Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27(kip1) translocation and retinoblastoma protein phosphorylation by 1'-acetoxychavicol acetate.

1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.

Suppressive activity of a fermented grain food product on dextran sulfate sodium-induced experimental colitis in mice.

Oxidative stress has been shown to play pivotal roles in the onset of inflammatory bowel disease. We evaluated the effects of a dietary anti-oxidant, Antioxidant Biofactor (AOB), a processed grain food, on dextran sulfate sodium (DSS)-induced colitis in mice. Female ICR mice were fed a diet containing 0.1% or 1% AOB for 2 weeks, during which they were given 5% DSS in drinking water for the latter 1 week to induce colitis. A diet containing 1% AOB, but not that with 0.1% AOB, attenuated DSS-induced body weight loss and colon shortening (each, P < 0.05), and dramatically improved colitis histologic scores. In addition, DSS-induced increases in colonic mucosal IL-1beta, but not TNF-alpha, protein levels were significantly abrogated in 1% AOB-fed mice (P < 0.05). Further, 1% dietary AOB abolished the expression of IL-1beta mRNA levels in colonic mucosa (P < 0.01). Our results suggest that AOB is effective for the prevention of DSS-induced colitis in mice.

Extract of vinegar "Kurosu" from unpolished rice inhibits the development of colonic aberrant crypt foci induced by azoxymethane.

The modifying effects of administrating an ethyl acetate extract of "Kurosu" (EK), a vinegar made from unpolished rice, on development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EK on proliferating cell nuclear antigen (PCNA) index in ACF, prostaglandin (PG) E2 expression in the colonic mucosa and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received drinking water containing 0, 0.05, 0.1 or 0.2% EK for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 140 +/- 23 ACF/rat at the end of the study (week 4). EK administration dose-dependently inhibited ACF formation and inhibition by 0.2% EK was statistically significant (P < 0.002). Treatment with EK significantly lowered PCNA index in ACF and reduced PGE2 content in the colonic mucosa, while EK elevated liver GST and QR activities. These findings suggest that EK may be effective for inhibiting colonic ACF, through induction of liver GST and QR and possibly alteration of PGE2 production.

Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet.

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84+/-13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72+/-17 (14% reduction) at a dose of 0.01% and 45+/-18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These findings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACF.

Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora.

Five bufadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana x tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC50 = 0.4 microM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.

Occurrence of the insecticidal 16,17-didehydro-16(E)-stemofoline in Stemona collinsae.

The occurrence of two alkaloids, 16,17-didehydro-16(E)-stemofoline and its isomer at C-4, 16,17-didehydro-4(E)-16(E)-stemofoline, were found together with a known insecticidal compound, stemofoline, in Stemona collinsae. The 16,17-didehydro-16(E)-stemofoline displayed higher insecticidal and antifeedant activities against the diamondback moth larvae than stemofoline.

Actinidic acid, a new triterpene phytoalexin from unripe kiwi fruit.

Seven phytoalexins (1-7), including a new compound, were isolated from the peel of unripe kiwi fruit (Actinidia deliciosa cv. Golden King) that had been wounded and inoculated with Colletotrichum musae. The new phytoalexin (1) was identified as 2alpha,3beta,23-trihydroxy-12,20(30)-ursadien-28-oic acid, and named actinidic acid. Phytoalexins 2-6 are known triterpenes but have not previously been described as phytoalexins. Phytoalexin 7 is the same triterpene as the phytoalexin of nectarine fruit.

Changes in the content and biosynthesis of phytoalexins in banana fruit.

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-(13)C] and [2-(13)C]cinnamic acids, and [2-(13)C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4'-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.

Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice.

The intake of citrus fruits has been suggested as a way to prevent the development of some types of human cancer. Nitric oxide (NO) is closely associated with the processes of epithelial carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and superoxide (O2-) generation was induced by a combination of lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible NO synthase and cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of edema formation, epidermal thickness, leukocyte infiltration, hydrogen peroxide production, and the rate of proliferating cell nuclear antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation. Nobiletin significantly inhibited two distinct stages of skin inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of cyclooxygenase-2 and inducible NO synthase proteins and prostaglandin E2 release. Nobiletin inhibited dimethylbenz[a]anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin tumor formation at doses of 160 and 320 nmol by reducing the number of tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that nobiletin is a functionally novel and possible chemopreventive agent in inflammation-associated tumorigenesis.

Triterpene phytoalexins from strawberry fruit.

Strawberry cv. Houkouwase is resistant to infection by Colletotrichum fragariae. The formation of antifungal compounds was observed in unripe fruit which had been wounded and inoculated with conidia of C. musae. Three antifungal compounds were isolated and identified as euscaphic acid, tormentic acid and myrianthic acid. Myrianthic acid inhibited the growth of C. musae at 3 microg, and euscaphic and tormentic acids showed inhibitory effects at 100 microg. A quantitative analysis of their contents showed that the triterpenes increased in wounded fruit, and in wounded and inoculated fruit, but not in non-treated fruit. These findings indicate that unripe fruit of Houkouwase produced the triterpenes as phytoalexins. The triterpene phytoalexins seem to be involved in the resistance of strawberry to the fungus.

Molluscicidal saponins from Anagallis arvensis against schistosome intermediate hosts.

The molluscicidal activity of saponins isolated from the plant Anagallis arvensis (Primulaceae) was studied against schistosome intermediate hosts, Biomphalaria glabrata and Oncomelania quadrasi. @Strong molluscicidal activity was found in two compounds called desglucoanagalloside B and anagalloside B. Their structures were identified on the basis of chemical and spectroscopic analyses and their activities are comparable to that of the synthetic molluscicide, niclosamide.

Screening for the in vitro anti-tumor-promoting activities of edible plants from Malaysia.

A total of 114 methanol extracts from 42 plant families of edible Malaysian plants were screened for their inhibitory activities toward tumor promoter 12-O-hexadecanoylphorbol-13-acetate (HPA)-induced Epstein-Barr virus (EBV) activation in Raji cells. By testing at a concentration of 200 micrograms/ml, 74% of the 114 extracts inhibited EBV activation by 30% or more. This rate is comparable to those observed in the previous tests on edible Thai (60%) and Indonesian (71%) plants, and, importantly, much higher than that (26%) observed for Japanese edible plants. Approximately half of the Malaysian plants did not taxonomically overlap those from the other three countries, suggesting that Malaysian plants, as well as Thai and Indonesian plants, are an exclusive source of effective chemopreventive agents. Further dilution experiments indicated an extract from the leaves of Piper betle L. (Piperaceae) to be one of the most promising species. The high potential of edible Southeast Asian plants for cancer chemoprevention is collectively discussed.

Identification of zerumbone in Zingiber zerumbet Smith as a potent inhibitor of 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation.

Zerumbone was isolated from the rhizomes of Zingiber zerumbet Smith as a potent inhibitor of tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation. The IC50 value of zerumbone (0.14 microM) is noticeably lower than those of the anti-tumor promoters we have hitherto obtained. Interestingly, alpha-humulene lacking the carbonyl group at the 8-position in zerumbone was inactive (IC50 > 100 microM), while 8-hydroxy-alpha-humulune was markedly active (IC50 = 0.95 microM).

Identification of coumarins from lemon fruit (Citrus limon) as inhibitors of in vitro tumor promotion and superoxide and nitric oxide generation.

Three coumarins were isolated as siginificant inhibitors of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation in Raji cells from the peel of lemon fruit. They were identified as 8-geranyloxypsolaren (LE-1), 5-geranyloxypsolaren (bergamottin, LE-2), and 5-geranyloxy-7-methoxycoumarin (LE-3), respectively, by spectroscopic analysis. Three isolates had no potential O(2)-scavenging and markedly suppressed TPA-induced superoxide (O(2)(-)) generation in differentiated human promyelocytic HL-60 cells. Furthermore, LE-1 and LE-3 reduced both lypopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Similarly, they were found to be NO generation inhibitors rather than scavengers by measuring the extracellular L-citrulline levels. The occurrence of these coumarins in a lemon fruit was abundant in the flavedo of the peel based on quantitative analysis using high-performance liquid chromatography (HPLC). The present study suggests that the coumarins in lemon fruit are promising chemopreventive agents by inhibiting radical generation.

A diacetylenic spiroketal enol ether epoxide, AL-1, from Artemisia lactiflora inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion possibly by suppression of oxidative stress.

The inhibitory effects of the diacetylenic spiroketal enol ether epoxide AL-1 from Artemisia lactiflora on a variety of tumor promoter-induced biological responses such as oxidative stress as well as tumor promotion in ICR mouse skin were investigated. AL-1 inhibited TPA-induced intracellular peroxide formation in differentiated HL-60 cells, suggesting that this suppression might be attributable to the inhibition of O2- generation. In a double TPA application system in mouse skin, double pretreatments of AL-1 (810 nmol) significantly suppressed double TPA application-induced H2O2 generation. Pretreatment of AL-1 only before the second TPA treatment was sufficient to inhibit, while only with first treatment was not. From these results we concluded that AL-1 is a specific inhibitor of the activation phase in H2O2 production induced by double TPA treatments. In addition, AL-1 strongly inhibited tumor promoter-induced Epstein-Barr virus (EBV) activation in Raji cells (IC50 = 0.5 microM), which was comparable to or even stronger than that of curcumin, a well-known antioxidative chemopreventer from turmeric. In a two-stage carcinogenesis experiment with TPA (topical application at 1.6 nmol) and 7,12-dimethylbenz[a]anthracene (DMBA, at 0.19 micromol) in ICR mouse skin, topical application of AL-1 (at 160 nmol) significantly reduced tumor incidence, the numbers of tumors per mouse, and edema formation by 58% (P < 0.01 in t-test), 20% (P < 0.005 in chi2-test) and 42% (P < 0.01), respectively. These results together indicate that an inhibitor of O2 generation is an effective chemopreventer of mouse skin carcinogenesis by their antioxidative property.

Citrus auraptene exerts dose-dependent chemopreventive activity in rat large bowel tumorigenesis: the inhibition correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes.

In our previous short-term experiment, Citrus auraptene inhibited the development of azoxymethane (AOM)-induced aberrant crypt foci, which are precursor lesions for colorectal carcinoma. In the present study, the possible inhibitory effect of dietary administration of auraptene was investigated using an animal colon carcinogenesis model with a colon carcinogen AOM. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce colon neoplasms. They also received diets containing 100 or 500 ppm auraptene for 4 weeks in groups of "initiation" feeding, starting 1 week before the first dosing of AOM. The diets containing auraptene were also given to rats for 38 weeks in groups of "postinitiation" feeding. At the termination of the study (38 weeks), dietary administration of auraptene caused dose-dependent inhibition in AOM-induced large bowel carcinogenesis. Auraptene feeding during the initiation phase reduced the incidence of colon adenocarcinoma by 49% at 100 ppm (P = 0.099) and 65% at 500 ppm (P = 0.0075). Auraptene administration during the postinitiation phase inhibited the incidence of colon adenocarcinoma by 58% at 100 ppm (P = 0.021) and 65% at 500 ppm (P = 0.0075). Also, the multiplicity of colon carcinoma was significantly reduced by initiation feeding at a dose level of 500 ppm (P < 0.01) and postinitiation feeding at a level of 100 and 500 ppm (P < 0.05 and P < 0.01, respectively). Feeding of auraptene suppressed the expression of cell proliferation biomarkers (ornithine decarboxylase activity and polyamine content) in the colonic mucosa and reduced the production of aldehydic lipid peroxidation [malondialdehyde and 4-hydroxy-2(E)-nonenal]. In addition, auraptene increased the activities of Phase II drug-metabolizing enzymes (glutathione S-transferase and quinone reductase) in the liver and colon. These findings suggest that the inhibitory effects of auraptene on AOM-induced colon tumorigenesis at the initiation level might be associated, in part, with increased activity of Phase II enzymes, and those at the postinitiation stage might be related to suppression of cell proliferation and lipid peroxidation in the colonic mucosa.

Niaziminin, a thiocarbamate from the leaves of Moringa oleifera, holds a strict structural requirement for inhibition of tumor-promoter-induced Epstein-Barr virus activation.

Three known thiocarbamate (TC)- and isothiocyanate (ITC)-related compounds have been isolated from the leaves of Moringa oleifera, a traditional herb in southeast Asia, as inhibitors of tumor promoter teleocidin B-4-induced Epstein-Barr virus (EBV) activation in Raji cells. Interestingly, only niaziminin among 10 TCs including 8 synthetic ones showed considerable inhibition against EBV activation. The structure-activity relationships indicated that the presence of an acetoxy group at the 4'-position of niaziminin is important and indispensable for inhibition. On the other hand, among the ITC-related compounds, naturally occurring 4-[(4'-O-acetyl-alpha-L-rhamnosyloxy)benzyl]ITC and commercially available allyl- and benzyl-ITC significantly inhibited activation, suggesting that the isothiocyano group is a critical structural factor for activity.

Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly through suppression of superoxide generation in leukocytes.

Coumarin-related compounds, auraptene and umbelliferone, have been isolated from the cold-pressed oil of natsumikan (Citrus natsudaidai HAYATA), and tested as inhibitors of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus activation in Raji cells. The 50% inhibitory concentration (IC50) of auraptene (18 microM) was almost equal to that of genistein. Umbelliferone, which lacks a geranyloxyl group present in auraptene, was less active (IC50 = 450 microM). In a two-stage carcinogenesis experiment with 7,12-dimethylbenz[a] anthracene (topical application at 0.19 mumol) and TPA (topical application at 1.6 nmol) in ICR mouse skin, topical application of auraptene (at 160 nmol) significantly reduced tumor incidence and the numbers of tumors per mouse by 27% (P < 0.01) and 23% (P < 0.05), respectively. Auraptene at a concentration of 50 microM markedly suppressed superoxide (O2-) generation induced by 100 microM TPA in differentiated human promyelocytic HL-60 cells. Having no O2(-)-scavenging potential, auraptene may inhibit the multicomponent NADPH oxidase system. Inhibition of intracellular hydroperoxide formation in differentiated HL-60 cells by auraptene was also confirmed by flow-cytometric analysis using 2',7'-dichlorofluorescein diacetate as a fluorescence probe. Quantitative analyses using high-performance liquid chromatography showed the occurrence of auraptene not only in both the peels and sarcocarps of natsumikan, but also in those of hassaku orange (C. hassaku) and grapefruit (C. paradisi), and even in their bottled fresh juice form. These results indicate that auraptene is a chemopreventer of skin tumorigenesis, and implies that suppression of leukocyte activation might be the mechanism through which it inhibits tumor promotion.

Regional chemotherapy to prevent hepatic metastasis after resection of pancreatic cancer.

BACKGROUND/AIMS: Liver metastasis is one of the major causes of cancer death after resection of pancreatic cancer. To deal with this problem, we developed a liver perfusion chemotherapy via the hepatic artery and portal vein. The present paper is designed to introduce the technique of this method and report on the promising results. METHODOLOGY: Between 1987 and 1995, 98 consecutive patients with adenocarcinoma of the pancreas received an extended pancreatectomy, in which a wide range of lymphatic and connective tissue clearance was performed in addition to the conventional pancreatectomy, at Osaka Medical Center for Cancer and Cardiovascular Diseases. All patients were followed-up to determine whether and where cancer recurrence developed. RESULTS: Excluding 4 patients who died of postoperative complications, our liver perfusion chemotherapy was performed on 27 patients. The other 67 patients did not receive this treatment. In the group that underwent liver perfusion chemotherapy, no patients experienced complications such as leucocytopenia (< 3000/mm3), thrombocytopenia (< 50,000/mm3), or liver disfunction. The cumulative survival rate differed remarkably between the two groups: 92% vs 62% at one year (p < 0.05); 51% vs 35% at three years (p < 0.05); and 41% vs 25% at five years (ns). CONCLUSIONS: When this method was performed after an extended pancreatectomy for adenocarcinoma of the pancreas, our preliminary report (Am J Surg. 1994; 168:361-364) indicated that it was useful not only for preventing hepatic metastasis, but also for improving patients' survival rate. Since then, further positive and supportive results have been obtained in an additional investigation.

[Adjuvant therapies in combination with pancreatectomy for carcinoma of the pancreas].

In the treatment of adenocarcinoma of the pancreas, surgical resection is the only curative strategy. However, the long-term survival rate after pancreatectomy remains poor, and most patients died of loco-regional and/or hepatic recurrence. Thus, we should perform effective adjuvant therapies in combination with surgery, in order to completely prevent these two types of cancer relapse. The present article is designed to introduce the recent reports on the adjuvant chemo-and/or radio-therapies for this cancer. As for loco-regional control, extended pancreatectomy plus chemoradiation seems to be most promising, and preoperative chemoradiation will be more popular in the near future. In order to decrease hepatic metastasis, our "2-channel chemotherapy", a continuous infusion of 5-FU via both hepatic artery and portal vein, is very promising. If postoperative survival is improved by combining these two types of regional therapy, the role of pancreatectomy will be enlarged and more widely understood in the near future.