Influence of 13-cis-retinoic acid on mouse skin tumor initiation and promotion

Tumor initiation in CD-1 mice by benzo[a]pyrene (BaP) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was unaffected by topical pretreatment with 13-cis-retinoic acid (13-cis-RA). Likewise, anthralin-induced tumor promotion in SENCAR mice was unaffected by pretreatment with 13-cis-RA. These results suggest that the action of retinoids in preventing either tumor initiation or promotion is very carcinogen or cocarcinogen specific.     

Modulation of mouse skin tumor promotion by dietary 13-cis-retinoic acid and {alpha}-difluoromethylornithine

The effects of dietary supplementation of 13-cis-retinoic acid(13-cis-RA) and -difluoromethylornithine (DFMO) in the drinkingwater on 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotedskin tumor formation was determined. Administration of 13-cis-RAin the diet and DFMO in the drinking water was started 1 weekand 2 days before the first TPA application to the dimethylbenz[a]anthracene-initiatedskin of either female CD-I or SENCAR mice, respectively. Dietary13-cis-RA failed to inhibit both the tumor yield and the incidence;papillomas per mouse at 0, 5, 50, 100 and 200 mg/kg diet 13-cis-RAdoses were 25, 30, 22, 28 and 25 respectively at 18 weeks ofpromotion treatment and at all doses 100% of the mice bore papillomas.However, dietary 13-cis-RA dramatically reduced the size ofskin tumor promoted with TPA. 13-cis-RA at doses of 5, 50, 100and 200 mg/kg diet inhibited skin papillomas (> 4 mm diameter)per mouse by 28, 55, 76 and 93%, respectively. Retinoid treatmentdid not affect body weight gains and the survival was more than80% in all groups. In accord with our previous findings, DFMOwhen given in drinking water, was a very effective inhibitorof mouse skin tumor promotion by TPA; DFMO at 0.25% concentrationinhibited the number of papillomas by 50%. Inhibition of skintumor promotion by combined treatments with dietary 13-cis-RA(100 mg/kg) and DFMO (0.25%) in the drinking water was possiblyadditive. The retinoid and DFMO preclude TPA-increased ornithinedecarboxylase (ODC) activity and the accumulation of putrescineby differential effects on ODC, an enzyme associated with skintumor promotion by TPA.     

Effects of dietary retinyl palmitate or 13-cis-retinoic acid on the promotion of tumors in mouse skin

The present study was designed to determine the effects of dietary 13-cis-retinoic acid and retinyl palmitate on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Female CD-1 mice were initiated with 150 nmol of 7,12-dimethylbenz(a)anthracene and promoted twice weekly with 8 nmol of TPA. Diets supplemented with retinyl palmitate to yield 60,000 or 200,000 IU or 700,000 for 5 wk followed by 350,000 IU per kg of diet (700,000/350,000) fed to mice during tumor promotion resulted in 9%, 37%, and 65% inhibition of the papilloma yield, respectively, at 21 wk of promotion. Although topical applications of 13-cis-retinoic acid have been almost as effective as retinoic acid in preventing the appearance of mouse skin tumors, dietary 13-cis-retinoic acid at 200,000 or 700,000 IU per kg of diet resulted in no reduction in papilloma yield but did result in a dose-dependent decrease in the tumor burden (weight of tumors per mouse). Therefore, dietary retinyl palmitate yielded a dose-dependent inhibition of the number and weight of tumors promoted by TPA, whereas dietary 13-cis-retinoic acid resulted in a decrease in weight but not in number of tumors promoted by TPA.     

Inhibition of both stage I and stage II mouse skin tumour promotion by retinoic acid and the dependence of inhibition of tumor promotion on the duration of retinoic acid treatment

Retinoic acid is a potent inhibitor of mouse skin tumor promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have further evaluated the effect of retinoic acid on the stages of tumor promotion and also analyzed the effect of duration of retinoic acid treatment on mouse skin tumor promotion by TPA. In a number of independent experiments, either with female CD-1 or SENCAR mice, we failed to observe a specificity of inhibition by retinoic acid of either Stage I or Stage II tumor promotion. In a typical experiment with SENCAR mice, application of 34 nmol of retinoic acid concurrently with each application of either TPA (3.2 nmol) or mezerein (3.2 nmol) to initiated (with 10 nmol 7,12-dimethylbenz(a)anthracene) skin equally inhibited promotion of skin papilloma formation. Furthermore, sustained inhibition of tumor promotion by retinoic acid required a continuous application of retinoic acid in conjunction with each promotional treatment with TPA; if retinoic acid treatment was discontinued, TPA treatment elicited tumor formation. These results indicate: (a) retinoic acid inhibits both Stage I and Stage II of tumor promotion; and (b) inhibition of tumor promotion exhibits retinoic acid dependency.     

Inhibition of mouse skin tumor promotion by tenuazonic acid.

Tenuazonic acid (TA) was topically applied to the interscapular region of Swiss albino mice at different doses before the application of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Skin from the painted area was examined for ornithine decarboxylase (ODC) enzyme estimation. It was observed that TA inhibited TPA induced ODC activity. The inhibitory effect of TA was also found in mouse skin tumor promotion in the two stage initiation promotion protocol. There was a remarkable delay in the latency period and decrease in the number of tumors developed and the percentage of tumor bearing animals after TA treatment.     

Inhibitory effect of ailanthoidol on 12-O-tetradecanoyl-phorbol-13-acetate-induced tumor promotion in mouse skin

Many components derived from dietary or medicinal plants showing antioxidant and anti-inflammatory potential have been found to possess chemopreventive properties. In our previous study, we achieved the total synthesis of ailanthoidol (AT), a neolignan from Zanthoxylum ailanthoides or Salvia miltiorrhiza Bunge, which are used in Chinese traditional herbal medicine. In the present study, preliminarily, AT exhibited a radical quenching property by DPPH assay. Following this, we assessed the effect of AT on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation in female CD-1 mouse skin which was closely linked to tumor promotion. The topical application of AT (0.5-2.5 mM; 200 microl) reduced the formation of hydrogen peroxide and inhibited the myeloperoxidase (MPO) activity in the mouse skin when compared with that of the TPA-treated alone group. In addition, AT presented a suppression effect on the TPA-induced hyperplasia and leukocyte infiltration in the epidermis and edema of mouse ears. Furthermore, it showed that AT inhibited the TPA-induced expression of COX-2 protein and ornithine decarboxylase (ODC) activity in epidermis. Finally, AT was evaluated for its ability to inhibit the TPA-induced promotion in skin tumors of female CD-1 mice. Topical application of AT 5 min prior to TPA (5 nmol) three times weekly for 12 weeks to mice which were initiated with benzo[a]pyrene (B[a]P) inhibited the incidence of skin tumors in mice and the average number of tumors per mice as compared to TPA-treated alone. These results indicate that AT possesses potential as a chemopreventive agent against tumor promotion.     

Reversal of food restriction-induced inhibition of mouse skin tumor promotion by adrenalectomy.

Restricting the food intake of laboratory mice and rats markedly reduces the incidence of spontaneous and experimentally induced cancers. Using the two-stage skin tumorigenesis model in CD-1 mice, we report now that food restriction suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of epidermal [3H]thymidine incorporation as well as TPA promotion of skin papillomas, whereas adrenalectomy completely reverses the inhibition in [3H]thymidine incorporation and tumor development. These results suggest that the adrenal gland may play an important role in mediating the tumor inhibitory effect of food restriction.     

13-cis-retinoic acid in the treatment of recurrent glioblastoma multiforme

Basic science and clinical investigations have demonstrated that 13-cis-retinoic acid (cRA) has activity against malignant gliomas. To assess its effectiveness in the setting of recurrent glioblastoma multiforme (GBM), we performed a retrospective analysis of the medical records and neuroimaging results of patients with recurrent GBM who were treated with cRA. The toxicity profile of cRA, response, and effect on progression-free survival from initiation of treatment were end points of our analysis. Eighty-two of 85 patients with a median age of 51 years received at least 1 full cycle of cRA. At the initiation of cRA treatment, the median Karnofsky performance score was 80. All patients had failed conventional radiotherapy. Seven patients were chemona?ve, whereas 75 patients had received some form of chemotherapy. Radiographic partial responses, minor responses, and stable disease were seen in 4%, 8%, and 34% of patients, respectively. Two patients were not assessable. Progression-free survival and overall survival after initiation of cRA were 10.0 and 24.6 weeks, respectively. Six-month progression-free survival was 19% for the entire group. Grade 3 or 4 toxicity developed in 14 patients (16%), one of whom developed pancreatitis and died. The results of this study demonstrate only modest efficacy for cRA therapy in this cohort of heavily pretreated patients with recurrent GBM. This data supports the use of cRA in such patients, but its further evaluation in larger, prospective, controlled studies with or without other noncytotoxic and cytotoxic agents may be warranted.     

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

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

Mechanism of mouse skin tumor promotion by n-dodecane

Application of the alkane n-dodecane to the dorsal skin of 6–8week old female SENCAR mice initiated with 10 nmol dimethylbenz[a]anthraceneled to papilloma formation in the majority of treated animals.Compared to the potent phorbol diester 12-O-tetradecanoylphorbol-13-acetate(TPA), n-dodecane was several orders of magnitude less potenton a dose basis, and maximal papilloma response required moreextended application (22 weeks for 50 mg dodecane compared to12 weeks for 2 µg TPA). In two-stage promotion experimentsn-dodecane appeared to act as a stage II promoting agent atappropriate doses, being comparable in activity to mezerein—anagent with well-characterized activity of this type. Dodecane,unlike mezerein, did not induce the formation of a significantnumber of pyknotic cells, however, suggesting that the weakpromoting activity of dodecane in stage 1 was not a result oftoxicity. In comparison with TPA, both mezerein and n-dodecaneat promoting doses induced lesa sustained hyperplasia in SENCARmouse skin, a finding also consistent with the proposal thatn-dodecane is principally active in stage II of two-stage promotionmodels. Both agents induced ornithine decarboxylase activityin SENCAR mouse skin, the maximal induction being observed atapparently the same time after a single application.     

Inhibition of mouse skin tumor promotion by several inhibitors of arachidonic acid metabolism

12-O-Tetradecanoylphorbol-13-acetate promotion of skin tumorsin mice can be inhibited by topical application of either thephospholipase A₂ inhibitor dibromoacetophenone or the cyclooxygenase-lipoxygenaseinhibitors 5,8,11,14-eicosatetrayonic acid or 1-phenyl-3-pyrazolidinone.The phospholipase A₂ inhibitors in particular appear to be amongthe most potent inhibitors of skin tumor promotion known. Theseresults support the hypothesis that at least some of the productsof arachidonic acid transformation are essential for tumor promotion.     

Mechanism of mouse skin tumor promotion by chrysarobin

The skin tumor-promoting ability of 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) was compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,8-dihydroxy-9-anthrone (anthralin) in SENCAR mice. Although dose-response comparisons indicated that chrysarobin was several orders of magnitude less potent than TPA for promoting papilloma formation, this anthrone was 1.5 to 2 times more potent than anthralin. Maximal papilloma responses were achieved by 15 weeks of promotion with TPA whereas at least 25 weeks of promotion were necessary to achieve maximal papilloma responses with chrysarobin or anthralin indicating marked differences in tumor latency between the two classes of compounds. Interestingly, at optimal promoting doses, chrysarobin gave a carcinoma response (22% with 0.3 carcinomas per mouse at 45 weeks) similar to that of TPA suggesting that this compound may be more efficient at promoting carcinomas than papillomas. In two-stage promotion experiments, chrysarobin was incapable of functioning independently as a Stage I or II promoter despite its complete promoting activity. Chrysarobin and TPA were compared at optimal promoting doses for their ability to induce: (a) skin edema, (b) epidermal hyperplasia, and (c) epidermal ornithine decarboxylase. In each case, distinct differences were noted between the two compounds. When taken together, the data support the hypothesis that anthracene-derived skin tumor promoters work at least in part by a mechanism different from the phorbol esters.     

Dermal collagen metabolism during tumor promotion with 12-O-tetradecanoylphorbol-13-acetate in mouse skin

Treatment of the skin with the tumor promoting phorbol ester12-O-tetradecanoylphorbol-13-acetate leads to a reduction ofthe collagen content of the dermis of the mouse. The collagendegradation as well as synthesis is affected by the tumor promoter,the effect on degradation being more pronounced at the beginningof the treatment. The collagenolytic activity that can be extractedfrom the dermis is increased 5- to 6-fold during the first fiveapplications and {small tilde} 2-fold at later times. The uptakeof [¹⁴C]proline in both total protein and NaCl-soluble collagenis doubled, but the increase of collagen synthesis does notrestore a normal collagen content in the dermis, as it is accompaniedby an elevated level of collagenolytic activity.     

Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate

The effects of topically applied curcumin, chlorogenic acid, caffeic acid, and ferulic acid on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal ornithine decarboxylase activity, epidermal DNA synthesis, and the promotion of skin tumors were evaluated in female CD-1 mice. Topical application of 0.5, 1, 3, or 10 mumol of curcumin inhibited by 31, 46, 84, or 98%, respectively, the induction of epidermal ornithine decarboxylase activity by 5 nmol of TPA. In an additional study, the topical application of 10 mumol of curcumin, chlorogenic acid, caffeic acid, or ferulic acid inhibited by 91, 25, 42, or 46%, respectively, the induction of ornithine decarboxylase activity by 5 nmol of TPA. The topical application of 10 mumol of curcumin together with 2 or 5 nmol of TPA inhibited the TPA-dependent stimulation of the incorporation of [3H]-thymidine into epidermal DNA by 49 or 29%, respectively, whereas lower doses of curcumin had little or no effect. Chlorogenic acid, caffeic acid, and ferulic acid were less effective than curcumin as inhibitors of the TPA-dependent stimulation of DNA synthesis. Topical application of 1, 3, or 10 mumol of curcumin together with 5 nmol of TPA twice weekly for 20 weeks to mice previously initiated with 7,12-dimethylbenz[a]anthracene inhibited the number of TPA-induced tumors per mouse by 39, 77, or 98%, respectively. Similar treatment of mice with 10 mumol of chlorogenic acid, caffeic acid, or ferulic acid together with 5 nmol of TPA inhibited the number of TPA-induced tumors per mouse by 60, 28, or 35%, respectively, and higher doses of the phenolic acids caused a more pronounced inhibition of tumor promotion. The possibility that curcumin could inhibit the action of arachidonic acid was evaluated by studying the effect of curcumin on arachidonic acid-induced edema of mouse ears. The topical application of 3 or 10 mumol of curcumin 30 min before the application of 1 mumol of arachidonic acid inhibited arachidonic acid-induced edema by 33 or 80%, respectively.     

Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Ursolic acid (UA) and oleanolic acid (OA), which had been isolated from Glechoma hederacea as inhibitors of Epstein-Barr virus (EBV) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), were tested against inhibitory effect on tumor promotion by TPA in vivo. They inhibited effectively the tumor promotion in mouse skin and the activities were comparable to that of a known inhibitor of tumor promotion, retinoic acid (RA). Interestingly, UA was more effective on a single application before initial TPA-treatment than on a continuous application before each TPA-treatment, while OA and RA were ineffective in the same treatment. These data suggest that the role of UA for inhibitory action on tumor promotion differs slightly from those of RA and OA.     

The effect of 13-cis-retinoic acid on dibutylnitrosamine induced polyploidy changes in mouse urothelium

The epithelium in the normal urinary bladder contains cells with diploid to octoploid DNA-content. The carcinogen dibutylnitrosamine (DBN), given subcutaneously in repeated doses causes a loss of polyploidy prior to cancer development. In this study the changes in polyploidy caused by DBN was followed by use of flow cytometry. 13-cis-retinoic acid did not prevent this loss of polyploidy, and did not affect the polyploidy in the normal urothelium.     

Hyperthermia and phorbol ester tumor promotion in mouse skin

In a two-stage skin tumorigenesis protocol [7, 12-dimethyl-benz[a]anthracene(DMBA) initiation followed by twice weekly 12-O-tetradecanoylphorbol-13-acetate(TPA) promotion], SENCAR mice developed an average of 8.5 papillomasper animal. Hyperthermia treatments of the initiated skin (44^°C,30 min) immediately before or after each TPA application (for90 days) reduced papilloma frequency 80–90%. Animals whoseinitiated skin was made thermo-tolerant at the time of TPA application(by hyperthermia treatment 24 h prior to each application ofpromoter) showed slightly less protection (70% reduction infrequency). Multiple 44^°C hyperthermia treatments alone(27 x, twice a week) had no promoting activity in DMBA-initiatedskin. The usual responses of skin to TPA promotion, includingan increase in dark cells, epidermal thickening, reddening anderosion were all suppressed in animals treated with hyperthermianear the time of TPA application. The effect of hyperthermiaon tumorigenesis was at the promotion stage and the survivalof initiated cells was not affected, since the normal numberof papillomas was produced when TPA promotion was delayed untilafter the multiple (27 x, twice a week) hyperthermia treatmentswere completed. Hyperthermia treatments (44^°C, 30 min, twiceweekly for 90 days) given near the time of TPA application alsosuppressed the incidence of carcinomas appearing within 300days. About 40% of the DMBA-initiated, TPA-promoted animalsdeveloped a carcinoma, compared with only 10% of a similargroup which received hyperthermia treatments near each TPA application.Papillomas appearing in spite of hyperthermia treatments duringpromotion were not more likely to progress into carcinomas thanthose appearing in unheated animals. Such hyperthermia treatmentsgiven to animals bearing pre-existing papillomas did not markedlyalter the subsequent development of carcinomas compared withunheated controls. The results demonstrated that 44^°C hyperthermiaapplied near the time of TPA promotion acted as a powerful antipromoterand suppressed the appearance of both papillomas and carcinomas,apparently by acting at an early stage of promotion.     

Dose and frequency effect in mouse skin tumor promotion

In order to study the influence of both dose and application frequency of tumor-promoting agents on tumor development, we conducted a large-scale mouse skin two-stage carcinogenesis experiment. The back skins of 1110 CD-1 mice were painted once with 50 micrograms benzo(a)pyrene. These mice were divided into 24 groups according to subsequent schedules of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Mice were treated with TPA at five different frequencies, i.e., daily, or every second, 4th, 8th, or 16th day, and six different TPA doses per application were used (0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 micrograms), which allowed groups to be established with the same total dose of TPA applied per time unit. Six of the 30 frequency/dose combinations at extreme low or high frequency and dose were excluded. At each fixed frequency of TPA application, there was a good dose-response of TPA in mouse skin papilloma incidence. There was also a good application frequency-response relationship at fixed doses of TPA application. Within the set of groups in which animals received the same total dose of TPA per time unit, some variation was observed with respect to frequency of application. In general, TPA applications every 4th and 8th day tended to yield a small number of tumors.     

Inhibitory effect of topical application of a green tea polyphenol fraction on tumor initiation and promotion in mouse skin.

A green tea polyphenol fraction was evaluated for its ability to inhibit tumor initiation by polycyclic aromatic hydrocarbons and tumor promotion by a phorbol ester in the skin of CD-1 mice. Topical application of the green tea polyphenol fraction inhibited benzo[a]pyrene- and 7,12-dimethylbenz[a]-anthracene-induced tumor initiation as well as 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion. Topical application of the green tea polyphenol fraction also inhibited TPA-induced inflammation, ornithine decarboxylase activity, hyperplasia and hydrogen peroxide formation. Studies with individual polyphenolic compounds in green tea indicated that topical application of (-)-epigallocatechin gallate, (-)-epigallocatechin and (-)-epicatechin gallate inhibited TPA-induced inflammation in mouse epidermis.