Pendolmycin, a new tumor promoter of the teleocidin A class on skin of CD-1 mice.

Pendolmycin, isolated from Nocardiopsis, is a compound structurally similar to teleocidin A, one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters. Pendolmycin has a C5 dimethyl allyl group attached to C-7 of (-)-indolactam-V, whereas teleocidin A has a C10 linalyl group attached to the molecule. The structure-activity relationships of a hydrophobic moiety attached to (-)-indolactam-V were studied in four compounds, (-)-indolactam-V, pendolmycin, teleocidin A and newly synthesized 7-(nerolidyl)-(-)-indolactam-V in tests on inhibition of the specific [3H]TPA binding to a particulate fraction of mouse skin, activation of protein kinase C and induction of both adhesion of HL-60 cells and ornithine decarboxylase in mouse skin. The potencies of the compounds for these activities increased mainly depending on the length of the hydrophobic group. Pendolmycin had a tumor-promoting activity on mouse skin initiated with a single application of 7,12-dimethyl-benz[a]anthracene, and its potency was just between those of (-)-indolactam-V and teleocidin A. The role of the hydrophobic moiety is discussed with particular emphasis on the results obtained with 7-(nerolidyl)-(-)-indolactam-V.

Differential effects of cryptoporic acids D and E, inhibitors of superoxide anion radical release, on tumor promotion of okadaic acid in mouse skin.

Cryptoporic acids D and E, isolated from the fungus Cryptoporus volvatus, are inhibitors of superoxide anion radical release. Cryptoporic acid E inhibited tumor promotion of okadaic acid in two-stage carcinogenesis experiments on mouse skin, initiated with 7,12-dimethylbenz[a]anthracene. Treatment with cryptoporic acid E using two different doses per application, 1 (1.2 mumol) and 5 mg (5.9 mumol), reduced the percentage of tumor-bearing mice from 73.3 to 53.3% and 20.0%, and the average number of tumors per mouse from 4.2 to 2.3 and 0.5 respectively in week 20 of tumor promotion. However, cryptoporic acid D slightly enhanced tumor promotion rather than inhibition of okadaic acid. Cryptoporic acid D was expected to have additional biochemical activities, such as activation of protein kinases. Cryptoporic acid D at concentrations of up to 100 microM activated protein kinase C and stimulated other protein kinase activity in vitro, whereas cryptoporic acid E did not. These two compounds provided differential effects on tumor promotion of okadaic acid on mouse skin.

Structure-activity relationship within a series of okadaic acid derivatives.

Okadaic acid (OA) is a potent non-12-O-tetradecanoyl-phorbol-13-acetate (non-TPA) type tumor promoter on mouse skin. OA acts on cells through inhibiting the activity of protein phosphatases and results in the increase of phosphorylation of proteins. Seventeen OA derivatives were evaluated as possible tumor promoters by means of three biochemical tests: inhibition of specific [3H]OA binding to a particulate fraction of mouse skin containing protein phosphatases, inhibition of protein phosphatase activity, and induction of ornithine decarboxylase in mouse skin. Potency in each of these biochemical tests correlated well for each of these derivatives. We present results indicating that the carboxyl group as well as the four hydroxyl groups at C-2, C-7, C-24 and C-27 of OA are important for activity. Acanthifolicin, which gave positive responses in these three biochemical tests as strong as those of OA and dinophysistoxin-1, is predicted to be an additional member of the OA class of tumor promoters.

Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin.

Staurosporine, which is a potent inhibitor of protein kinases, such as protein kinase C, inhibited both inductions of adhesion of human promyelocytic leukemia cells (50% effective dose = 9.0 nM) and Epstein-Barr virus early antigen in Raji cells (50% effective dose = 3.4 nM) by teleocidin. However, staurosporine induced irritation on mouse ear and histidine decarboxylase activity in mouse skin. It did not induce ornithine decarboxylase activity in mouse epidermis. The two-stage carcinogenesis experiments of staurosporine were carried out at two different doses. Experiment 1 revealed that the group treatment with a single application of 100 micrograms of 7,12-dimethylbenz(a)anthracene, followed by repeated applications of 50 micrograms of staurosporine, resulted in 85.7% of tumor-bearing mice at Wk 30, whereas group treatment with staurosporine alone or 7,12-dimethylbenz(a)anthracene alone gave 6.7% and 0%, respectively. Experiment 2 showed that group treatment with 7,12-dimethylbenz(a)anthracene followed by applications of 10 micrograms of staurosporine resulted in 33% of tumor-bearing mice at Wk 30. In addition, staurosporine treatment reduced the percentages of tumor-bearing mice treated with teleocidin from 100% to 67% in Wk 15. These results demonstrated that staurosporine is a weak tumor promoter of mouse skin compared with teleocidin, but staurosporine has some potency to inhibit tumor promotion by teleocidin.

Calyculin A, an inhibitor of protein phosphatases, a potent tumor promoter on CD-1 mouse skin.

Calyculin A, isolated from a marine sponge, has a novel spiro ketal skeleton. Structurally unrelated to okadaic acid, calyculin A bound to the okadaic acid receptors in particulate and soluble fractions of mouse skin. The biochemical and tumor-promoting activities of calyculin A were studied with those of okadaic acid. Calyculin A inhibited the activity of protein phosphatases, which serve as the okadaic acid receptors. The effective dose of calyculin A for 50% inhibition was 0.3 nM, similar to that of okadaic acid. Like okadaic acid, calyculin A induced ornithine decarboxylase in mouse skin and hyperphosphorylation of a Mr 60,000 protein in human papilloma virus type 16-transformed human keratinocytes. A two-stage carcinogenesis experiment on mouse skin, initiated by 100 micrograms (390 nmol) of 7,12-dimethylbenz(a)anthracene and followed by 1 microgram (1.0 nmol) of calyculin A, revealed that calyculin A is an additional member of the okadaic acid class of tumor promoters. The percentages of tumor-bearing mice in the groups treated with DMBA plus calyculin A, and with DMBA followed by 1 microgram (1.2 nmol) of okadaic acid were 86.7 and 80.0%, respectively, in week 30. The mechanisms of action of calyculin A and okadaic acid, in addition to dinophysistoxin-1 (35-methylokadaic acid), are discussed. Calyculin A is the first tumor promoter to be screened by the okadaic acid receptor binding test.

New antitumor promoters: (-)-epigallocatechin gallate and sarcophytols A and B.

EGCG, the main constituent of green tea, and sarcophytols A and B, isolated from a soft coral, inhibited tumor promotion by teleocidin in a two-stage carcinogenesis experiment on mouse skin. EGCG and sarcophytols showed inhibition of tumor development by chemical carcinogenesis. A possibility of developing these compounds as cancer chemopreventives for human beings is discussed.

Specific binding of okadaic acid, a new tumor promoter in mouse skin.

The tumor promoter okadaic acid binds specifically to a particulate as well as a cytosolic fraction of various mouse tissues, e.g., skin, brain, lung and colon. The KD value was 21.7 nM for receptors in the particulate fraction and 1.0 nM for those in the cytosolic fraction of mouse skin. The specific binding of [3H]okadaic acid to the particulate fraction of mouse skin was inhibited dose-dependently by okadaic acid, but not okadaic acid tetramethyl ether, an inactive compound, or by other tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin. The results suggest a new pathway of tumor promotion mediated through the okadaic acid receptor(s).

Apparent "activation" of protein kinases by okadaic acid class tumor promoters.

A cytosolic fraction of mouse brain gave two peaks of protein kinase activity on DEAE-cellulose column chromatography. The first peak of protein kinase corresponded to protein kinase C. The second peak contained protein kinases that were "activated" dose-dependently by the okadaic acid class tumor promoters, okadaic acid and dinophysistoxin-1. This "activation" was not achieved by other tumor promoters, such as 12-0-tetradecanoyl-phorbol-13-acetate, teleocidin, aplysiatoxin, or palytoxin. In addition, the second peak contained phosphatases. The phosphate liberation from phosphorylated histone type III-S by incubation with the second peak was inhibited by okadaic acid or dinophysistoxin-1, dose-dependently. The resulting apparent "activation" of protein kinases by okadaic acid is indicated and would imply a new pathway of tumor promotion on mouse skin.

Sarcophytols A and B inhibit tumor promotion by teleocidin in two-stage carcinogenesis in mouse skin.

Sarcophytols A and B, isolated from a soft coral, Sarcophyton glaucum, are cembrane-type diterpenes with different numbers of hydroxyl groups. Sarcophytols A and B inhibited tumor promotion by teleocidin in two-stage carcinogenesis experiments on mouse skin. The inhibitory effect of sarcophytol A was demonstrated with two different initiating doses of 7,12-dimethylbenz[a]anthracene (DMBA): 50 micrograms (experiment 1) and 100 micrograms (experiment 2). In experiment 1, three groups of mice were treated with DMBA, and then twice a week with doses (1.6 micrograms, 16 micrograms, and 82 micrograms) of sarcophytol A followed by 2.5 micrograms teleocidin. In week 25, the incidences of tumors in these groups were only 7.1%, 20.0%, and 13.3%, respectively, whereas that in the control group treated with DMBA plus teleocidin was 53.3%. Moreover, at this time, the average numbers of tumors per mouse in these groups were 0.1, 0.3, and 0.3, respectively, while that in the control group was 2.1. In experiment 2 an amount of sarcophytol A (1.6 micrograms) or B (1.7 micrograms) equimolar to 2.5 micrograms teleocidin was applied twice a week, as in experiment 1, and results showed that sarcophytol B also inhibited tumor promotion by teleocidin. Both sarcophytols A and B caused delay in onset of tumor formation, and reduced the percentage of tumor-bearing mice and the average number of tumors per mouse. The effective concentrations of sarcophytols A and B were in the microgram range with an equimolar amount of teleocidin.

A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol.

A new type of phorbol ester, which has a macrocyclic dicarboxylic acid diester structure, was isolated from the seed oil of Jatropha curcas L. (Euphorbiaceae). Based on the results of spectroscopic analyses of the compound and its chemical degradation products, its structure is proposed to be an intramolecular 13,16-diester of 12-deoxy-16-hydroxyphorbol, 12-deoxy-16-hydroxyphorbol-4'-[12',14'-butadienyl]-6'-[16',18',20' - nonatrienyl]-bicyclo[3.1.0]hexane-(13-O)-2'-[carboxylate]-(16-O)-3 '- [8'-butenoic-10']ate (DHPB). DHPB showed slightly weaker biological and biochemical activities than 12-O-tetradecanoylphorbol-13-acetate (TPA). DHPB induced ornithine decarboxylase in mouse skin (2.8 nmol CO2/30 min/mg protein/34 nmol application), inhibited the specific binding of [3H]-12-O-tetradecanoylphorbol-13-acetate to phorbol ester receptors (50% effective dose, 17.0 nM), and activated protein kinase C in vitro (50% effective dose, 36.0 nM). Also, a weak tumor-promoting activity of DHPB was found in a two-stage carcinogenesis experiment on mouse skin. One week after initiation of mice with 100 micrograms of 7,12-dimethyl-benz(a)anthracene, topical application, twice a week, of 2 micrograms of DHPB until week 17, followed by application of 5 microgram of DHPB until week 30 at the same rate, resulted in 46.7% incidence of tumors by week 30. The groups treated with 7,12-dimethylbenz(a)anthracene alone or DHPB alone did not produce significant numbers of tumors. These results indicate that the new phorbol ester, DHPB, is a tumor promoter with weaker activity than 12-O-tetradecanoylphorbol-13-acetate.

Diarrhetic shellfish toxin, dinophysistoxin-1, is a potent tumor promoter on mouse skin.

Dinophysistoxin-1, 35-methylokadaic acid, is a causative agent of diarrhetic shellfish poisoning. The biological activities and tumor-promoting activity of dinophysistoxin-1 were studied together with those of okadaic acid and 7-O-palmitoyl okadaic acid. Dinophysistoxin-1 is a skin irritant and induces ornithine decarboxylase in mouse skin with the same potency as okadaic acid. 7-O-Palmitoyl okadaic acid induced a lower activity than the other compounds. Dinophysistoxin-1 inhibited the specific [3H]okadaic acid binding to a particulate fraction of mouse epidermis. The binding affinities of dinophysistoxin-1 and okadaic acid to a particulate fraction were almost the same. Dinophysistoxin-1 showed a tumor-promoting activity as strong as that of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The percentages of tumor-bearing mice in the groups treated with 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed by 5 micrograms of dinophysistoxin-1, twice a week, and with DMBA followed by 5 micrograms of okadaic acid twice a week were 86.7% and 80.0% in week 30, respectively. The average number of tumors per mouse was 4.6 in the former group and 3.9 in the latter. Dinophysistoxin-1 and okadaic acid act on cells through different pathways from the 12-O-tetradecanoylphorbol-13-acetate-type tumor promoters.

Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter.

Okadaic acid is a polyether compound of a C38 fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase (OrnDCase; 3-hydroxyl-L-glutamate 1-carboxy-lyase, EC 4.1.1.17) in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and followed by application of 10 micrograms of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of [3H]TPA to a mouse skin particulate fraction when added up to 100 microM or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 microM. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.