Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth.

Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53(R175), one of the most frequent 'hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53(R175) mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53(R175) mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53(R175) mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53(R175) and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

Synthesis of (-)-[4-3H]epigallocatechin gallate and its metabolic fate in rats after intravenous administration.

Because a great deal of attention has been focused on the metabolism of (-)-epigallocatechin gallate (EGCg), quantitative analysis of this compound is required. For this purpose we developed a method of chemical synthesis of [4-(3)H]EGCg. Synthesized [4-(3)H]EGCg showed 99.5% radiochemical purity and a specific activity of 13 Ci/mmol. To clarify the excretion route of EGCg, the radioactivity levels of bile and urine were quantified after intravenous administration of [4-(3)H]EGCg to bile-duct-cannulated rats. Results showed that the radioactivity of the bile sample excreted within 48 h accounted for 77.0% of the dose, whereas only 2.0% of the dose was recovered in the urine. The excretion ratio of bile to urine was calculated to be about 97:3. These results clearly showed that bile was the major excretion route of EGCg. Time-course analysis of the radioactivity in blood was also performed to estimate the pharmacokinetic parameters following intravenous administration of [4-(3)H]EGCg. In addition, EGCg metabolites excreted in the bile within 4 h after the intravenous dose of [4-(3)H]EGCg were analyzed by HPLC. The results showed that 4',4"-di-O-methyl-EGCg was present in the conjugated form and made up about 14.7% of the administered radioactivity.

The prevention of lung cancer induced by a tobacco-specific carcinogen in rodents by green and black Tea.

A growing body of evidence from studies in laboratory animals indicates that green tea protects against cancer development at various organ sites. We have previously shown that green tea, administered as drinking water, inhibits lung tumor development in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone (NNK), a potent nicotine-derived lung carcinogen found in tobacco. The inhibitory effect of green tea has been attributed to its major polyphenolic compound, epigallocatechin gallate (EGCG), and, to a lesser extent, to caffeine. We have also demonstrated that while levels of O6-methylguanine, a critical lesion in NNK lung tumorigenesis, were not affected in lung DNA. However, the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, were significantly suppressed in mice treated with green tea or EGCG. These studies underscore the importance of the antioxidant activity of green tea and EGCG for their inhibitory activity against lung tumorigenesis. Unlike green tea, the effect of black tea on carcinogenesis has been scarcely studied, even though the worldwide production and consumption of black tea far exceeds that of green tea. The oxidation products found in black tea, thearubigins and theaflavins, also possess antioxidant activity, suggesting that black tea may also inhibit NNK-induced lung tumorigenesis. Indeed, bioassays in A/J mice have shown that black tea given as drinking water retarded the development of lung cancer caused by NNK. However, data on the relationship of black tea consumption with the lung cancer risk in humans are limited and inconclusive. There is a need for additional tumor bioassays in animal models to better examine the protective role of black tea against lung cancer. The development of adenocarcinomas and adenosquamous carcinomas in F344 rats upon chronic administration of NNK provides an important and relevant model for lung carcinogenesis in smokers. Thus far, no information was previously available regarding the effects of tea on this model. We conducted a 2-year lifetime bioassay in F344 rats to determine whether black tea and caffeine are protective against lung tumorigenesis induced by NNK. Our studies in both mice and rats have generated important new data that support green and black tea and caffeine as potential preventive agents against lung cancer, suggesting that a closer examination of the roles of tea and caffeine on lung cancer in smokers may be warranted.

Inhibition of lung carcinogenesis by black tea in Fischer rats treated with a tobacco-specific carcinogen: caffeine as an important constituent.

Here, we examined the effect of black tea and caffeine on lung tumorigenesis in F344 rats induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in a 2-year bioassay. NNK was administered s.c. at a dose of 1.5 mg/kg body weight three times weekly for 20 weeks. Animals were given either black tea as drinking water at concentrations of 2%, 1%, or 0.5%, or caffeine in drinking water at concentrations identical to those in 2% and 0.5% tea infusions for 22 weeks. The treatment period began 1 week before and ended 1 week after the NNK administration. The animals were sacrificed on week 101 for the examination of tumors in target organs, including lung, liver, nasal cavity, and other major organs. The NNK-treated group, given 2% black tea, showed a significant reduction of the total lung tumor (adenomas, adenocarcinomas, and adenosquamous carcinomas) incidence from 47% to 19%, whereas the group given 1% and 0.5% black tea showed no change. The 2% tea also reduced liver tumor incidence induced by NNK from 34% in the group given only deionized water to 12%. The tumor incidence in the nasal cavity, however, was not affected by either black tea or caffeine at any of the concentrations tested. The most unexpected finding was the remarkable reduction of the lung tumor incidence, from 47% to 10%, in the group treated with 680 ppm caffeine, a concentration equivalent to that found in the 2% tea. This incidence is comparable to background levels seen in the control group. This study demonstrated for the first time in a 2-year lifetime bioassay that black tea protects against lung tumorigenesis in F344 rats, and this effect appears to be attributed, to a significant extent, to caffeine as an active ingredient of tea.

Dietary control of cancer.

Many laboratory studies and human epidemiological data suggest that most cancer deaths are attributable to lifestyle, including nutritional factors and tobacco and alcohol consumption. Tobacco consumption is causally related to cancer of the lung, mouth, larynx, esophagus, bladder, kidney, and pancreas. Nutrients and non-nutrient dietary components probably account for cancer of the colon, breast, prostate, and stomach. This report is based on literature and our own data pertaining to the role of dietary fat, calories, and fiber in the development of colon and breast cancer. We also discuss the evidence from epidemiological, mechanistic, and preclinical efficacy studies indicating a protective effect of micronutrients, non-nutrients, and certain antioxidants in food against oral and lung cancers. Given the continuing cancer burden and the relatively slow impact of proven cancer treatment strategies in reducing cancer mortality, it is essential to evaluate promising nutrients and non-nutrients in foods as chemopreventive agents in persons at increased risk for cancer. Development of reliable intermediate biomarkers is valuable for clinical chemoprevention intervention trials. The purpose of this report is to provide the reader with plausible approaches to cancer control.

Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard.

Allyl isothiocyanate (AITC) is a constituent of cruciferous vegetables. It occurs widely in the human diet as a natural ingredient or food additive. AITC possesses numerous biochemical and physiological activities. It is cytotoxic and tumorigenic at high doses and also is a modulator of enzymes involved in metabolism of xenobiotics, including carcinogens. It is plausible that the wide consumption of dietary AITC may have profound effects on human health. To facilitate investigations of the effects of dietary AITC in humans, a method of measuring its uptake is needed. In this study, a urinary marker was developed for quantifying AITC uptake in humans. Four adult volunteers were asked to eat a meal containing brown mustard as the source of AITC. The 48-h urine samples were collected from these individuals and analyzed by reverse phase high performance liquid chromatography. A major urinary metabolite was found, which was identified as N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine, the N-acetylcysteine conjugate of AITC, by comparing its retention time and UV, nuclear magnetic resonance, and mass spectra with those of the synthetic standard. After ingestion of mustard, the AITC conjugate was detected in urine collected from 0 to 12 h. No conjugate was found in urine samples collected after 12 h. The major portion of this metabolite was excreted within 8 h. The average total excretion of AITC conjugate was 5.4 +/- 1.7 (SD) mg after consumption of 10 g of mustard and 12.8 +/- 2.0 mg when 20 g of mustard was consumed. Thus, a dose-dependent excretion of this metabolite was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis by compounds derived from cruciferous vegetables and green tea.

We have shown that PEITC and I3C, both of cruciferous origin, inhibited lung tumor formation induced by the tobacco-specific nitrosamine NNK. The inhibition by PEITC is due largely to its inhibitory effect on the enzymes of NNK metabolism, whereas; the inhibition by I3C may be attributed to its ability to induce hepatic enzyme activity of NNK metabolism, which resulted in decreased availability of NNK to the lung. On a molar basis, PEITC is considerably more effective than I3C. PEITC was released upon consumption of watercress. The N-acetylcysteine conjugate of PEITC is a promising urinary marker for quantitating uptake of this dietary anticarcinogen in humans. These studies also showed that green tea polyphenol EGCG inhibited the NNK-induced lung tumorigenesis, probably due to its antioxidant property. These studies provide for the first time evidence for the involvement of free radicals in nitrosamine tumorigenesis. The mechanism by which free radicals are generated by NNK treatment is not yet known. The reduced levels of oxidative lesions in lung as a result of EGCG treatment may be related to its ability to reduce reactive oxygen species and/or to chelate iron ion resulting in a decreased production of hydroxyl radicals. Overall, these studies have identified ingredients in cruciferous vegetables and green tea that are inhibitory against lung tumorigenesis induced by NNK in rodents.

Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants.

In this study we examined the effects of green tea and its major components, (-)-epigallocatechin gallate (EGCG) and caffeine, on the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. We also studied the effects of green tea and EGCG on O6-methylguanine and 8-hydroxydeoxyguanosine (8-OH-dGuo) formation in lung tissues caused by NNK treatment. Mice were given 2% tea, 560 ppm EGCG, or 1120 ppm caffeine in drinking water for 13 weeks. During this time, NNK (11.65 mg/kg body weight) was administered by gavage three times weekly for 10 weeks from weeks 3 to 12. The bioassay was terminated 6 weeks after the last NNK treatment. Mice treated with NNK developed 22.5 lung adenomas per mouse, whereas NNK-treated mice that drank green tea or EGCG as drinking water developed only 12.2 (P less than 0.01) and 16.1 (P less than 0.05) tumors per mouse, respectively. Mice that drank green tea or caffeine solution showed lower body weight gains, although little difference in water and diet consumption was noted in these groups. While green tea and EGCG exerted little effect on the formation of O6-methylguanine, a critical DNA lesion in NNK lung tumorigenesis, both treatments suppressed the increase of 8-OH-dGuo levels in mouse lung DNA. The inhibition of 8-OH-dGuo formation in lung DNA by green tea and EGCG is consistent with their ability to inhibit lung tumorigenesis by NNK. Because 8-OH-dGuo is a DNA lesion caused by oxidative damage, these results suggest that the mechanism of inhibition by green tea and EGCG in NNK-induced lung tumorigenesis is due at least partly to their antioxidant properties.

Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice.

Phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC), and the newly synthesized 5-phenylpentyl isothiocyanate (PPeITC), 6-phenylhexyl isothiocyanate (PHITC), and 4-(3-pyridyl)butyl isothiocyanate (PyBITC) were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of A/J mice. Mice were administered isothiocyanates by gavage for 4 consecutive days at doses of 5, 1, or 0.2 mumol/day prior to administration of 10 mumol of NNK by i.p. injection. Mice were sacrificed 16 weeks after NNK administration and pulmonary adenomas were quantitated, PEITC effectively inhibited NNK-induced lung tumors at a dose of 5 mumol/day but was not inhibitory at doses of 1 or 0.2 mumol/day. PPITC, PBITC, PPeITC, and PHITC were all considerably more potent inhibitors of NNK lung tumorigenesis than PEITC. While virtually no differences in inhibitory activity could be ascertained for PPITC, PBITC, and PPeITC, PHITC appeared to be the most potent tumor inhibitor of all of the compounds. At a dose of 0.2 mumol/day, PHITC pretreatment reduced tumor multiplicity by 85%. PyBITC, an analogue of both NNK and PBITC, was ineffective as an inhibitor. Using the same protocol, the compounds were found to have qualitatively similar inhibitory effects on NNK-induced DNA methylation when administered at 1 mumol/day. These results extend our previous findings that increased alkyl chain length enhances the inhibitory activity of an arylalkyl isothiocyanate toward NNK lung tumorigenesis and demonstrate the exceptional chemopreventive potentials of two new isothiocyanates, PPeITC and PHITC.