Markers Of Tumor Promotion Research Articles

PROTECTION AGAINST ULTRAVIOLET B RADIATION‐INDUCED EFFECTS IN THE SKIN OF SKH‐1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA

In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-1 hairless mice (Wang et al., Carcinogenesis 12, 1527-1530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wt/vol) as the sole source of drinking water for 30 days to SKH-1 hairless mice followed by irradiation with UVB (900 mJ/cm2) resulted in significant protection against UVB radiation-caused cutaneous edema (P < 0.0005) and depletion of the antioxidant-defense system in epidermis (P < 0.01-0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC (P < 0.005-0.01) and cyclooxygenase activities (P < 0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one of the possible mechanisms of chemopreventive effects associated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.

Inhibitory effects of semisynthetic flavonoid derivatives on the biochemical markers of tumor promotion in mouse epidermis in vivo

Two sets of flavonoid derivatives were synthesized from condensed tannins (CTs) or catechin, and compared with the procyanidin monomer models, (+)-catechin and (−)-epicatechin, for their abilities to inhibit the biochemical effects of the potent tumor promoter 12- O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse epidermis in vivo. Topical applications of the semisynthetic flavonoids, catechin dialkyl ketals and epicatechin-4-alkylsulphides inhibit TPA-induced ornithine decarboxylase (ODC) activity to a much greater degree than catechin or epicatechin. Moreover, they reduce TPA-stimulated hydroperoxide (HPx) production, a response that cannot be inhibited by catechin or epicatechin. These compounds also inhibit the sequential stimulations of protein and DNA synthesis linked to TPA promotion. The remarkable effectiveness of these synthetic compounds, especially against the ODC marker of skin tumor promotion, suggests that they may be effective anti-tumor promoters.

Possible tumor-initiating and -promoting activity of p-methylcatechol and methylhydroquinone in the pyloric mucosa of rat stomach.

The possible tumor‐promoting and tumor‐initiating activities of p‐methylcatechol and methylhydroquinone in the pyloric mucosa of male F344 rats were studied. Ornithine decarboxylase (ODC) and replicative DNA synthesis (RDS) were used as markers of tumor promotion and DNA single strand scission and unscheduled DNA synthesis (UDS) as markers of tumor initiation. The compounds were administered by gastric intubation and results were compared with those after administration of catechol. p‐Methylcatechol at doses of 60 to 180 mg/kg body weight dose‐dependently induced up to 20‐fold increase in ODC activity and 9‐fold increase in RDS with maxima 24 h after its administration, but it did not induce significant DNA single strand scission or UDS. Methylhydroquinone at a dose of 200 mg/kg body weight induced up to 6‐fold increase in ODC activity 24 h, and 5‐fold increase in RDS 16 h after its administration, but the induction was not dose‐dependent. At a dose of 200 mg/kg body weight it induced DNA single strand scission, but not UDS. These results and previous findings show that the possible tumor‐promoting activities of catechol are several times higher than those of p‐methylcatechol and 10 times higher than those of methylhydroquinone.

Differential co-promoting activities of alpha, beta and gamma interferons in the murine skin two-stage carcinogenesis model.

SENCAR mice develop more papillomas in two-stage skin carcinogenesis protocols if gamma interferon (IFN-gamma) is co-administered with 12-O-tetradecanoylphorbol-13-acetate (TPA) during the promotion phase. In the current study preparations of murine alpha, beta and gamma IFNs were surveyed for their abilities to modulate TPA-dependent promotion and induction of epidermal hyperplasia, inflammation and ornithine decarboxylase activity (ODC). Single or multiple i.p. administrations of IFN-alpha, -beta or -gamma (< or = 2500 units) did not induce epidermal hyperplasia, inflammation or ODC activity. Single or multiple i.p. administrations of IFN-alpha, -beta or -gamma (2500 units) to mice being topically promoted with 0.1 or 1 microgram of TPA did not alter the epidermal hyperplasia induced by the phorbol ester. The vascular permeability of the skin, as evaluated by the extravasation of Evans blue dye, was increased in a dose-dependent fashion by TPA over the range of 0.1-1 microgram. Treatment of mice promoted with 0.1 microgram of TPA with IFN-gamma (> or = 2500 units) significantly increased the skin's vascular permeability. Comparable effects were not obtained with IFN-beta (IFN-alpha not tested). Treatment of TPA-promoted mice with IFN-gamma, and to a lesser extent IFN-beta, weakly potentiated the TPA-dependent induction of epidermal ODC activity. Under conditions in which IFN-gamma had co-promoting activities in an initiation-promotion protocol, co-treatment of initiated mice with 1 microgram of TPA and IFN-alpha or -beta (100-5000 units) did not reproducibly alter tumor latency., or papilloma and carcinoma multiplicities. These findings suggest that the co-promoting activities of IFNs are restricted to the gamma class, and are not uniformly reflected by parameters commonly employed as short-term markers of tumor promotion.

Antitumor-promoting activities of tannic acid, ellagic acid, and several gallic acid derivatives in mouse skin.

Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the biochemical and biological effects of the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse epidermis in vivo. When applied topically to mouse skin, tannic acid (TA), ellagic acid, and several gallic acid derivatives all inhibit TPA-induced ornithine decarboxylase activity, hydroperoxide production, and DNA synthesis, three biochemical markers of skin tumor promotion. Moreover, in the two-step initiation-promotion protocol, the same phenolic compounds also inhibit the incidence and yield of skin tumors promoted by TPA. TA is the most effective of these treatments. Since they are already known to inhibit tumor initiation, the plant phenols protecting against skin tumor promotion by TPA may be universal inhibitors of multistage carcinogenesis. TA and other polyphenols, therefore, might be valuable in cancer therapy and/or prevention.

(-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin.

Recently, we and others showed that the components of green tea may be useful cancer chemopreventive agents. It has been suggested that (-)-epigallocatechin-3-gallate (EGCG), the major constituent in green tea, may possess antitumor-promoting and/or anticarcinogenic effects in rodent tumor bioassay systems. During the chemical analysis of various green tea products, we found a traditionally preserved preparation of green tea used by tribes in the Himalayan region of Sikkim, India that was rich in EGCG. EGCG was isolated from this tea product, and its inhibitory effects were evaluated against the binding of topically applied 3H-labeled polycyclic aromatic hydrocarbons (PAHs) to epidermal DNA and 12-O-tetradecanoylphorbol-13-acetate (TPA) caused induction of epidermal ornithine decarboxylase (ODC) activity in Sencar mice, the short-term markers of tumor initiation and tumor promotion, respectively. Preapplication of EGCG resulted in significant inhibition (p less than 0.05) in the binding of [3H]PAH to epidermal DNA. Similarly, the topical application of EGCG resulted in significant inhibition (p less than 0.005) in TPA-caused induction of epidermal ODC activity. In further studies, we assessed the anti-skin tumor-initiating effect of EGCG in Sencar mice in an initiation-promotion protocol. The application of EGCG before challenge with 7,12-dimethylbenz[a]anthracene as tumor initiator resulted in significant reduction both in percentage of mice with tumors and number of tumors per mouse compared with a non-EGCG-pretreated group of animals. The results of the present study suggest that the green tea preparation from Sikkim may be a good source for the isolation of EGCG and that this compound may have significant potential as a cancer chemopreventive agent.

Inhibitory effect of dietary perilla oil rich in the n-3 polyunsaturated fatty acid alpha-linolenic acid on colon carcinogenesis in rats.

The inhibitory effect of dietary perilla oil rich in the n‐3 polyunsaturated fatty acid α‐linolenic acid against colon carcinogenesis was investigated in rats. Four groups of 26 F344 rats each received an intrarectal dose of 2 mg of N‐methyl‐N‐nitrosourea 3 times a week for 2 weeks, and received a diet containing 12% perilla oil, 6% or 12% safflower oil (rich in the n‐6 polyunsaturated fatty acid linoleic acid), or 12% palm oil (rich in saturated and monounsaturated fatty acids). At week 35, the incidence of colon cancer was significantly lower in perilla oil‐fed rats than in other dietary groups; 19% vs. 46%, 56% and 58%. When examined at week 10, the concentration of fecal bile acids, known to be tumor promoters, was not significantly different among the dietary groups, and the intrarectal deoxycholic acid‐induced colonic mucosal ornithine decarboxylase activity, a marker of tumor promotion, was significantly lower in perilla oil‐fed group than in other groups. The serum and colonic mucosal fatty acid compositions and the blood plasma prostaglandin E2 level directly reflected the fatty acid composition of each dietary fat. The results suggest that the anti‐tumor‐promoting effect of dietary perilla oil was a result of a decreased sensitivity of colonic mucosa to tumor promoters arising from the altered fatty acid composition in membrane phospholipid of colonic epithelial cells, and was not a consequence of a decrease of promoters such as bile acids.

Free radical-derived quinone methide mediates skin tumor promotion by butylated hydroxytoluene hydroperoxide: expanded role for electrophiles in multistage carcinogenesis.

Free radical derivatives of peroxides, hydroperoxides, and anthrones are thought to mediate tumor promotion by these compounds. Further, the promoting activity of phorbol esters is attributed, in part, to their ability to stimulate the cellular generation of oxygen radicals. A hydroperoxide metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHTOOH), has previously been shown to be a tumor promoter in mouse skin. BHTOOH is extensively metabolized by murine keratinocytes to several radical species. The primary radical generated from BHTOOH is a phenoxyl radical that can disproportionate to form butylated hydroxytoluene quinone methide, a reactive electrophile. Since electrophilic species have not been previously postulated to mediate tumor promotion, the present study was undertaken to examine the role of this electrophile in the promoting activity of BHTOOH. The biological activities of two chemical analogs of BHTOOH, 4-trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH, were compared with that of the parent compound. 4-Trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH have a reduced ability or inability, respectively, to form a quinone methide; however, like the parent compound, they both generate a phenoxyl radical when incubated with keratinocyte cytosol. The potency of BHTOOH, 4-trideuteromethyl-BHTOOH, and 4-tert-butyl-BHTOOH as inducers of ornithine decarboxylase, a marker of tumor promotion, was commensurate with their capacity for generating butylated hydroxytoluene quinone methide. These initial results were confirmed in a two-stage tumor promotion protocol in female SENCAR mice. Together, these data indicate that a quinone methide is mediating tumor promotion by BHTOOH, providing direct evidence that an electrophilic intermediate can elicit this stage of carcinogenesis.

Induction of short-term markers of tumor promotion by organic peroxides

Experiments from different laboratories have shown that benzoyl peroxide (BzPo) and other organic peroxides are effective tumor promoters in the mouse skin two-stage carcinogenesis system. In the present paper we have studied the short-term effect of six other organic peroxides, which have not been previously assayed as skin tumor promoters. These compounds were chosen for their molecular diversity, the type of radical predicted to be formed, solubility and availability. The parameters evaluated in this study include a series of short-term markers of tumor promotion, hyperplasia, induction of dark basal keratinocytes and induction of ornithine decarboxylase activity. After single applications the biological activity of the compounds was: m-chloroperoxybenzoic acid greater than di-m-methylbenzoyl peroxide greater than dicumyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)mono-peroxycarbonate greater than benzoyl peroxide greater than di-m-chlorobenzoyl peroxide greater than di-t-butyl peroxide greater than t-butyl hydroperoxide. After multiple applications, the order of activity of the compounds was: dicumyl peroxide greater than di-m-methyl-benzoyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)monoperoxy carbonate greater than m-chloroperoxybenzoic acid greater than di-m-chlorobenzoyl peroxide greater than t-butyl hydroperoxide greater than benzoyl peroxide greater than di-t-butyl peroxide. The difference of activity among the different compounds did not seem to correlate directly with the chemical stability of the compound; it is more likely that the activity depends on different factors such as percutaneous absorption, metabolism, and the rate of free radical formation in vivo. The data presented here further support the association between free radicals and tumor promotion since all of the compounds, with the exception of one, were active in inducing the short-term markers of tumor promotion. It will also establish conditions for future tumor experiments.

Mouse skin ornithine decarboxylase induction and tumor promotion by cyclohexane

Cyclohexane, a frequently used solvent in industry, was assessed for its tumorigenic potential on mouse skin following multistage initiation-promotion protocols. The activity of ornithine decarboxylase (ODC), a marker of tumor promotion was found to be induced by the topical application of cyclohexane. This ODC induction was dependent on the dose of cyclohexane used and the duration of application. Effect of protein synthesis inhibitors and the modifiers of tumor promotion on the cyclohexane induced ODC activity was also studied. ODC induction was inhibited by cycloheximide and also, up to some extent, by actinomycin D. Inhibitors of stage II tumor promotion showed more effect on the ODC induction by cyclohexane as compared to the inhibitors of stage I tumor promotion. In chronic animal bioassay experiments topical application of cyclohexane to DMBA initiated mouse skin resulted in just 10% of tumor bearing animals while prior application of TPA for two weeks resulted in 45% of tumor bearing animals. Collectively, the present study demonstrates that cyclohexane is more effective as a stage II tumor promoter over mouse skin and possibly affects the biochemical events at the molecular level.

Characterization of the induction of ornithine decarboxylase activity by benzoyl peroxide in SENCAR mouse epidermis.

The induction of epidermal ornithine decarboxylase (ODC) activity by benzoyl peroxide (BPO) was characterized to evaluate the usefulness of this effect as a short-term marker of BPO-induced mouse skin tumor promotion. The maximal induced levels of ODC specific activity, after a single topical dose of BPO, were greater than 2-fold higher when a cold scraping method was used to prepare epidermis rather than the commonly used heat treatment method. Therefore, the cold scraping method was used for all the work reported here. Application of a single 20 mg dose of BPO to the dorsal skin of SENCAR mice caused a relatively small induction of epidermal ODC activity, to a level less than 1/40 that induced by a single 2 micrograms dose of 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the time-courses of induction were different after single doses of TPA and BPO, with peak activities observed at approximately 6 and approximately 24 h after treatment respectively. In contrast, after a total of five 20 mg doses of BPO were topically applied (one dose every 2 days), ODC activity was transiently induced to an average level greater than 15 times after a single dose. Additionally, on this dosing regimen, the peak of ODC activity shifted to approximately 4 h after the last treatment, so that the time-course of ODC induction resembled that after multiple applications of TPA. The extent of epidermal ODC induction by BPO was found to be a complex function of the frequency of dosing and the number of treatments. However, when BPO treatments were administered from 1 to 7 days apart, similar maximal induced levels of ODC activity were eventually achieved after application of multiple doses. Importantly, the dose-response for the induction of ODC activity by five doses of BPO (applied one dose every 2 days) was highly correlated with published data on the dose-response for tumor promotion by this organic peroxide, indicating that ODC induction is a good short-term marker of BPO-induced tumor promotion in SENCAR mice.

Involvement of prostaglandin E2 in bile acid-caused promotion of colon carcinogenesis and anti-promotion by the cyclooxygenase inhibitor indomethacin.

The mechanism of the anti-promoting effect of the prostaglandin (PG) synthesis inhibitor indomethacin in colon carcinogenesis was investigated. Male Sprague-Dawley rats received 0.002% water solution of indomethacin as drinking water freely for 3 days, then a subcutaneous injection of various doses of PGE2 and/or an intrarectal instillation of 12 mumol of sodium deoxycholate as a colon tumor promoter. Ornithine decarboxylase (ODC), a marker of tumor promotion, in the distal colonic mucosa was assayed at 4 hr after deoxycholate instillation. Indomethacin significantly suppressed the deoxycholate-augmented increase of ODC activity, while exogenous PGE2 restored or further increased the augmented ODC activity. The amount of PGE2 and the level of ODC activity were well correlated. However, PGE2 alone without deoxycholate did not increase the activity. Deoxycholate markedly increased colonic mucosal PGE2 at 1 hr after the instillation, and indomethacin decreased it. The results indicate that PGE2, the production of which is stimulated in the colonic mucosa by deoxycholate, is involved in the induction of colonic mucosal ODC. This is probably why PG synthesis inhibitors may inhibit the tumor promotion and prevent cancer development in the colon.