Nexrutine inhibits azoxymethane-induced colonic aberrant crypt formation in rat colon and induced apoptotic cell death in colon adenocarcinoma cells.

Colon cancer is the third most common cause of death in the United States. Therefore, new preventive strategies are warranted for preventing colon cancer. Nexrutine (NX), an herbal extract from Phellodendron amurense, has been shown to have anti-inflammatory, anti-microbial and anti-cancer activity for various tissue specific cancers, but its chemopreventive efficacy has not been evaluated against colon cancer. Here, we explored the mechanism of chemopreventive/chemotherapeutic efficacy of NX against colon cancer. We found that dietary exposure of NX significantly reduced the number of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. In addition, significant inhibition in AOM-induced cell proliferation and reduced expression of the inflammatory markers COX-2, iNOS as well as the proliferative markers PCNA and cyclin D1 were also seen. Moreover, NX exposure significantly enhanced apoptosis in the colon of AOM treated rats. Furthermore, in in vitro studies, NX (2.5, 5, 10 µg/ml, 48 h) decreased cell survival and colony formation while inducing G0/G1 cell cycle arrest and apoptosis in colon adenocarcinoma cells COLO205 and HCT-15. However, NX had minimal cytotoxic effect on IEC-6 normal rat intestinal cells, suggesting its high therapeutic index. NX treatment also modulates the level of Bax and Bcl-2 proteins along with cytochrome c release, cleavage and enhanced expression of poly (adenosine diphosphate-ribose) polymerase as well as the catalytic activity of caspase 3 and caspase 9 in both COLO205 and HCT-15 cells. Based on these in vivo and in vitro findings, we suggest that NX could be useful candidate agent for colon cancer chemoprevention and treatment. © 2015 Wiley Periodicals, Inc.

Protective effect of topical application of α-tocopherol and/or N-acetyl cysteine on argemone oil/alkaloid-induced skin tumorigenesis in mice.

Since bioantioxidants in plasma of Epidemic Dropsy patients [a condition caused by consumption of adulterated mustard oil with argemone oil (AO)] were found to be significantly decreased, the beneficial effect of N-acetyl cysteine (NAC) and α-tocopherol (TOCO) against AO- or sanguinarine (SANG)-induced tumorigenicity was undertaken in mice. Topical application of TOCO and NAC either alone or in combination showed significant protection against AO/TPA- and SANG/TPA-induced skin tumorigenicity. Histopathological findings suggest that papillomatous growth in AO/TPA- and SANG/TPA-treated animals were substantially protected following topical application of TOCO or NAC. Further, treatment of TOCO and NAC either alone or in combination to AO/TPA- or SANG/TPA-induced mice significantly decreased lipid peroxidation, along with significant revival in glutathione (GSH) content and activities of tyrosinase, histidase, catalase, SOD, GSH peroxidase, and GSH reductase in skin. In vitro studies showed that TOCO and/or NAC significantly decreased the AO and SANG induced cell proliferation and activation of ERK, p38, JNK MAPKs and NF-κB signaling in HaCaT cells. In summary, TOCO and NAC may be useful in preventing the tumorigenic response of AO and SANG probably by acting as scavenger of free radicals and inhibiting MAPKs and NF-κB signaling.

Nexrutine(R) inhibits tumorigenesis in mouse skin and induces apoptotic cell death in human squamous carcinoma A431 and human melanoma A375 cells.

Nexrutine(®) (NX), a herbal extract from Phellodendron amurense, has been shown to possess antitumor, antimicrobial, anti-inflammatory and other biological activities. In the present investigation, we explored the mechanism of chemopreventive/chemotherapeutic efficacy of NX against skin cancer. Single application of NX (1.0mg/mouse) prior to 12-O-tetradecanoylphorbol 13-acetate (TPA) application significantly inhibited TPA-induced skin edema, hyperplasia, thymidine incorporation and ornithine decarboxylase (ODC) activity; expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS); phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38 and c-jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs); and activation of I kappa B kinase (IKK), IκBα and nuclear factor-kappa B (NF-κB) in mouse skin. In a two-stage mouse skin tumorigenesis model, it was found that twice-weekly treatment of NX prior to TPA application in 7,12-dimethylbenz[α]anthracene (DMBA)-initiated animals showed reduced tumor incidence, lower tumor body burden and significant delay in latency period compared with DMBA-initiated and TPA-promoted animals. Furthermore, the therapeutic efficacy of NX was assessed against human squamous carcinoma (A431) and human melanoma (A375) cells. A431 and A375 cells treated with NX (2.5-10.0 µg/ml, 48h) showed a decrease in viability and enhanced cell cycle arrest at the G(0)/G(1) phase and apoptosis; however, NX had minimal cytotoxic effect on HaCaT cells and primary murine keratinocytes, suggesting its high therapeutic index. In addition, NX treatment also modulates the levels of Bax and Bcl-2 proteins along with cytochrome c release, cleavage and enhanced expression of poly (adenosine diphosphate-ribose) polymerase as well as catalytic activities of caspases 3 and 9 in both A431 and A375 cells. Based on our in vivo and in vitro studies, NX could be useful in the management (chemoprevention as well as chemotherapy) of skin cancer.

Role of ErbB2 mediated AKT and MAPK pathway in gall bladder cell proliferation induced by argemone oil and butter yellow. Argemone oil and butter yellow induced gall bladder cell proliferation.

The effect of noncytotoxic doses of argemone oil (AO) and butter yellow (BY), the common adulterants in edible oil, on free radical generation and signaling pathway for cell proliferation in primary cells of gall bladder (GB) was undertaken. AO and BY showed no cytotoxicity at 0.1 µl/ml and 0.1 µg/ml concentration, respectively. AO caused significant increase in ROS after 30 min and RNS after 24 h in GB cells while no change was observed following BY treatment. Enhanced level of COX-2 was observed following AO (0.1 µl/ml) and BY (0.1 µg/ml) treatment to cells for 24 h. AO treatment caused phosphorylation of ErbB2, AKT, ERK, and JNK along with increased thymidine uptake indicating cell proliferation ability in GB cells. BY treatment also showed significant expression of these proteins with the exception of phosphorylated JNK. These results suggest that AO and BY have cell proliferative potential in GB cells following up-regulation of COX-2 and ErbB2; however, their downstream signaling molecules and free radical generation have differential response, indicating that the mechanism of proliferation is different for both compounds and may have relevance in gall bladder cancer.

Edible oil adulterants, argemone oil and butter yellow, as aetiological factors for gall bladder cancer.

Carcinogenic potential of argemone oil (AO) and butter yellow (BY), the adulterants encountered in edible oil, in gall bladder of Swiss albino mice was undertaken to investigate the potential aetiological factors of gall bladder carcinoma (GBC) in the Indo-Gangetic basin. Twice weekly intraperitoneal (ip) administration of AO (5 ml/kg body wt) and BY (25 mg/kg body wt) to Swiss albino male and female mice for 30 and 60 days indicated that females were more vulnerable to these adulterants in terms of responses to inflammatory markers. Subsequent experiments with dietary exposure of AO (1%) and BY (0.06%) for 6 months in female mice showed symptoms related to cachexia, jaundice and anaemia. High levels of total cholesterol, low density lipoprotein (LDL), TG, bilirubin and low level of high density lipoprotein (HDL) as well as gallstone formation was shown by AO exposure only, leading to the development of adenocarcinoma. BY exposure resulted in adenoma and hyperplasia without stone formation. The cyclooxygenase (COX-2) overexpression was found to be related to prostaglandin E2 (PGE2) production in AO treated mice but not in BY exposed animals, thereby indicating a differential pathway specific carcinogenicity. PGE2 stimulates the secretion of secreted mucins (MUC5AC), which is involved in stone formation following AO exposure. Enhanced secretion of membrane bound mucins (MUC4) in BY and AO exposed mice resulted in the activation of ErbB2 and downstream signalling such as p-AKT, p-ERK and p-JNK, which ultimately affects the target proteins, p53 and p21 leading to adenoma and adenocarcinoma, respectively. The study suggests that AO and BY are responsible for producing GBC in mice along with stone formation in the AO exposed animals.

Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis.

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300µl) or SANG (4.5µmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100µl) or SANG (1.5µmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45-49%), histidase (30-32%), superoxide dismutase (53-56%), catalase (41%), GSH reductase (37-40%) and GSH-peroxidase activity (29-33%) compared to control. Furthermore, significant decrease of epidermal GSH (64-66%) content and enhanced formation of lipid peroxides (96-121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.

Activation of inflammatory response and apoptosis of polymorphonuclear leukocytes in patients with argemone oil poisoning.

In the present study, the role of ROS and RNS in activation of inflammatory response and associated molecular events during apoptosis of polymorphonuclear leucocytes (PMNs) in patients from an outbreak of argemone oil (AO) poisoning leading to epidemic dropsy in Lucknow, India was undertaken. It was observed that generation of superoxide radical, nitrite formation and phagocytosis (103-429%) were significantly increased in PMNs of dropsy patients. Furthermore, activities of superoxide dismutase and glutathione peroxidase (GPx) (47-79%) were found to be increased while that of catalase and glutathione reductase (GR) (56-57%) were decreased. Lipid and protein oxidation, nitrotyrosine formation and 8-hydroxydeoxyguanosine (8-OHdG) excretion were significantly enhanced with concomitant depletion of GSH levels (67%) in dropsy patients. In addition, significant elevation of IL-6, IL-8 and TNF-alpha (68-406%) in plasma was observed. Apoptosis was enhanced (1.5 folds) with increased (2.0-3.6 folds) caspases 3, 8 and 9 activities along with DNA fragmentation (119%). The results suggest that generation of ROS and RNS along with enhancement of secretion of inflammatory mediators leading to DNA damage followed by apoptosis may have an effect on immune system, which in turn may be responsible for histopathological changes in target organs of dropsy patients.

Skin tumor promotion by argemone oil/alkaloid in mice: evidence for enhanced cell proliferation, ornithine decarboxylase, cyclooxygenase-2 and activation of MAPK/NF-kappaB pathway.

Consumption of argemone oil (AO) contaminated edible oil causes "Epidemic Dropsy". Previously, we have shown that AO and isolated sanguinarine possess genotoxicity and skin tumor initiating activity. Here, we evaluate tumor-promoting potential of AO/sanguinarine alkaloid and investigate the molecular mechanisms involved therein. Single topical application of AO (50-400 microl/mouse) or sanguinarine alkaloid (1.5-12.0 micromol/mouse) afforded significant increase in (i) ornithine decarboxylase (ODC) activity, (ii) uptake of [(3)H]-thymidine in DNA, (iii) cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA) and ODC protein expressions, (iv) phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-jun-N-terminal kinase (JNK)1/2 and p38 mitogen-activated protein (MAP) kinases, (v) increased NF-kappaB activation and (vi) no significant increase in dark basal keratinocytes. Subsequently, when AO and sanguinarine alkaloid was tested either as complete or stage I or stage II tumor promoter in 7, 12-dimethyl benz(a)anthracene (DMBA)-initiated mice, there was enhanced tumor incidence, tumor body burden and higher % of mice with tumors, when AO (0.1 ml) or isolated sanguinarine (1.5 micromol) was tested as stage II tumor promoter. However, no tumors were found when AO or sanguinarine alkaloid was tested either as complete or stage I tumor promoter. These results indicate that AO/ sanguinarine alkaloid possesses tumor-promoting potential at stage II level involving MAPK/NF-kappaB pathway.

Alterations in redox potential of glutathione/glutathione disulfide and cysteine/cysteine disulfide couples in plasma of dropsy patients with argemone oil poisoning.

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. In the present study, we determined the relationship between redox potentials (E(h)) of glutathione/glutathione disulfide (GSH/GSSG), cysteine/cysteine disulfide (Cys/CySS) couples and non-enzymatic antioxidants such as alpha-tocopherol and ascorbic acid status in plasma of dropsy patients (n=14) from an outbreak of argemone oil poisoning in Lucknow (March, 2005), India. Depleted GSH (55%) and concomitant enhancement (163%) of plasma GSSG content was observed in patients (P<0.05). Furthermore, lower content of Cys (42%) and CySS (25%) was noticed in patients (P<0.05) when compared to control subjects. Eh GSH and Eh Cys values were shifted by +46 mV and +12 mV towards more oxidizing environment in patients (P<0.05). In addition, alpha-tocopherol and ascorbic acid contents were found to be depleted significantly (P<0.05) in plasma of patients (59-58%). The alterations in redox potentials and antioxidants in plasma, which are synthesized in liver, may be responsible for histopathological changes in hepatic tissue of patients showing swelling of hepatocytes, fluid accumulation in spaces of Desci along with mild kupfur cell hyperplasia. Over all the present study shows that redox state of GSH/GSSG and Cys/CySS pools become oxidized which inturn causes depletion of alpha-tocopherol and ascorbic acid, thus providing a strategy to distinguish pro-oxidant and antioxidant events in patients.

Protective effect of bioantioxidants on argemone oil/sanguinarine alkaloid induced genotoxicity in mice.

Our prior studies have shown that argemone oil (AO) and its alkaloid sanguinarine causes DNA damage in mice and Epidemic Dropsy patients. Since some of the bioantioxidants including riboflavin and alpha-tocopherol offered protection to Epidemic Dropsy patients, a combination of riboflavin and alpha-tocopherol was evaluated on AO and sanguinarine induced genotoxicity using alkaline comet assay. Single administration of combination of riboflavin (50mg/kg) and alpha-tocopherol (150mg/kg) to mice, 24h prior to or immediately after AO (2.0ml/kg) exposure showed significant decrease in tail moment (70-72%), tail length (37-44%), and tail DNA (49-53%) in bone marrow cells. Single or multiple doses of antioxidants given after 24h of AO exposure resulted in substantial (P<0.05) decrease in all the parameters of comet assay in bone marrow cells. Single dose of antioxidants given either 24h prior to or immediately after sanguinarine (21.6mg/kg) exposure caused significant decrease in tail moment (56-62%), tail length (69%) and tail DNA (34-42%) in bone marrow cells of mice. Single or multiple doses of antioxidants given after 24h of sanguinarine treated resulted in decrease in tail moment (50-71%), tail length (54-63%) and tail DNA (29-43%) in bone marrow cells. Similar protective response of combination of antioxidants was observed in blood cells of mice treated either with AO or sanguinarine alkaloid. Further, the frequency of bone marrow and blood cells in Olive tail moment category of 8 and onwards were found to be substantially reduced in antioxidants treated animals as compared to respective AO or sanguinarine exposed mice. Based on these results, it can be suggested that a combination of riboflavin and alpha-tocopherol provides protection against AO and sanguinarine induced genotoxicity.

Correlation of DNA damage in epidemic dropsy patients to carcinogenic potential of argemone oil and isolated sanguinarine alkaloid in mice.

In recent times, a higher incidence of gall bladder carcinoma in the Indo-Gangetic basin has been linked with the consumption of contaminated mustard oil. Consumption of mustard oil contaminated with argemone oil (AO) is well known to cause clinical manifestation referred to as "epidemic dropsy." Because sanguinarine, an active alkaloid of AO, has been shown to intercalate DNA, a possible correlation of DNA damage in epidemic dropsy patients to tumorigenic potential of AO and isolated sanguinarine alkaloid in mice was investigated in the present study. Single topical application of AO (0.15-0.3 ml) or isolated sanguinarine (4.5-18 micromol) followed by twice-weekly application of tetradecanoylphorbolmyristate acetate (TPA) for 25 weeks resulted in formation of tumors. Histopathologically these tumors were of squamous cell carcinoma type and similar to those found in the positive control group using dimethylbenzanthracene (DMBA)/TPA. The activities of cutaneous gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase P (GST-P), marker enzymes of tumorigenesis, were found to exhibit higher expression in AO or isolated sanguinarine/TPA treated groups when compared to control. The higher expression of p53 and p21/WAF1 in skin after single topical application of AO or isolated sanguinarine further confirms the tumorigenic response. Single topical application of AO or isolated sanguinarine alkaloid to mice showed significant DNA damage in terms of Olive tail moment (89-129%), tail length (54%) and tail DNA (153-205%) using Comet assay in skin cells. Further, the extent of DNA damage in blood cells of epidemic dropsy patients in alkaline Comet assay was found to be significantly higher as compared to normal population, indicating the genotoxic response of AO exposure. Although the genotoxic lesions may be repaired to some extent on withdrawal of consumption of AO contaminated mustard oil and the residual genotoxic effects caused by AO may not be expressed as signs of carcinogenesis. Environmental factors or hormonal changes during aging process may lead to stimulate/promote the genetically altered latent cells to form neoplastic lesions and can act as one of the etiological factors responsible for higher incidence of gall bladder carcinoma in the population of Indo-Gangetic basin.

In vivo DNA damaging potential of sanguinarine alkaloid, isolated from argemone oil, using alkaline Comet assay in mice.

Consumption of mustard oil contaminated with argemone oil is well known to cause clinical manifestation referred to as "Epidemic Dropsy". Our prior studies have shown that argemone oil produces genotoxic effects in mice [Ansari, K.M., et al., 2004. Int. J. Cancer 112, 890]. Since, sanguinarine alkaloid is the major component of argemone oil, the in vivo DNA damaging potential of the isolated alkaloid was investigated in blood and bone marrow cells of mice using alkaline Comet assay. Swiss albino male mice were given single intraperitoneal administration of 1.35, 2.70, 5.40, 10.80 and 21.60 mg sanguinarine alkaloid/kg b wt., while controls were treated with saline in the same manner. The results revealed a dose dependent increase in DNA damage in blood and bone marrow cells following 24 h treatment of sanguinarine alkaloid. All the three parameters of Comet assay including olive tail moment (OTM), tail length and tail DNA showed significant (p<0.05) increases in blood and bone marrow cells at respective doses of 10.80 and 5.40 mg alkaloid/kg b wt. However, some of the parameters were significantly increased even at lower doses of sanguinarine alkaloid (2.70 mg/kg b wt.). The frequency of cells exhibiting greater DNA damage were found to be increased by sanguinarine alkaloid in a concentration dependent manner. These results indicate that single exposure of sanguinarine alkaloid causes DNA damage in blood and bone marrow cells of mice, which could be responsible for the genotoxicity of argemone oil.

Unequivocal evidence of genotoxic potential of argemone oil in mice.

Consumption of mustard oil adulterated with argemone oil leads to a clinical condition, commonly referred to as "Epidemic Dropsy." Since in vitro studies have shown that sanguinarine, an active benzophenanthridine alkaloid of argemone oil, intercalates DNA molecule, the in vivo clastogenic and DNA damaging potential of argemone oil was investigated in mice. Swiss albino mice were intraperitoneally administered 0.5, 1.0, 2.0 and 4.0 ml/kg body wt. of argemone oil to analyze chromosome aberrations and micronucleus test, while 0.25, 0.5, 1.0 and 2.0 ml/kg body wt. were given for alkaline comet assay. The frequencies of chromosomal aberrations and micronucleated erythrocytes formation in mouse bone marrow cells increased in a dose-dependent manner following argemone oil treatment. However, significant induction in chromosomal aberrations (83%) and micronucleated erythrocytes formation (261%) were observed at a minimum dose of 1.0 ml/kg. The results of comet assay revealed DNA damage in blood, bone marrow and liver cells following argemone oil treatment. Olive tail moment (OTM) and tail DNA showed significant increase in bone marrow (35-44%) and blood cells (25-40%) even at a dose of 0.25 ml/kg body wt. of argemone oil. In liver cells, OTM was significantly increased (20%) at a dose of 0.25 ml/kg, while all the comet parameters including OTM, tail length and tail DNA showed significant increase (31-101%) at a dose of 0.5 ml/kg. These results clearly suggest that single exposure of argemone oil even at low doses produces genotoxic effects in mice.