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Effects of Nobiletin on PhIP-Induced Prostate and Colon Carcinogenesis in F344 Rats
Abstract
The current study was designed to investigate the effects of nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced prostate and colon carcinogenesis. PhIP was administered to 6-wk-old F344 male rats intragastrically (100 mg/kg) twice a wk for 10 wk. The animals were given 0.05% nobiletin or the basal diet for 50 wk. At the end of the experiment, serum testosterone, estrogen, and leptin did not differ between the 2 groups. The body weights of nobiletin-treated rats were significantly higher than controls (P<0.05), and feeding of nobiletin significantly reduced the relative prostate (P<0.05) and testes (P<0.05) weights as well as the Ki67 labeling index in the normal epithelium in the ventral prostate (P<0.01). The incidence and multiplicity of adenocarcinomas in nobiletin-treated ventral prostate were 50% and 36%, respectively, of controls, but the differences were not statistically significant. However, nobiletin did significantly reduce the total number of colonic aberrant crypt foci (ACF) compared to the control value (P<0.05). Nobiletin, therefore, may have potential for chemoprevention of early changes associated with carcinogenesis in both the prostate and colon.
... Investigatory studies developed with the use of citrus flavonoids are mainly focusing on in vitro studies to demonstrate the mechanistic activity such as enzyme inhibition and anti-proliferative and cancer cell attenuation. They have a synergistic approach for the treatment of colon cancer and can act as a potent chemotherapeutic agent and also as a P-gp inhibitor (Tang et al. 2011). ...
... When nobiletin from citrus fruits (100 ppm) was given through the diet to mice with induced colon cancer, within a period of 20 weeks, they showed a noteworthy decrease in the incidence of colon cancer; hence, the researchers suggested that nobiletin has a chemopreventive action on colon tumor cells. When the experiment was done by using the nobiletin in the obese mice in the early stage of colon cancer, the reports that they had shown were a decrease in the proliferation of colon cancer cells (Tang et al. 2011). ...
... Nobiletin, a polymethoxy flavonoid found in citrus fruits, is proved with a potent anti-cancer effect on mice and with an inhibitory effect on human prostate carcinoma cells (Tang et al. 2011). The mode of action exhibited was through downregulating the protein expression levels (MMP-2 and MMP-9) which reasons a drastic reduction in tumor weight and tumor volume, deprived of any observed toxic outcome in the meantime. ...
The current state of the art for the use of natural ingredients for cancer therapy is by reviewing the publications and findings associated with cancer research with the employment of flavonoids. Cancer is the most furious disease making fear in the eyes of mankind. Though various treatment methods are prevalent, the patient’s choices are shifting from synthetic treatment strategy to the natural ones. The plant-based metabolites are used very often in our life as a food additive and also as a medicine for primary health care. The safety profile and its efficacy add on advantage for the incorporation of the natural products separately or in combination as a remedy for cancer. Flavonoids, the plant-based metabolites are proven for their anti-inflammatory, anti-oxidant, and anti-cancer properties. Their chemotherapeutic and chemosensitizing power had made it interesting for the researchers to dig more on the health benefits of the flavonoids and incorporating it in a holistic approach, with its natural benefits to relieve the pain and the symptoms of the patient suffering from various medical conditions. The predominant approach for the management of cancer is by following safe and effective treatment modality. In this review, we mentioned the benefits of the flavonoids for the management of various cancers and its potency as a chemotherapeutic agent and as the chemosensitizer. Our mother nature had given remedies to cure various diseases in both human beings and animals by it; we just need to find out the sources and access to them.
... Nobiletin (Nob) is a bioactive polymethoxylated flavone that is derived from different citrus varieties [19]; it has multiple targets and many biological functions, such as antioxidant, anti-inflammatory, antithrombotic, antilipidemia, and anticancer [20][21][22][23][24][25][26]. Many studies have shown that Nob has protective effects on the brain, myocardium, and vascular endothelium, suggesting that Nob has an excellent application prospect in prevention and treatment of cardiocerebrovascular diseases [27][28][29][30][31][32]. ...
... It is an important bioactive compound in traditional Chinese medicine, Pericarpium Citri Reticulatae and Fructus Aurantii [19,20], Chinese usually use Pericarpium Citri Reticulatae to make tea, wine and soup (25-30 g per time) [42]. Nob possesses antitumor, antithrombotic, anti-inflammatory, and antiatherosclerotic activities, which not only has strong antioxidant capacity but also could be a multi-target and multimechanism phytochemical that changes the expression and function of some proteins, which is important for their specific functions [20][21][22][23][24][25][26]. Studies found that Nob could alter HIF-1α, IL-1β, IFN-γ, TNF-α, and Bcl-2/Bax expression; regulate PI3K/Akt, SIRT-1/FOXO3a, Nrf-2/HO-1/MMP, JNK/ERK1/ 2, abd Akt/mTOR pathway; protect myocardium, neuron, and hepatocyte; improve endothelial function and so on [43][44][45][46][47]. ...
... The addition of antioxidants to the relevant treatment regimens has been suggested as a viable therapeutic approach for attenuating tissue damage induced by oxidative stress. Nob is the main antioxidant flavonoid in the drug-food homologous food, which belongs to polymethoxy flavonoids, and flavonoids are relatively non-toxic to animal cells [19][20][21][22][23][24][25][26]. In addition, Nob also possesses the properties of multi-target, multi-mechanism, and multi-function. ...
Iron overload is harmful to health and associates with intracellular excessive reactive oxygen species (ROS) generation. Nobiletin (Nob) is known to be antioxidant and anti-inflammatory. However, whether Nob can protect endothelial cells against iron overload has not been studied, and the specific mechanism has not yet been elucidated. In this study, we have identified the protective effects of Nob, and its underlying molecular mechanism in human umbilical vein endothelial cells (HUVECs) suffered from iron overload via ROS/ADMA/DDAHII/eNOS/NO pathway. We found that compared with 50 μM iron dextran treatment, co-treatment with 20 μM Nob increased cell viability and decreased lactate dehydrogenase activity. Besides, Nob could upregulate DDAHII expression and activity, promote eNOS phosphorylation to produce more NO, reduce ADMA content, and therefore increase superoxide dismutase, catalase, and glutathione peroxidase activities, and decrease malondialdehyde level and ROS generation. Nob also inhibited mitochondrial permeability transition pore (mPTP) openness and cleaved caspase-3 expression, and decreased apoptosis induced by iron overload. These results were consistent when Nob was replaced by the positive control reagents L-arginine (a competitive substrate of ADMA), cyclosporin A (an mPTP closing agent), or edaravone (a free radical scavenger). The addition of pAD/DDAHII-shRNA adenovirus reversed the above effects of Nob. These data suggested that the protective mechanism of Nob was to inhibit ROS burst, upregulate DDAHII expression and activity, promote eNOS phosphorylation, produce NO, reduce ADMA content, and ultimately alleviate iron overload damage in vascular endothelium.
... DMBA is able to induce the development of carcinomas in ventral prostate lobe through a subcutaneous injection (27). PhiP is a heterocyclic amine whose active metabolites form DNA adducts, inducing development of cancer in prostate, mammary gland, and intestine (44)(45)(46). It can be administered in the diet or administered by gavage (47,48). ...
Prostate cancer (PCa) is among the most frequent cancers worldwide. Nowadays, several therapeutic strategies are available for PCa treatment, namely chemotherapy, immunotherapy, radiotherapy, and hormonal therapy. Despite existing therapeutic approaches, in vitro and in vivo models are essential to better understand cancer development and search for more effective therapies, with a positive impact in cancer patient survival and quality of life. Among several models available, the rat model is the one most frequently used, since it shares anatomical, physiological, pathological, and behavioral features with humans. Animal models can be classified as: spontaneous, chemically-induced; hormonally-induced; implantation of cancer cell lines obtained from humans or from the same species, in the place of disease development or in a different place; and genetically-modified models. The chemically-induced models are among the most frequently used for PCa research. This manuscript provides a comprehensive overview of PCa models, presenting their application, advantages, and disadvantages, and their importance for the development of current therapies for prostate cancer.
... Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone, NOB) is one of the major polymethoxyflavones (PMFs) mostly found in citrus fruits [1]. In recent years, there has been increasing interest in the investigation of the biological properties, biotransformation and bioavailability of PMFs, due to their wide range of health-promoting effects, including anticancer [2][3][4][5], anti-inflammation [6,7], neuroprotection [8] and antiobesity [9]. Previously, we and others have investigated the anticarcinogenic effects of NOB against colon cancer. ...
... NOB can also activate autophagy and mitochondrial function through the sirtuin-1(SIRT-1)/ Forkhead box O3 (FOXO3α)and peroxisome proliferator-activated receptor γ coactiva-tor-1 alpha (PGC-1α) pathways in hepatic ischemia and reperfusion injury 11 . In addition, a considerable number of reports have shown that NOB exhibits anticarcinogenic activity against various cancer cells [12][13][14] , including colon 15,16 , prostate 17 and lung 18,19 cancers. For example, NOB can promote cell-cycle arrest, induce apoptosis, and profoundly modulate signaling proteins associated with cell proliferation and cell death to significantly inhibit the growth of human colon cancer cells 20 . ...
Objectives
This study was to investigate whether nobiletin (NOB) can inhibit the proliferation of oral squamous cell carcinoma (OSCC) cell by promoting apoptosis, oxidative stress (ROS), and DNA damage.
Study Design
OSCC were treated with different concentrations of NOB (25, 50, and 100 µM) for different amounts of time (0, 24, 48, and 72 hours). The viability of NOB was assessed using MTT-based cell viability assays. Flow cytometry was used to assess cell apoptosis, and the expressions of capase-3 and PARP were assessed by quantitative real-time PCR (RT-PCR) and western blotting analyses. The intensity of ROS fluorescence was measured using a spectrophotometer. The expression of γH2AX and 8-oxodG were assessed to determine the degree of DNA damage.
Results
We observed that NOB decreased OSCC cell viability in a dose- and time-dependent manner but had little effect on primary normal human oral epithelial cells (H0ECs). Moreover, with the increase in NOB concentration and treatment time, capase-3, PARP mRNA and protein levels gradually increased, as did annexin V- and 7ADD-mediated apoptosis. In addition, NOB also increased the levels of ROS and DNA damage in a concentration- and time-dependent manner.
Conclusions
NOB can inhibit OSCC cell by promoting apoptosis, ROS production and DNA damage.
... Nobiletin, a major PMF of particular interest has been reported to inhibit carcinogenesis in colon, prostate, and breast cancer models (C. Chen, Ono, Takeshima, & Nakano, 2014;Tang et al., 2011). Notably, 3',4'-didemethylnobiletin (DDMN), one of the major metabolites of nobiletin in mice and also the most potent metabolite, is reported to be present at a much higher concentration in mouse colonic mucosa than nobiletin; therefore, these findings suggest many of the observed positive effects of nobiletin could be attributed to its metabolite DDMN . ...
Chemoprevention strategies employing the use of multiple dietary bioactive components and their metabolites in combination offer advantages due to their low toxicity and potential synergistic interactions. Herein, for the first time, we studied the combination of curcumin and 3',4'‐didemethylnobiletin (DDMN), a primary metabolite of nobiletin, to determine their combinatory effects in inhibiting growth of human colon cancer cells. Isobologram analysis revealed a synergistic interaction between curcumin and DDMN in the inhibition of cell growth of HCT116 colon cancer cells. The combination treatment induced significant G2–M cell‐cycle arrest and extensive apoptosis, which greatly exceeded the effects of individual treatments with curcumin or DDMN. Proteins associated with these heightened anticarcinogenic effects were p53, p21, HO‐1, c‐poly(ADP‐ribose) polymerase, Cdc2, and Cdc25c; each of the proteins was confirmed to be substantially impacted by the combination treatment, more than by individual treatments alone. Interestingly, an increase in the stability of curcumin was also observed with the presence of DDMN in cell culture medium, which could offer an explanation in part for the synergistic interaction between curcumin and DDMN. This newly identified synergy between curcumin and DDMN should be explored further to determine its chemopreventive potential against colon cancer in vivo.
Practical Application
This study identifies for the first time the synergistic inhibition of colon cancer cell growth by the dietary component curcumin present in turmeric, in combination with a metabolite of nobiletin, a unique citrus flavonoid. The synergism of the combination may be due to cell‐cycle arrest and apoptosis induced by the combination as well as an improvement in the stability of curcumin as a result of the antioxidant property of the nobiletin metabolite. These significant findings of synergism between curcumin and the nobiletin metabolite could offer potential chemopreventive value against colon cancer.
... [6][7][8] As one of the most abundant polymethoxyflavones (PMFs), nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone, NBT, Fig. 1) is specifically found in the peel of Citrus depressa and Citrus aurantium and provides a natural resistance against pathogenic fungi. 9 Since it was discovered in 1967, NBT has been demonstrated to exert a wide range of beneficial activities, including anti-inflammation, 10,11 anti-carcinogenesis, [12][13][14][15][16][17] antidementia 18,19 and anti-atherosclerosis. 20 Most notably, some previous studies had showed that treatment with NBT suppressed lung carcinogenesis both in vitro and in vivo. [21][22][23] Luo et al. reported that NBT induced apoptosis and cycle arrest at the G2/M phase in A549 cells and suppressed the lung tumor growth in nude mice. ...
Nobiletin (NBT), a citrus flavonoid, has been associated with various health benefits. Herein, we investigated the chemopreventive actions of NBT and its metabolites in a pulmonary carcinogenesis mouse model and human lung cancer cells. In 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)-treated mice, oral administration of NBT significantly suppressed lung tumorigenesis evidenced by reduced tumor volume compared to the control mice. NBT also greatly attenuated cell proliferation in the lung of NNK-treated mice. Our previous study has identified three major metabolites of NBT, namely, 3’-demethylnobiletin (M1), 4’- demethylnobiletin (M2), and 3’,4’ -didemethylnobiletin (M3). In this study, we further determined the inhibitory effects of NBT and its metabolites on human non-small cell lung cancer (NSCLC) cells and the underlying mechanisims of action. Interestingly, we found that M2 and M3 exerted much stronger growth inhibition on both H460 and H1299 cells, compared to their parent compound NBT. Flow cytometry and western blotting analysis revealed that M2 and M3 caused significant cell cycle arrest and cellular apoptosis, and profoundly modulated multiple proteins associated with cell proliferation and cell death, including p21, cyclin B1, CDK1, cyclin D1, CDK6, CDK4, Bax, cleaved caspase-1, cleaved PARP. Overall, our results demonstrated that oral administration of NBT significantly inhibited lung carcinogenesis in mice, and these chemopreventive effects could be attributed to its metabolites that showed potent anti-cancer effects.
Colorectal cancer is one of the most common gastrointestinal malignancies in humans, affecting approximately 1.8 million people worldwide. This disease has a major social impact and high treatment costs. Animal models allow us to understand and follow the colon cancer progression; thus, in vivo studies are essential to improve and discover new ways of prevention and treatment. Dietary natural products have been under investigation for better and natural prevention, envisioning to show their potential. This manuscript intends to provide the readers a review of rodent colorectal cancer models available in the literature, highlighting their advantages and disadvantages, as well as their potential in the evaluation of several drugs and natural compounds’ effects on colorectal cancer.
Nobiletin is a citrus flavonoid with poor water solubility, which limits its application. To investigate the effects of emulsions on the bioavailability of nobiletin, long-chain triglycerides (LCT, i.e., corn oil) and medium-chain triglycerides (MCT) were used in combination with bergamot oil at different mixing ratios (i.e., 1: 0, 3: 1, 1: 1, 1: 3, and 0: 1) as the oil phase to produce nobiletin–encapsulating emulsions by high-pressure homogenization. The emulsion stability, digestion fate, and bioavailability of nobiletin were systematically investigated. The results showed that triacylglycerol–bergamot oil at the ratio of 1: 3 was the most stable. The hydrolysis rate of triacylglycerol–bergamot oil emulsions increased with increasing triacylglycerol: bergamot oil ratio. The total bioavailability of nobiletin in corn oil–bergamot oil emulsions was higher than that of pure bergamot oil and MCT–bergamot oil emulsions during in vitro digestion, due to the higher hydrolysis rate of corn oil–bergamot oil and greater solubilization capacity of micelles formed by bile salts and long-chain fatty acids (LCFAs) from corn oil. Nobiletin bioavailability was highest in the 3: 1 corn oil: bergamot oil emulsion. This simulated digestion study may be useful for designing emulsion-based delivery systems to control the bioavailability of hydrophobic nutrients.
Cooked food contains a variety of mutagenic heterocyclic amines. All the mutagenic heterocyclic amines tested were carcinogenic in rodents when given in the diet at 0.01-0.08%. Most of them induced cancer in the liver and in other organs. It is noteworthy that the most abundant heterocyclic amine in cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, produced colon and mammary carcinomas in rats and lymphomas in mice but no hepatomas in either. 2-Amino-3-methylimidazo[4,5-f]quinoline induced liver cancer in monkeys. Formation of adducts with guanine by heterocyclic amines is presumably involved in their carcinogenesis. Quantification of heterocyclic amines in cooked foods and in human urine indicated that humans are continuously exposed to low levels of them in the diet. These low levels of heterocyclic amines are probably insufficient to produce human cancers by themselves. However, a linear relationship between DNA adduct levels and a wide range of doses of a heterocyclic amine was demonstrated in animals. It suggests that even very low doses of heterocyclic amines form DNA adducts and may be implicated in the development of human cancer under conditions in which many other mutagens-carcinogens, tumor promoters, and factors stimulating cancer progression exist.
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced during cooking and is mutagenic to bacteria and cultured mammalian cells, was found to induce high incidences of colon and mammary carcinomas in F344 rats when administered at a concentration of 400 p.p.m. in the diet for 52 weeks. Since PhIP is the most abundant of the mutagenic heterocyclic amines in cooked meat and fish, the compound might be related to malignancies of the colon and breast in humans.
Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds found in meats cooked at high temperatures. Although chicken is consumed in large quantities in the United States, there is little information on its HAA content. The objective of this study was to measure the five predominant HAAs (IQ, MeIQ, MeIQx, DiMeIQx, and PhIP) in chicken cooked by various methods to different degrees of doneness. Chicken breasts were panfried, oven-broiled, or grilled/barbecued. Whole chickens were roasted or stewed. Skinless, boneless chicken breasts were cooked to three degrees of doneness: just until done, well done, or very well done. High levels of PhIP (ranging from 12 to 480 ng/g cooked meat) were found in chicken breasts when panfried, oven-broiled, and grilled/barbecued but not in while roasted or stewed chicken. PhIP concentration increased in skinless, boneless chicken breast with longer cooking time, higher internal temperature, and greater degree of surface browning. PhIP concentration was also high in chicken breasts cooked with skin and bones. MeIQx and DiMeIQx levels increased with the degree of doneness, whereas IQ and MeIQ were not detectable in any of these chicken samples. Certain cooking methods produce PhIP, a known colon and breast carcinogen in rodents and possibly a human carcinogen, at substantially higher levels in chicken than has been reported previously in red meat.
Prostate tissues obtained from rats given a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), at a dose of 400 ppm in the diet for 52 weeks were histopathologically evaluated and found to contain prostate carcinomas limited to the ventral lobe in 18 of 27 cases. Atypical hyperplasias were also detected in the ventral and anterior prostate as well as the seminal vesicles. 32P-Postlabeling analysis of DNA demonstrated that PhIP-DNA adducts are produced in all lobes of the prostate of rats receiving PhIP. The findings indicate that PhIP is carcinogenic to rat prostate in addition to the previously demonstrated targeting of the colon and mammary glands, providing evidence of a possible role of PhIP in human prostate carcinogenesis and highlighting the potential importance of PhIP for man.
A polyclonal antibody against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts was raised for their immunohistochemical demonstration in paraffin-embedded sections. Specificity of this antibody was confirmed by competitive ELISA. Positive signals were immunohistochemically detected in acetone-fixed but not in formalin- or ethanol-fixed sections from F344 rats treated by gavage with a single dose of PhIP at 37.5-300 mg/kg and killed at 1, 2, and 7 days thereafter. Dose-dependent positive staining was observed in almost all organs of both sexes, including the colon, prostate, and mammary gland but largely independent of the tumor response. Repair activity, judged by disappearance of adducts with time, differed according to the organ or cell type. One exception was hepatocytes, the liver incidentally being a nontarget organ. The results suggest that the generated antibody is applicable for detection of cells targeted by PhIP in paraffin-embedded sections and also for the investigation of the mechanisms of PhIP-carcinogenesis.
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.
We have generated a transgenic rat with the SV40 T antigen under probasin promoter control, allowing prostate-specific gene expression. Males demonstrate atypical epithelial cell proliferation in the prostate from 4 weeks of age and develop prostate carcinomas at 100% incidence before they are 15 weeks old. Castration at 5 weeks of age was found to inhibit the prostate tumor formation completely, whereas testosterone propionate administration induced marked cell proliferation as well as microinvasion in prostate carcinomas. Castration at 20 weeks of age, after tumor development, even with testosterone propionate treatment, induced complete tumor involution within 5 weeks. To investigate the underling processes, sequential histological changes were monitored 1, 2, 3, 7, 14, and 21 days after castration. At days 1-3, many apoptotic bodies and inflammatory cells, including foam cells, were observed, and clear glandular structures were no longer evident in the tumors. Seven days after castration, most glands were involved, and nuclei of the cells did not show atypia. After 14 and 21 days, only atrophic glands were observed. During this process, expression of caspase 3, caspase 6, BAX, bcl-x, TRPM-2, and MMP7 genes was apparently increased. Comparison of the gene expression profile between a prostate carcinoma in a transgenic animal and a normal prostate of a wild-type rat by a cDNA array technique was also conducted. The results suggested that our model is suitable to investigate mechanisms of carcinogenesis, including androgen dependence, involution, and apoptosis.
Dietary flavonoids have been shown to reduce risk of cardiovascular disease, but the underlying molecular mechanisms are not known. The objective of this study was to investigate the effect of nobiletin, a dietary phytochemical belonging to polymethoxy flavonoid from the peel of Citrus fruit, on vascular smooth muscle cells (VSMCs) proliferation and its mechanisms. VSMCs proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and [(3)H]thymidine incorporation assay. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were determined by western blotting. [Ca(2+)](i) was measured by laser scanning confocal microscopy. Our results showed that angiotensin II-induced VSMCs proliferation was inhibited by nobiletin. While no effect on ERK1/2 and p38 MAPK, nobiletin markedly inhibited angiotensin II-induced activation of JNK. Anthra[1-9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, decreased the [(3)H]thymidine incorporation induced by angiotensin II. Nobiletin also attenuated both the intracellular Ca(2+) mobilization and the extracellular Ca(2+) influx induced by angiotensin II. Furthermore, intracellular Ca(2+) chelation by BAPTA-AM, extracellular Ca(2+) chelation by EGTA or blockade of L-type Ca(2+) channel with verapamil inhibited angiotensin II-induced JNK activation. These findings suggest that the preventing effect of nobiletin on angiotensin II-induced VSMCs proliferation is attributed, in part, to its inhibitory effect on Ca(2+)-dependent JNK activation in VSMCs. Thus, inhibition of JNK by nobiletin may imply its usefulness for the treatment of cardiovascular diseases relevant to VSMCs growth.
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant mutagenic heterocyclic amine by weight in cooked foods. This mutagen was found to produce DNA adducts in all ten tested organs of rats using the 32P-postlabeling method. The level of DNA adducts in the pancreas, kidney and liver increased dose-dependently and feeding time-dependently up to four weeks. When diet containing 0.05% PhIP was given to rats for four weeks, levels of PhIP-DNA adducts were relatively high in the lung, pancreas and heart, being around 20 per 10(7) nucleotides, and lowest in the liver, being 2.20 per 10(7) nucleotides. Thus, PhIP showed a unique feature in the formation of DNA adducts compared to other mutagenic and carcinogenic heterocyclic amines, which produce the highest level of DNA adducts in the liver.
Polyclonal antibodies against 3,2'-dimethyl-4-aminobiphenyl (DMAB)- and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts were raised for their immunohistochemical demonstration in paraffin-embedded sections. Both carcinogens target multiorgans in rats with colon, mammary glands and prostate as common tumor sites. PhIP is particularly important because it is a cooked food-derived carcinogen to which man is exposed on a daily bases. Dose-related nuclear staining in various tissue of rats was observed 24 h after single applications of both carcinogens but no observable decrease in staining intensity was evident in most organs by 168 h. Staining specificity in terms of sites of tumor development was lacking. However, in target organs, semiquantitative data on adduct formation correlated with alteration in tumor response by modifying factor(s). Furthermore, human prostate tissue implanted into nude mice showed positive staining. Thus, the antibodies can be applied as tools to clarify tissue or cell-specific carcinogenesis, carcinogen-exposure levels and metabolic activation in various species, including man after tissue transplantation.