Effect of ellagic acid and 3-O-decylellagic acid on the formation of benzo[a]pyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice

The effect of ellagic acid and its more lipophilic derivative,3-O-decylellagic acid, on the amount of DNA-bound adducts inthe epidermis or lung of CD-I mice treated with [³H]benzo-[a]pyrene([³H]B[a]P) was evaluated using several different treatmentprotocols. The i.v. administration of 50µmol/kg of ellagicacid or 3-O-decyIellagk acid either together with or 5 min beforea 0.2 µmol/kg i.v. dose of [³H]B[a]P did not inhibit theformation of pulmonary DNA-bound adducts. Feeding mice a dietthat contained 1% ellagic acid for 10 days or the i.p. administrationof 120 µmol/kg of ellagic acid 30 min before the i.v.administration of 0.2 µmol/kg of [³H)B(a)P did not inhibitthe formation of DNA-bound adducts in the lung. The applicationof 2500 nmol of ellagic acid or 3-O-decylellagic acid to mouseskin 5 min before the application of 2, 10 or 50 nmol of [³H]B[a]Phad little or no effect on the covalent binding of [³H]B[a]Pmetabolites to epidermal DNA. Feeding mice a diet containing1% ellagic acid for 10 days did not inhibit the formation ofepidermal DNA-bound adducts after a topical dose of 2 nmol of[³H]B[a]P. Similarly, the topical application of 2500 nmol ofellagic acid at 2 h, 1 h and 5 min before and at 10 min afterthe application of 2 nmol of [³H]B[a]P did not inhibit the formationof DNA-bound adducts, but the same dosing regimen of 3-O-decylellagicacid (total dose of 10 000 nmol) resulted in a modest inhibitionin the formation of DNA-bound adducts. The topical applicationof 1500 nmol of ellagic acid 1 h before the application of 1500nmol of 3-methylcholanthrene (3-MC) to CD-I or BALB/c mice twiceweekly did not inhibit the development of skin tumors. Our resultsindicate that ellagic acid and 3-O-decylellagic acid are noteffective in inhibiting [³H]B[a]P DNA adduct formation in mouseskin and lung and that ellagic acid does not inhibit 3-MC-inducedskin tumori-genesis in BALB/c or CD-I mice.     

Inhibition of 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis by the naturally occurring plant phenolics caffeic, ellagic, chlorogenic and ferulic acids

The modifying effects of dietary administration of the plantphenolic antioxidants caffeic acid (CA), ellagic acid (EA),chlorogenic acid (CGA) and ferulic acid (FA) during the initiationphase on 4-nitroquinoline-1-oxide (4-NQO)-induced tongue carcinogenesisand on the number and area of silver-stained nucleolar organizerregion proteins (AgNORs), a new cell proliferation marker, ofthe tongue squamous epithelium were investigated in male F344rats. Rats were fed the diet containing 500 p.p.m. CA, 400 p.p.m.EA, 250 p.p.m. CGA or 500 p.p.m. FA for 7 weeks. One week afterthe commencement of the diets, 4-NQO (20 p.p.m.) was administeredin the drinking water for 5 weeks. Feeding of four phenoliccompounds significantly reduced the incidences of tongue neoplasms(squamous cell papilloma and carcinoma) and preneoplastic lesions(hyperplasia and dysplasia) by 32 weeks, and rats fed CA orEA had no tongue neoplasms. The number and area of AgNORs pernucleus were decreased significantly by dietary treatment withthese four phenolics. Thus, CA, EA, CGA and FA inhibited thetongue carcinogenesis induced by 4-NQO when they were administeredconcurrently with the carcinogen. These results might suggestpossible application of these natural substances for cancerchemoprevention in tongue in addition to other tissues (skin,lung, liver and esophagus).     

Inhibition of epidermal xenobiotic metabolism in SENCAR mice by naturally occurring plant phenols

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid have been shown to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocarbons including benzo(a)pyrene (BP). The present study was designed to determine whether these plant phenols can alter epidermal cytochrome P-450-dependent monooxygenases in SENCAR mice. In vitro addition of these plant phenols to epidermal microsomal preparations inhibited aryl hydrocarbon hydroxylase (AHH) activity in a concentration-dependent manner. The 50% inhibitory concentrations for tannic acid, myricetin, quercetin, and anthraflavic acid ranged from 4.4 X 10(-5) M to 12.4 X 10(-5) M in microsomes prepared from control and 3-methylcholanthrene-pretreated animals. Of the plant phenols studied tannic acid was found to be the most potent inhibitor of epidermal AHH activity. Tannic acid, quercetin, myricetin, and anthraflavic acid exhibited a mixed type of inhibitory effect with Ki values of 81, 63, 135, and 165 microM, respectively. In vitro addition of these plant phenols (240 microM) to the incubation mixture prepared from control and 3-methylcholanthrene-treated animals resulted in varying degrees of inhibition of epidermal microsomal AHH (57-92%), ethoxycoumarin O-deethylase (19-58%), and ethoxyresorufin O-deethylase (33-85%) activities. High pressure liquid chromatographic analysis of the organic solvent-soluble metabolites of BP produced by epidermal microsomes indicated a substantial decrease in the formation of BP-diols (23-67%) and BP-phenols (29-57%) by each of the plant phenols. The formation of BP-7,8-diol was substantially inhibited (29-52%) by each of the plant phenols. Further in vivo studies showed that a single topical application of tannic acid, quercetin, and myricetin greatly diminished epidermal AHH (53-65%), ethoxycoumarin O-deethylase (30-68%), and ethoxyresorufin O-deethylase (66-97%) activities whereas anthraflavic acid was ineffective in this regard even when repeatedly applied. Our results indicate that plant phenols have substantial though variable inhibitory effects on epidermal monooxygenase activities and BP metabolism suggesting that these compounds may be capable of inhibiting the carcinogenic effects of polycyclic aromatic hydrocarbons in the skin.     

Exceptional activity of tannic acid among naturally occurring plant phenols in protecting against 7,12-dimethylbenz(a)anthracene-, benzo(a)pyrene-, 3-methylcholanthrene-, and N-methyl-N-nitrosourea-induced skin tumorigenesis in mice

Our recent studies have shown that naturally occurring dietary plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid are capable of inhibiting polycyclic aromatic hydrocarbon (PAH) metabolism and subsequent PAH-DNA adduct formation in epidermis of SENCAR mice (M. Das, et al., Cancer Res., 47: 760-766, 1987, and 47: 767-773, 1987). In this study these plant phenols were tested for their effects against PAHs and N-methyl-N-nitrosourea-induced skin tumorigenesis in mice. Each plant phenol was evaluated as a possible anticarcinogen in an initiation and promotion and a complete skin tumorigenesis protocol. In the two-stage tumor protocol in SENCAR mice using 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N-nitrosourea as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate as tumor promoter each plant phenol afforded significant protection against skin tumorigenicity. The protective effects were verified both by prolongation of latency period and by subsequent tumor development. In the complete carcinogenesis protocol in BALB/c mice using 3-methylcholanthrene as a tumorigen the applications of each of the plant phenols 30 min prior to each PAH application afforded significant protection by delaying the onset and the subsequent development of skin tumors. Our results suggest that these plant phenols have substantial though variable potential for modifying the risk of skin tumorigenicity induced by a wide variety of chemicals and of these tannic acid was shown to have maximal chemoprotective effects.     

Protective effects of ellagic acid and other plant phenols on benzo[a]pyrene-induced neoplasia in mice

The inhibitory effects of three phenolic compounds (ferulic,chlorogenic and ellagic acids) on benzo[a]pyrene- and 7,12-dimethylbenz[a]anthracene-inducedneoplasia have been investigated in mice. Ellagic acid was themost potent antagonist of tumorigenesis since this compoundis active, by i.p. administration or added in the diet, on benzo[a]pyrene-inducedpulmonary adenoma formation in A/J mice and, after topical application,on 7,12-dimethylbenz[a]anthracene-induced skin tumorigenesisin NMRI Swiss mice. If ellagic acid has little or no effecton the number of tumor bearing animals, the incidence of pulmonarytumors per animal is decreased by >50%. Ferulic acid andchlorogenic acid (5 x 100 mg/kg, by i.p. route) were also active,but less than ellagic acid, against the lung carcinogenesisby benzofa]-pyrene (100 mg/kg, i.p.) but were totally ineffectiveagainst the formation of skin tumors by 7,12-dimethylbenz[a]-anthracene.These results remarkably paralleled the in vitro antimutageniceffects of these compounds shown by Wood et al. on benzo[a]pyrene.It must be noted that ellagic acid only exerted, by i.p. route,a severe toxicity after four injections of 100 mg/kg, in oilsuspension, whereas the oral administration in the diet (a dailydose of 100 mg/kg during 15 days) did not cause any toxicity.     

Mutagenicity of the naturally occurring carcinogen cycasin and synthetic methylazoxymethanol conjugates in Salmonella typhimurium.

The aglycone methylazoxymethanol of the naturally occurring carcinogenic glucoside, cycasin, has previously been shown to be mutagenic, but cycasin per se has not. In this work, cycasin was demonstrated to be mutagenic using a modification of the Ames Salmonella test in which it was preincubated with beta-glucosidase and the tester strain in liquid medium. The mutagenicity of cycasin to six histine-depedent Salmonella strains varied considerably with strain HisG46 being the most susceptible. Methylazoxymethyl-beta-D-glucosiduronic acid, which also is nonmutagenic per se, similarly became mutagenic when preincubated with beta-glucuronidase. Methylazoxymethyl acetate, which is slightly mutagenic by the Ames standard pour plate method, became highly mutagenic on preincubation. The mutagenicity of free methylazoxymethanol was confirmed, and a linear dose-response relationship was observed. The common conditions required for activation of nonmutagenic methylazoxymethanol conjugates, the glucoside cycasin and methylazoxymethyl-beta-D-glucosiduronic acid, are 90-min preincubation at 30 degrees, pH 6.5, with an appropriate hydrolase and Salmonella typhimurium HisG46.     

Inhibition of polycyclic aromatic hydrocarbon-DNA adduct formation in epidermis and lungs of SENCAR mice by naturally occurring plant phenols

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid are known to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs). The binding of bay-region diol-epoxides of PAHs to target tissue DNA is thought to be essential for the initiation of cancer by these compounds. In this study we investigated the effect of these plant phenols on PAH-DNA adduct formation in the epidermis and lung of SENCAR mice. In vitro addition of tannic acid, quercetin, myricetin, and anthraflavic acid (25 microM) to an incubation system containing epidermal microsomes prepared from either control or 3-methylcholanthrene-pretreated mice inhibited benzo(a)pyrene binding to calf thymus DNA by 63-64, 38-43, 36-37, and 27-33%, respectively. A single topical application of tannic acid, quercetin, myricetin, and anthraflavic acid at a dose of 400 mumol/kg body weight resulted in the inhibition of [3H]benzo(a)pyrene binding to epidermal DNA (48-73%) and protein (51-63%). The same dose of these plant phenols (400 mumol/kg) caused even greater inhibition of (+/-)-[3H]-7 beta,8 alpha-dihydroxy-7,8-dihydrobenzo(a)pyrene and [3H]-7,12-dimethybenz(a)anthracene binding to epidermal DNA and protein. The formation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-deoxyguanosine adducts was substantially diminished in both epidermis (62-86%) and lungs (38-84%). These results indicate that tannic acid, quercetin, myricetin, and anthraflavic acid are potent inhibitors of carcinogen binding to epidermal and lung DNA and suggest that these plant phenols could prove useful in modifying the risk of tumor induction by PAHs such as benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in these two tissues.     

Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes

Naturally occurring compounds belonging to two chemical groups were studied for their capacities to inhibit N-nitrosodiethylamine (NDEA)-induced carcinogenesis in female A/J mice. One group consists of organosulfur compounds found in Allium species, including garlic, onions, leeks, and shallots, and the other, two monoterpenes, i.e., D-limonene and D-carvone. In an initial experiment, in which organosulfur compounds were investigated, diallyl disulfide, allyl mercaptan, and allyl methyl disulfide were found to produce a marked inhibition of NDEA-induced neoplasia of the forestomach when the test compounds were administered p.o. 96 and 48 h prior to NDEA. The most potent was diallyl disulfide which reduced forestomach tumor formation by more than 90%. Pulmonary adenoma formation also was inhibited but to a considerably lesser extent, i.e., about 30%. In three additional experiments, test compounds were given p.o. either 15 min or 1 h prior to NDEA. Under these conditions diallyl disulfide and allyl mercaptan again inhibited forestomach tumor formation substantially, i.e., greater than 75%, and pulmonary adenoma formation marginally, i.e., less than 20%. In these experiments D-limonene and D-carvone were tested and reduced forestomach tumor formation by slightly over 60% and pulmonary adenoma formation by about 35%. The results of these studies provide evidence of an increasing diversity of naturally occurring compounds having the capacity to inhibit nitrosamine carcinogenesis.     

Low frequency of H-ras activation in naturally occurring hepatocellular tumors of C3H/HeNCr mice

Previous reports from several laboratories have consistentlyshown that     

Induction of squamous metaplasia in organ cultures of hamster trachea by naturally occurring and synthetic fibers

Asbestos exhibits many properties of classical tumor promoters. These characteristics include the ability to stimulate proliferation and inhibit normal differentiation of cells. In organ cultures of trachea, crocidolite and amosite asbestos stimulate squamous metaplasia, a pathological process in which a rapidly proliferating squamous epithelium replaces the normal epithelium. We hypothesized that the induction of metaplasia depends upon the fibrous nature of asbestos. Accordingly, several naturally occurring and synthetic fibrous materials and their nonfibrous analogues were assessed for their ability to induce metaplastic changes in tracheal mucosa of the Syrian hamster. Exposure to both crocidolite asbestos and fiberglass resulted in significant increases (p less than 0.05) in squamous metaplasia over a range of dosages (1.0, 4.0, 16.0 mg/ml). Attapulgite (palygorskite) and both "long-" and "short-" fiber preparations of chrysotile asbestos had similar but less marked effects. Nonfibrous analogues of each material (riebeckite, antigorite, and glass particles) failed to produce metaplasia. Asbestos, and fibrous materials in general, appear to stimulate squamous metaplasia because of their fibrous geometry.     

Activation of lymphocytes of normal and tumor bearing mice by mangiferin, a naturally occurring glucosylxanthone

Mangiferin, a naturally occurring glucosylxanthone, was assessed for its immunomodulatory potential. The phytochemical induced extensive in vitro proliferation of murine splenocytes and thymocytes at the doses of 5-40 micrograms ml-1. Suppression of the proliferative response of the cells was observed with higher doses of mangiferin. Mangiferin also activated the splenocytes of tumor hosts at early and late stages of tumor growth. The Phytohemagglutinin (PHA) and Con A unresponsive splenocytes of advanced tumor bearer proliferated extensively in response to mangiferin. Mangiferin when used with Con A produced additive stimulatory effect and induced heightened DNA synthesis of normal and advanced tumor bearers' splenocytes.     

Effect of ellagic acid on hepatic and pulmonary xenobiotic metabolism in mice: studies on the mechanism of its anticarcinogenic action

Our recent studies have shown that ellagic acid, a naturallyoccurring dietary plant phenol, protects BALB/c mice against3-methylcholanthrene-induced skin tumorigenesis. To furtherelucidate the mechanism of the antineoplastic action of ellagicacid its effect on hepatic and pulmonary benzo[a]pyrene (BP)metabolism, cytochrome P-450-dependent monooxygenases and glutathioneS-transferase activities were studied in BALB/c mice. Chronicoral feeding of the compound in drinking water (0.3 mg/1 for16 weeks) or acute intraperitoneal administration (50 mg/kgfor five consecutive days) of ellagic acid resulted in 20–25%decreases in hepatic and pulmonary cytochrome P-450 levels.Hepatic and pulmonary aryl hydrocarbon hydroxylase and 7-ethoxycoumarinO-deethylase activities in both groups of ellagic acid-treatedanimals were 33–52% and 28–43% lower than theirrespective non-ellagic acid-treated controls. Hepatic as wellas pulmonary aminopyrine N-demethylase and epoxide hydrolaseactivities were unchanged in both groups of ellagic acid-treatedmice. Hepatic glutathione S-transferase activity towards BP-4,5-oxide or l-chloro-2, 4-dinitrobenzene as substrates was foundto be enhanced 51 – 79% and 38–58% in both groupsof animals. H.p.l.c. analysis of organic solvent-soluble metabolitesof BP by liver and lung microsomes indicated a substantial inhibitionof diol formation (including BP-7, 8-diol), as well as of phenolsand quinones. In liver, these inhibitory effects were more pronouncedafter oral feeding than after intraperitoneal administration.Our results indicate that both acute and chronic administrationof ellagic acid inhibits BP metabolism and/or enhances glutathioneS-transferase activity. Thus the modulation of polycyclic aromatichydrocarbon metabolism by ellagic acid may be related to theanticarcinogenic effects of this compound.     

Chemoprevention of 1,2-dimethylhydrazine-induced colon cancer in mice by naturally occurring organosulfur compounds

Organosulfur compounds (OSCs) present in garlic and onion oil have been shown to inhibit chemical carcinogenesis. In this study, we compared the chemopreventive efficacy of five lipid- and four water-soluble OSCs using the murine nuclear aberration assay. Administration of diallyl sulfide and S-allyl cysteine p.o. at a dose of 200 mg/kg 3 h prior to i.p. 1,2-dimethylhydrazine (DMH) injection (20 mg/kg) significantly inhibited colonic nuclear damage in female C57Bl/6J mice by 47% and 36%, respectively. The inhibitory effect of S-allyl cysteine was found to be dose dependent. The other OSCs did not affect the level of DMH-induced nuclear toxicity. Furthermore, the incidence and frequency of colonic tumors induced by DMH (20 mg/kg, 10 weekly i.p. injections) in female CF-1 mice were significantly inhibited by S-allyl cysteine pretreatment, given 3 h prior to each carcinogen injection. These data indicate that the allyl group coupled to a single sulfur atom might play an important structural role in inhibition of DMH-induced colonic nuclear toxicity and carcinogenesis. OSCs containing allyl groups stimulated glutathione S-transferase activity in both the liver and colon. However, their saturated analogues stimulated little or no hepatic and colonic glutathione S-transferase activity. Induction of hepatic and colonic glutathione S-transferase might assist in detoxification of carcinogens and could be necessary for some aspects of chemoprevention.     

Embryotoxicity of benzo(a)pyrene and some of its synthetic derivatives in Swiss mice

We have studied the teratogenicity of benzo(a)pyrene (BP), benzo(a)pyrene-4,5-oxide, and a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, a proximal metabolite and ultimate carcinogenic metabolite of BP, respectively, and of 6-methylbenzo(a)pyrene after direct injection into embryonal Swiss mice. The compounds were dissolved in acetone and trioctanoin (1:1) and injected at doses ranging from 0.4 to 16.0 nmol/embryo on days 10, 12, and 14 of development. The transplacental effects of BP given at the same gestational days and at comparable dose levels were also evaluated. The control groups received 0.5, 1.0, or 2.0 microliter/embryo of vehicle on days 10, 12, or 14 of pregnancy, respectively. The fetuses were examined when they were 18 days old. On the basis of gross external and internal malformations, 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene appeared to be the most potent embryotoxic and teratogenic compound tested, causing 85% of embryolethality and 100% of malformed fetuses in the group treated on day 10 of intrauterine development. There were 61 and 27% of malformed fetuses following 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene treatment on days 12 and 14 of gestation, respectively. The effects of this BP metabolite were very specific and malformations such as exencephaly, thoraco- and gastroschisis, phocomelia, and edema were found. The administration of BP (both transplacental and direct intraembryonal injection) and benzo(a)pyrene-4,5-oxide caused no significant increase of malformed fetuses in any of the developmental stages considered. 6-Methylbenzo(a)pyrene induced multiple malformations (among these a high percentage of protruding tongue) in 50, 46 and 31% of the fetuses treated on days 10, 12, and 14 of gestational age, respectively. These results combined with previous data concerning the induction of lung tumors by the tested compounds in 15-day-old Swiss mouse embryos, emphasize the requirement of a common metabolic derivative of BP to induce both teratogenesis and carcinogenesis in mice. Furthermore present data show that midgestation Swiss embryos are also highly sensitive to the 6-methyl derivative of BP.     

Spontaneous and induced metastasis of naturally occurring tumors in mice: analysis of cell shedding into the blood

The lung colonization capability (after iv inoculation) of each of 77 autochthonous virus-induced mammary tumors in C3H/Avy female mice was compared with its own spontaneous metastatic behavior. Twenty-four of these neoplasms had spontaneously metastasized to the lungs of the tumor bearer. The results were interpreted in the framework of dose-response studies on experimentally induced metastasis. These results indicate that tumors of high lung colonization potential (HCP) overgrow the lungs with iv doses as low as 10(4) cells, whereas tumors of low lung colonization potential (LCP) require at least 250 times this dose to achieve the same result. No general similarity was found between spontaneous and induced metastatic capability. Although some tumors showed good correspondence, others showed discrepancies and provided new information on mechanisms of cell shedding from primary tumors and on rate-limiting steps in metastasis. For example, from the dose-response studies, nonmetastatic tumors of HCP (iv) are deduced to be shedding very few cells. These findings suggest that escape from the primary tumor is an active process or else passive leakage of cells into vessels by hemorrhage, necrosis, or palpation should have resulted in spontaneous metastasis. Spontaneously metastatic tumors of LCP corroborate the view that metastasis is effected by a highly active subpopulation with special properties; otherwise, the required scale of passive cell release into the blood would be unrealistically large. Blood bioassay and time-course studies on pulmonary deposit formation indicate that shedding of metastatic cells occurs early in mammary tumor development.     

Modifying effects of the naturally occurring antioxidants gamma-oryzanol, phytic acid, tannic acid and n-tritriacontane-16, 18-dione in a rat wide-spectrum organ carcinogenesis model.

The modifying effects of the naturally occurring antioxidants gamma-oryzanol, phytic acid, tannic acid and n-tritriacontane-16, 18-dione (TTAD) were investigated in a rat wide-spectrum organ carcinogenesis model. Animals were initiated with two i.p. injections of 1000 mg/kg body wt 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) followed by two i.g. administrations of 1500 mg/kg body wt N-ethyl-N-hydroxy-ethylnitrosamine (EHEN), and then three s.c. injections of 75 mg/kg body wt 3,2'-dimethyl-4-aminobiphenyl (DMAB) during the first 3 weeks. Starting 1 week after the last injection, groups of rats received diet containing 1% gamma-oryzanol, 2% phytic acid, 0.2% TTAD or 1% tannic acid or basal diet alone for 32 weeks. Animals were then killed and complete autopsy was performed at the end of week 36. Histological examination revealed enhancement of lung carcinogenesis by gamma-oryzanol, and the incidence of urinary bladder papillomas to be increased by phytic acid. On the other hand, TTAD inhibited hepatic and pancreatic carcinogenesis. Phytic acid and tannic acid were marginally effective in inhibiting hepatic and colon carcinogenesis respectively. The results thus indicated that naturally occurring antioxidants each exert specific modifying effects depending on the organ site and indicate that wide-spectrum carcinogenesis models are useful for defining complex influences.     

Disposition of leucovorin and its metabolites in the plasma,intestinal epithelium,and intraperitoneal L1210 cells of methotrexate-pretreated mice

Leucovorin (LV or 5-CHOFH₄) has had longstanding clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH₂FH₄) plus tetrahydrofolate (FH₄) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH₄). The dihydrofolate (FH₂) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH₃FH₄) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH₄) in both intestinal epithelial and L1210 cells. The elevation of FH₂ in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH₂FH₄+FH₄. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.Abbreviations MTX methotrexate - LV or 5-CHOFH₄, leucovorin or 5-formyltetrahydrofolate - FU 5-fluorouracil - FdUMP 5-fluoro-2-deoxyuridine-5-monophosphate - FH₂ dihydrofolate - FH₄ tetrahydrofolate - CH₂FH₄ 5,10-methylenetetrahydrofolate - 5-CH₃FH₄ 5-methyltetrahydrofolate - 10-CHOFH₄ 10-formyltetrahydrofolate This research was supported in part by ACS grant CH461A (to D.G.P.) and USPHS grants CA22754 (to D.G.P.), CA08748 (to F.M.S.), CA22764 (to F.M.S.), CA18856 (to F.M.S.) and CA46153 (to F.M.S.) awarded by the National Cancer Institute     

A naturally occurring secreted human ErbB3 receptor isoform inhibits heregulin-stimulated activation of ErbB2, ErbB3, and ErbB4

A variety of receptor-mediated signaling pathways are controlled by both positive and negative extracellular regulators. In this study, we demonstrate that a naturally occurring secreted form of the human ErbB3 receptor, p85-soluble ErbB3 (sErbB3), is a potent negative regulator of heregulin (HRG)-stimulated ErbB2, ErbB3, and ErbB4 activation. We show that p85-sErbB3 binds to HRG with an affinity comparable to that of full-length ErbB3 and competitively inhibits high affinity HRG binding to ErbB2/ErbB3 heterodimers on the cell surface of breast carcinoma cells with an IC(50) of 0.5 nM. p85-sErbB3 inhibits HRG-induced phosphorylation of ErbB2, ErbB3, and ErbB4 in breast carcinoma-derived cell lines and can also block HRG-stimulated activation of mitogen-activated protein kinase, Akt, and association of ErbB3 with the phosphatidylinositol 3'-kinase p85 regulatory subunit. Cell growth assays show that exogenous addition of a 100-fold molar excess of p85-sErbB3 inhibits HRG-stimulated cell growth by as much as 90%. Whereas several potential mechanisms of p85-sErbB3 inhibition of ErbB receptor activation exist, our results suggest that at least one means of inhibition is competition for HRG binding. The IC(50) for both p85-sErbB3- and 2C4 (a monoclonal antibody specific for ErbB2)-mediated inhibition of HRG binding is approximately 0.5 nM, although the mechanism of inhibition by these two proteins is distinct. Together these results suggest that p85-sErbB3 is a naturally occurring negative regulator of HRG-stimulated signal transduction that may have important therapeutic applications in human malignancies associated with HRG-mediated cell growth such as breast and prostate cancer.