Chemopreventive Effects of Eryngium foetidum L. Leaves on COX-2 Reduction in Mice Induced Colorectal Carcinogenesis.

To investigate the potential effects of Eryngium foetidum Linn. leaves (EF) in colitis-induced colorectal carcinogenesis in mice by azoxymethane (AOM) and dextran sulfate sodium (DSS), 39 ICR male mice were studied and divided into 6 groups. The mice were received a modified AIN-76 diet in Group 1, whereas Group 2 was given an AOM, DSS, and AIN-76 diet. Groups 3 and 4 were fed with 0.8% and 3.2% freeze-dried EF with AIN-76 diets, for 5 wk. Groups 5 and 6 were fed with 0.8% and 3.2% EF diets for 5 wk during AOM/DSS administration. The mice were necropsied at Week 20 and their colons were collected. The results indicated that the incidences of tumors in Groups 2, 5, and 6 was 100%, 75%, and 88%, with multiplicities (mean ±SE) of 3.75 ±0.92, 2.38 ± 0.96 and 4.25 ± 0.79, respectively. Interestingly, there was a significant difference in COX-2 expression in mice received 3.2% EF in their diet, but the proliferative cell nuclear antigen index and iNOS protein expression were not significantly different. We concluded that EF at a dose level of 3.2% in their diet had a preventive effect on colorectal carcinogenesis via the proinflammatory cytokine, COX-2.

Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models.

BACKGROUND: Chemotherapy of cholangiocarcinoma (CCA), a devastating cancer with increasing worldwide incidence and mortality rates, is largely ineffective. The discovery and development of effective chemotherapeutics is urgently needed. METHODS/DESIGN: The study aimed at evaluating anticancer activities, toxicity, and pharmacological activities of the curcumin compound (CUR), the crude ethanolic extracts of rhizomes of Zingiber officinale Roscoe (Ginger: ZO) and Atractylodes lancea thung. DC (Khod-Kha-Mao: AL), fruits of Piper chaba Hunt. (De-Plee: PC), and Pra-Sa-Prao-Yhai formulation (a mixture of parts of 18 Thai medicinal plants: PPF) were investigated in animal models. Anti-cholangiocarcinoma (anti-CCA) was assessed using CCA-xenograft nude mouse model. The antihypertensive, analgesic, anti-inflammatory, antipyretic, and anti-ulcer activities and effects on motor coordination were investigated using Rota-rod test, CODA tail-cuff system, writhing and hot plate tests, carrageenan-induced paw edema test, brewer's yeast test, and alcohol-induced gastric ulcer test, respectively. Acute and subacute toxicity tests were performed according to the OECD guideline for testing of chemicals with modification. RESULTS: Promising anticancer activity against CCA in nude mouse xenograft model was shown for the ethanolic extract of AL at all oral dose levels (1000, 3000, and 5000 mg/kg body weight) as well as the extracts of ZO, PPF, and CUR compound at the highest dose level (5000, 4000, and 5000 mg/kg body weight, respectively). PC produced no significant anti-CCA activity. Results from acute and subacute toxicity tests both in mice and rats indicate safety profiles of all the test materials in a broad range of dose levels. No significant toxicity except stomach irritation and general CNS depressant signs were observed. Investigation of pharmacological activities of the test materials revealed promising anti-inflammatory (ZO, PPF, and AL), analgesic (CUR and PPF), antipyretic (CUR and AL), antihypertensive (ZO and AL), and anti-ulcer (CUR, ZO, and AL) activities. CONCLUSION: Plants used in Thai traditional medicine for the treatment of various ailments may provide reservoirs of promising candidate chemotherapeutics for the treatment of CCA.

Protein expression profiles that underpin the preventive and therapeutic potential of Moringa oleifera Lam against azoxymethane and dextran sodium sulfate-induced mouse colon carcinogenesis.

Previous studies in a mouse model have indicated the anticancer potential of boiled Moringa oleifera pod (bMO)-supplemented diets; however, its molecular mechanisms are still unclear. Therefore, the present study aimed to explore the protein expression profiles responsible for the suppressive effect of bMO supplementation on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced mouse colon carcinogenesis. Analysis by gel electrophoresis and liquid chromatography-tandem mass spectrophotometry demonstrated that there were 125 proteins that were differentially expressed in mouse colon tissues between 14 experimental groups of mice. The differentially expressed proteins are involved in various biological processes, such as signal transduction, metabolism, transcription and translation. Venn diagram analysis of the differentially expressed proteins was performed in six selected mouse groups, including negative control, positive control mice induced by AOM/DSS, the AOM/DSS groups receiving preventive or therapeutic bMO diets and their bMO-supplemented control groups. This analysis identified 7 proteins; 60S acidic ribosomal protein P1 (Rplp1), fragile X mental retardation, cystatin 9, round spermatids protein, zinc finger protein 638, protein phosphatase 2C (Ppm1g) and unnamed protein product as being potentially associated with the preventive and therapeutic effects of bMO in AOM/DSS-induced mouse colon cancer. Analysis based on the search tool for interactions of chemicals (STITCH) database predicted that Rplp1 interacted with the apoptotic and inflammatory pathways, whereas Ppm1g was associated only with inflammatory networks. This proteomic analysis revealed candidate proteins that are responsible for the effects of bMO supplementation, potentially by regulating apoptotic and inflammatory signaling networks in colorectal cancer prevention and therapy.

Brown rice and retrograded brown rice alleviate inflammatory response in dextran sulfate sodium (DSS)-induced colitis mice.

The present study was aimed to investigate the impacts of brown rice (BR) and retrograded brown rice (R-BR) consumption on colonic health and gut microbiota in dextran sulfate sodium (DSS) induced colitis mice. Thirty two female C57Bl/6Mlac mice were fed with modified AIN 93G diets by replacing cornstarch in the original composition with white rice (WR), BR and R-BR powder. The mice were divided into 4 groups and fed with the following experimental diets for 4 weeks: (1) negative control (WR: diet with WR), (2) positive control (DSS_WR: DSS and diet with WR), (3) DSS_BR: DSS and diet with BR, and (4) DSS_R-BR: DSS and diet with R-BR. BR and R-BR had a greater content of fat, dietary fiber, GABA, γ-oryzanol, γ-tocotrienol, ferulic acid and p-coumaric acid than WR (p < 0.05). No significant difference in the level of these bioactive compounds was noted between BR and R-BR. Nevertheless, R-BR had a 1.8 fold resistant starch (RS) content of BR (p < 0.05). The DSS_BR and DSS_R-BR groups showed a lower ratio of colonic weight to length, and a lower content of iNOS, COX-2, MPO, IL-6 and INF-γ in colonic homogenates than the DSS_WR group. However, the DSS treated mice fed with the R-BR diet had significantly milder histopathological inflammatory injury and lower colonic iNOS expression than the DSS_BR and DSS_WR groups. The percentage of mesenteric regulatory T cells significantly increased in the DSS_R-BR group compared to that in the DSS_WR group. The DSS treated mice fed with the R-BR diet showed a significant increase in cecal bacterial diversity and abundance of genera Prevotella, Ruminococcus, Dorea, Coprococcus and Dehalobacterium but a significant decrease in pathogenic bacteria including Bacteroides and Enterococcus compared to the DSS_WR group. Thus, the present data indicate that BR and R-BR ameliorate colonic inflammation in experimental colitis induced by DSS in mice by suppressing inflammatory mediators and modulating regulatory T cell responses as well as bacterial diversity in the cecum.

A 24-Weeks Toxicity Study of Eryngium foetidum Linn. Leaves in Mice.

Eryngium foetidum Linn. leaves (EF) are widely used in Thailand and many countries throughout Asia as a culinary seasoning and a traditional medicine. However, adverse effect of high dose consumption in long duration has not been evaluated. The aim of this study was to investigate chronic toxicity of EF in mice. Thirty-two ICR male mice were divided into 4 groups of 8 mice each. The mice were fed AIN-76 rodent diet, or AIN-76 rodent diet supplemented with ground freeze-dried EF at 0.8%, 1.6% and 3.2% that is equivalent to approximately 35, 73 and 155 times that of human consumption, respectively, at 97.5 percentile for a period of 24 weeks. At the end of experiment, the mice were euthanized and blood samples were collected for hematological and biochemical evaluations. Necropsy was performed while visceral organs such as lung, liver, kidneys, spleen etc. were collected, weighed and histopathologically examined. Blood urea nitrogen (BUN) results of mice in 1.6% and 3.2% EF diet groups were significantly higher than the BUN of control group. No significant difference was noted in other biochemical and hematological properties between the treatment groups and control; all results were within normal range. Histopathology of almost all visceral organs showed no significant changes. However, tubulonephrosis and chronic interstitial nephritis were observed in the groups treated with 1.6% and 3.2% EF diet. Body weight was reduced significantly at week 12 to week 20 when compared to the control group while relative kidney weights were significantly increased. In conclusion, the consumption of EF in diet at high doses illustrated the adverse effect on some biochemical parameters and histopathology in mice. Our findings suggested that EF daily consumption for 24 weeks, at higher doses than the 0.8% EF diet (35 times of human consumption), might cause adverse effect on kidney function in mice.

Anticlastogenic effect of Eryngium foetidum L. assessed by erythrocyte micronucleus assay.

The aim of this study was to investigate the anticlastogenicity as well as the clastogenicity of Eryngium foetidum leaf (EF) using the in vivo mouse peripheral blood erythrocyte micronucleus assay. Eighty ICR male mice were fed AIN-76 diet supplemented with ground freeze-dried EF at 0.0%, 0.8%, 1.6% and 3.2% for 2 weeks prior to the administration of both direct-acting, mitomycin C (MMC), and indirect-acting, 7, 12-dimethylbenz(a) anthracene (DMBA) clastogens. Peripheral blood samples were collected from mice just before administration of clastogen and at 24 and 48 h thereafter for MMC. Blood samples were collected at the same times and after 72 h for DMBA. Then, reticulocytes in blood samples were counted using fluorescent microscopy. The results indicated that EF had no clastogenic effect in mice. All doses of diets supplemented with EF decreased the number of micronucleated peripheral reticulocytes in all the MMC-treated groups in a dose dependent manner, but significant reduction was found only at 1.6% and 3.2% EF in the DMBA-treated groups. It can be concluded that EF has no clastogenicity, but possesses anticlastogenic potential against both direct- and indirect-acting types of clastogen in mice.

Anticlastogenic and anticarcinogenic potential of Thai bitter gourd fruits.

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possess phase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activating enzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined the anticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, in mice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesis in rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing 6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end of experiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. For anticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% ground freeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kg once a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleated peripheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstrated that rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as the multiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruits possesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potential against AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm when giving during the initiation stage.

Suppressive effects of Moringa oleifera Lam pod against mouse colon carcinogenesis induced by azoxymethane and dextran sodium sulfate.

Moringa oleifera Lam (horseradish tree; tender pod or fruits) is a major ingredient in Thai cuisine and has some medicinal properties. Previous studies have shown potentially antioxidant, antitumor promoter, anticlastogen and anticarcinogen activities both in vitro and in vivo. The present study was conducted to investigate chemopreventive effects on azoxymethane (AOM)-initiated and dextran sodium sulfate (DSS)-promoted colon carcinogenesis in mice. Male ICR mice were divided into 8 groups: Group 1 served as a negative control; Group 2 received AOM/DSS as a positive control; Groups 3-5 were fed boiled freeze-dried M. oleifera (bMO) at 1.5%, 3.0% and 6.0%, respectively supplemented in basal diets for 5 weeks; Groups 6-8 were fed with bMO diets at the designed doses above for 2 weeks prior to AOM, during and 1 week after DSS administration. At the end of the study, colon samples were processed for histopathological examination. PCNA indices, and iNOS and COX-2 expression were assessed by immunohistochemistry. The results demonstrated the incidences and multiplicities of tumors in Groups 6-8 to be decreased when compared to Group 2 in a dose dependent manner, but this was significant only in Group 8. The PCNA index was also significantly decreased in Group 8 whereas iNOS and COX-2 protein expression were significantly decreased in Groups 7 and 8. The findings suggest that M. oleifera Lam pod exerts suppressive effects in a colitis-related colon carcinogenesis model induced by AOM/DSS and could serve as a chemopreventive agent.

Nutritive evaluation and effect of Moringa oleifera pod on clastogenic potential in the mouse.

Moringa oleifera Lam (horseradish tree; tender pod or fruits) has been consumed as a vegetable and utilized as a major ingredient of healthy Thai cuisine. Previous studies have shown that M. oleifera pod extracts act as bifunctional inducers along with displaying antioxidant properties and also inhibiting skin papillomagenesis in mice. This study was aimed to determine the nutritive value, and clastogenic and anticlastogenic potentials of M. oleifera pod. The nutritive value was determined according to AOAC methods. The clastogenic and anticlastogenic potentials were determined using the in vivo erythrocyte micronucleus assay in the mouse. Eighty male mice were fed semi-purified diets containing 1.5%, 3.0% and 6.0% of ground freeze-dried boiled M. oleifera pod (bMO) for 2 weeks prior to administration of both direct-acting (mitomycin C, MMC) and indirect-acting (7, 12-dimethylbenz(a)anthracene, DMBA), clastogens. Blood samples were collected at 0, 24, 48 and 72 h, dropped on acridine orange-coated slides, and then counted for reticulocytes both with and without micronuclei by fluorescence microscopy. The nutritive value of 100 g bMO consisted of: moisture content, 8.2 g; protein, 19.2 g; fat, 3.9 g; carbohydrate (dietary fiber included), 60.5 g; dietary fiber, 37.5 g; ash, 8.1 g and energy, 354 kcal. Freeze-dried boiled M. oleifera had no clastogenic activity in the mouse while it possessed anticlastogenic activity against both direct and indirect-acting clastogens. Freeze-dried boiled M. oleifera pod at 1.5%, 3.0% and 6.0% in the diets decreased the number of micronucleated peripheral reticulocytes (MNRETs) induced by both MMC and DMBA. However, the effect was statistically significant in the dose dependent manner only in the MMC-treated group. In conclusion, the present study demonstrated that bMO has no clastogenicity and possesses anticlastogenic potential against clastogens, and particularly a direct-acting clastogen in the mouse.

Effects of Rhinacanthus nasutus kurz on colon carcinogenesis in mice.

Rhinacanthus nasutus Kurz, a Thai medicinal plant which possess antiproliferative and pro-apoptotic effects on human cancer cells, was examined for chemopreventive potential against colonic neoplasms induced by azoxymethane (AOM) combined with dextran sodium sulfate (DSS) in mice. Male ICR mice were given a single intraperitoneal administration of AOM (10 mg/kg body weight) followed by 2% DSS in their drinking water for a week. Water extract of the roots of R. nasutus (RNR) was given to the animals intragastrically daily in the initiation and promotion phases. The one hundred mice were divided into 8 groups, one group treated with AOM plus DSS serving as a control. Four other groups received AOM/DSS and RNR at 100 or 500 mg/kg body weight for 5 weeks (initiation phase study) and for 14 weeks (promotion phase study). Another two groups were given RNR alone at 100 and 500 mg/kg body weight and the last group was maintained untreated. At the end of the study, we found that the incidence and multiplicity of colonic tumors in mice fed with RNR both at 100 and 500 mg/kg body weight in initiation phase were higher than those in the control group. Moreover, RNR feeding during the promotion phase also gave similar results. Our results suggest that water extract of the roots of R. nasutus Kurz. has no preventive potential against colon carcinogenesis induced by AOM/DSS in mice, rather increasing the incidence of colonic tumors when given during initiation and promotion phases. Further study on RNR should provide more information on mechanisms of its tumor promotion activity.

Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo.

Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes.

Chemopreventive Potential of Neem Flowers on Carcinogen-Induced Rat Mammary and Liver Carcinogenesis.

We have previously reported that dietary neem flowers (Azadirachta indica A. Juss var. siamensis Valeton) caused a marked increase in glutathione S-transferase (GST) activity in the liver, while resulting in a significant reduction in the activities of some hepatic P450-dependent monooxygenases. These results strongly indicate that neem flowers may have chemopreventive potential. In the present study, we examined the inhibitory effects of neem flowers on 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague Dawley rats and on aflatoxin B(1)(AFB(1))-induced hepatocarcinogenesis in male Wistar rats. Young animals were fed with AIN-76 purified diets containing either 10-12.5% ground freeze-dried neem flowers for 1 week prior to, during, and for 1 week after the administration of each carcinogen. Interestingly, it was found that neem flowers resulted in a marked reduction of the incidence of mammary gland (about 35.2%) and liver tumors (61.7% and 80.1% for benign and malignant tumors, respectively). Furthermore, the multiplicity of tumors per rats was also lower in the neem flower groups, i.e. those for mammary gland tumors and benign and malignant liver tumors were reduced to 44.0%, 87.9% and 88.9%, respectively. These results clearly demonstrated that neem flowers contain some chemopreventive agents capable of inhibiting AFB(1) and DMBA induced liver and mammary gland carcinogenesis in rats.

Carcinogenicity Testing of the Cosmetic Dye: D&C Red No. 36.

D&C Red No. 36, a drug and cosmetic dye commonly used for coloring lipsticks, was evaluated for its carcinogenic potential in male and female Wistar rats. This dye has been shown to exhibit mutagenic activity towards Salmonella typhimurium TA 98 in the presence of S9 mix. In the present study, 50 male and 50 female rats in each group were given diets containing D&C Red No. 36 at 2 different concentrations, 1,000 ppm and 2,000 ppm, for 78 weeks and sacrificed at week 98. It was found that dye treatment had no significant effect on the survival of either male or female animals as well as the body weight gain in males. However, body weight gain of treated females was slightly lower than that of the control group. Histopathological assessment demonstrated a number of benign and malignant tumors to have developed in various organs of both dye treated and control groups. In male rats, benign liver tumors were found at incidences of 16.7% and 18.8% of the low (1,000 ppm) and high (2,000 ppm) dose groups, respectively, similar to the 20% for the control group. Malignant tumors of the thyroid gland were observed only in the low dose and control groups, at 4.2% and 2%, respectively. In the high dose group, the incidences of lung, liver, urinary bladder and soft tissue cancers were 4.2%, 2.1%, 2.1% and 2.1%, respectively, only one soft tissue cancer being observed in a control group animal. In females, benign tumors were observed in the liver and mammary glands. The incidences of liver tumors in the low and high dose groups were 12.8% and 16%, respectively, and 6% in the control group. Values for mammary gland tumors were 10.6%, 10%, and 18% respectively. Malignant tumors were also observed in various other organs, including the uterus, lung, kidney, thyroid, thymus and salivary gland, but the incidences were very low (about 2-4%) and in dye treated male and female rats were not statistically different from those in the control animals. The results of the present study thus demonstrated that D&C Red No. 36 at the concentrations of 1,000 ppm and 2,000 ppm in the diet is not carcinogenic either to male or female Wistar rats. While the occurrence of benign liver tumors in female rats may be related to dye treatment, the lack of any apparent dose-dependence or any statistically significant difference from the control group (P = 0.06) suggests that this is unlikely.