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with from 1 to 5 micrograms PMA in 40 microliters acetone 2X/week. Forty microliters of toluene 2X/week per se was a weak promoter (6-13% of control ANT/M), and produced mild skin irritation at the application site but behavior and body weights were normal. The toluene inhibition of tumorigenesis was not a direct chemical action on PMA since similar effects occurred whether toluene was the vehicle for PMA or whether it was applied up to 1 day before PMA (i.e., prepromotion). Prepromotion with acetone had no effect on tumorigenesis, substantiating its use as control vehicle and suggesting that the toluene inhibition was a specific tissue reaction. The inhibitory effect appeared to be on PMA promotion rather than on initiation since toluene and acetone produced similar numbers of tumors when used as the vehicle for BaP or DMBA in two-stage or BaP in single-stage trials. The inhibition was not permanent since tumorigenesis returned to control rates 2-3 weeks after prepromotion with toluene ceased but promotion with PMA in acetone continued. Toluene may be unique among reported promotion inhibitors in that it is a widely used commercial chemical which sometimes serves as a vehicle in cancer-screening trials. Since its metabolism is reasonably well defined, it may be of value in exploring further the process of tumor promotion.

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate.

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.

Lung vascular injury after Escherichia coli endotoxin and phorbol myristate acetate infusion in dogs.

The effects of Escherichia coli endotoxin and phorbol myristate acetate (PMA), a potential stimulator of polymorphonuclear leukocyte (PMN), on circulating PMN counts, gas exchange, protein concentration of lavage fluid, pulmonary hemodynamics and pathology of the lung were studied in ten anesthetized dogs. Six dogs were infused with 1 microgram/kg endotoxin plus 10 micrograms/kg of PMA; four other dogs were infused with the same amount of endotoxin but 5 micrograms/kg of PMA. After administration of endotoxin plus 10 micrograms/kg PMA, the number of circulating PMN (per mm3) decreased dramatically from 4081 +/- 1041 to 303 +/- 119, arterial oxygen partial pressure (PaO2) dropped to 49.1 +/- 2.4 mmHg and the arterial alveolar oxygen partial pressure difference (A-a DO2) increased significantly above baseline. Lungs from this group appeared to be grossly damaged: edema with distinct petechial hemorrhage and areas of hemorrhagic consolidation; frothy edema fluid often emanated from the tracheas. The group infused with endotoxin plus 5 micrograms/kg PMA showed no significant decrease in the number of PMN; PaO2 and A-a DO2 maintained comparatively stable. Protein concentration of lavage fluid and lung wet/dry weight ratios in dogs of 10 micrograms/kg PMA group were significantly increased (P less than 0.05) as compared to those of 5 micrograms/kg PMA group. Our study showed that the magnitude of leukopenia after endotoxin and PMA was paralleled with the severity of lung vascular injury. These results support the potential role of PMN in the pathogenesis of acute edematous lung injury.

Differing patterns of cytotoxicity of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in various human cell strains.

Inhibition of colony formation by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) over a wide range of concentrations (10(-4)-10(2) ng/ml) was examined in two normal human diploid fibroblast strains and eight cell lines derived from various human tumors. Three dose-response patterns were observed: (i) no killing at any dose, which is characteristic of rodent cells; (ii) increasing cytotoxicity with TPA doses of 0.1 ng/ml or greater; and (iii) a biphasic response with maximal cytotoxicity at 1.0 ng/ml, and minimal effects at much lower or higher concentrations. The latter response group included both normal and tumor cell strains. When normal cells were incubated concurrently with superoxide dismutase or CuDIPS, survival was enhanced in a dose-dependent manner. Specific binding of [3H]PDBu to cells from each of the three response categories was studied to determine whether the cells might contain two classes of specific phorbol ester receptors. Scatchard plots yielded straight lines, consistent with one class of binding sites. The possible significance of this cytotoxic effect of TPA in human cells at dose levels usually considered typical for specific phorbol ester responses is discussed.

Involvement of histamine in vascular permeability increase of the rat pleurisy induced by phorbol myristate acetate.

Rat pleurisy was induced by an intrapleural injection of 1 nmol (0.1 ml) of phorbol myristate acetate (PMA). Accumulation of pleural fluid peaked at a volume of 1 ml at 1 hr, and exudation rate peaked at 30 min when measured by the amount of dye leaked into the pleural cavity during 20 min. Histamine contents decreased in the pleural cell pellets markedly at 30 min and increased in the supernatant fluid simultaneously, reflecting histamine release that occurred in 30 min after PMA. Pretreatment with mepyramine maleate or methysergide 30 min prior to the PMA-injection suppressed the fluid volume significantly. The result indicates that there are involvements of 5-hydroxytryptamine and the H1-receptor of histamine in the vascular permeability increase at the early stage of PMA-pleurisy.

Specific growth response of ras-transformed embryo fibroblasts to tumour promoters.

Chemical carcinogenesis is a process involving multiple steps, as shown in several in vivo experimental systems. Two early steps have been well characterized: initiation, achieved by a single, subthreshold dose of a carcinogen, and promotion, induced by repetitive treatments with a non-carcinogenic tumour promoter. At the cellular level, establishment of the transformed phenotype is also a multi-step process and activation of several, independent genes appears to be required. Here we show that, like initiated cells, primary rat embryo fibroblasts (REFs) containing a ras but not a myc oncogene, are strongly and specifically stimulated to grow by tumour promoters. In the presence of these promoters, ras-containing REFs acquire the ability to overgrow normal cells in the monolayer and to form foci with 100% efficiency. Similar to the in vivo situation, promoter effects can be blocked by the concomitant application of retinoic acid.

Keratin gene expression in mouse skin tumors and in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate.

Alterations in the pattern of epidermal differentiation and proliferation occur during mouse skin carcinogenesis. We have used cDNA clones corresponding to the major keratin subunits synthesized in differentiating epidermal cells (Mr 67,000 and 59,000) and in proliferating epidermal cells (Mr 60,000, 55,000, and 50,000) to study changes in keratin gene transcript levels in mouse epidermis exposed to tumor promoters. The same probes were used to characterize the keratin expression patterns in benign and malignant skin tumors. A single topical treatment with 12-O-tetradecanoylphorbol-13-acetate caused a rapid initial decrease in the epidermal transcript levels corresponding to the Mr 67,000 and 59,000 keratin subunits. By 48 h the transcript level for the Mr 67,000 keratin subunit was restored to control values, whereas the transcript levels for the Mr 59,000 subunit returned to control at a slower rate. In contrast, the transcript level for the Mr 55,000 subunit was increased substantially 12- 48 h after treatment, the Mr 50,000 subunit transcript increased to a lesser extent, and the Mr 60,000 subunit message was transiently decreased at 12 h but returned to the level of solvent-treated skin by 24 h. Single exposure to the incomplete tumor promoters 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, the ionophore A23187, and mezerein induced changes in keratin gene transcripts similar to those of 12-O-tetradecanoylphorbol-13-acetate. The antipromoter fluocinolone acetonide, administered with 12-O-tetradecanoylphorbol-13-acetate, partially inhibited the decrease in the Mr 59,000 and 67,000 transcripts and completely inhibited the increase in the Mr 55,000 transcript. In skin papillomas produced by initiation and promotion, keratin gene expression was similar to normal skin, with the exception of a two-fold increase in the transcript levels for the Mr 55,000 keratin subunit. However, in carcinomas, the transcript levels for the Mr 67,000 and 59,000 subunits were only 1-3% of those observed in untreated mouse epidermis. In concert with other data, the rapid and selective loss of transcripts for differentiation-related keratins after exposure to both complete and incomplete tumor promoters is most consistent with an accelerated rate of maturation in differentiating keratinocytes, resulting in the rapid production of transcript-depleted fully mature squames. The enhanced level of Mr 55,000 transcripts suggests a concomitant increase in the number of all cells or a subset of cells in the proliferative compartment.(ABSTRACT TRUNCATED AT 400 WORDS)

Dose and schedule of oral retinoic acid and indomethacin needed to effectively inhibit phorbol ester-induced epidermal ornithine decarboxylase activity.

Currently there is no well-defined biological parameter or marker to help define agents, doses, and dose schedules for human cancer chemoprevention trials. Induction of ornithine decarboxylase, the rate limiting enzyme in the polyamine biosynthetic pathway, has been shown to be an essential aspect of mouse skin tumor promotion. Supplementary information suggest that this enzyme is an important aspect of carcinogenesis in other organ systems and in other animals (including humans). We have developed an assay system which effectively measured tumor promoter (TPA)-induced ornithine decarboxylase activity on 3-4 mm skin samples from mice and humans. Using this system we evaluated the doses and dose schedules of retinoic acid and indomethacin needed to effectively inhibit ornithine decarboxylase activity. Our data suggest that the doses and schedules of these compounds needed to inhibit ornithine decarboxylase activity would be toxic in humans.

An examination of the DNA damaging and repair inhibitory capacity of phorbol myristate acetate in human diploid fibroblasts.

The effects of the tumor promoter, phorbol myristate acetate (TPA) on DNA and DNA repair in human diploid fibroblasts was investigated. TPA induced detectable DNA single-strand breaks at doses as low as 1.3 microM using a sensitive nick-translation assay and at doses as low as 40 microM using alkaline sucrose velocity sedimentation analysis. This report provides the first indication of TPA-induced DNA damage in any cell other than a leucocyte. Experiments utilizing enhancers or inhibitors of active oxygen generation suggest that the DNA damage is related to free radical formation. In addition, we have investigated the effects of TPA on survival following u.v.-irradiation and on removal of pyrimidine dimers from cellular DNA and find no indication of enhancement of u.v. sensitivity or inhibition of normal DNA repair processes in these cells. In fact, TPA treatment enhances colony-forming ability of u.v.-irradiated, but not unirradiated cells.

12-O-tetradecanoylphorbol-13-acetate promotes tumors prior to initiation in two-stage promotion.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the non-promoter mezerein both induce ornithine decarboxylase activity in mouse epidermis by a route which can be blocked by indomethacin. In two-stage tumor promotion experiments in mice with mezerein as the stage II promoter, TPA was effective as the stage I promoter whether it was applied before or after an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA). There appear to be at least 4 events in promotion, only 3 of which are caused by second stage promoters.