CYLD Inhibits the Development of Skin Squamous Cell Tumors in Immunocompetent Mice.

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.

Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its potential usage as a therapeutic target.

In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response.

BACKGROUND: Mesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity. METHODS: We established multiple stages of MET by in vitro transformation of NIH3T3 mouse embryonic fibroblasts. Nutritional deprivation followed by repetitive treatment cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate with frequent isolation of foci established three progressive strains (NIH3T3.1, NIH3T3x3, NIH3T3x8x3) depicting MET, and one strain (NIH3T3x12) with partial reversion. Alterations in morphology, cell adhesion properties, expression/intracellular localization of cell adhesion proteins, microRNA expression and cellular radiosensitivity were studied in these stably transformed cell strains. RESULTS: All four transformants had increased proliferation rate, saturation density, bipolarity, E-cadherin expression; coupled with reduced cell size/spreading, pseudopodia/migration, and fibroblast marker protein and vimentin. The most aggressive trans-differentiated (phenotypically epithelial) cell strain, NIH3T3x8x3 acquired ~30% higher growth potential associated with more than two-fold reduction in cell size and migration. These phenotypic changes accompanied ~40% reduction in endogenous or radiation-induced connexin-43 expression/mitochondrial translocation. Incidentally, all three progressive strains displayed prominent HRS (αs/αr: 7.95-37.29) whereas parental (NIH3T3) and reverting (NIH3T3x12) strains lacked HRS and had distinct radiation-induced Cx43 translocation into mitochondria. CONCLUSION: Our study shows that trans-differentiating fibroblasts progressively acquiring epithelial features during MET process, display low-dose hyper-radiosensitivity associated with altered Cx43 behaviour. GENERAL SIGNIFICANCE: This study demonstrates that MET progression triggers low-dose hyper-radiosensitivity in trans-differentiating cells, which has significant therapeutic implications.

Variation on Composition and Bioactivity of Essential Oils of Four Common Curcuma Herbs.

Chemical compositions, antioxidative, antimicrobial, anti-inflammatory, and cytotoxic activities of essential oils extracted from four common Curcuma species (Curcuma longa, Curcuma phaeocaulis, Curcuma wenyujin, and Curcuma kwangsiensis) rhizomes in P. R. China are comparatively studied. In total, 47, 49, 35, and 30 compounds are identified in C. longa, C. phaeocaulis, C. wenyujin, and C. kwangsiensis essential oils by GC/MS, and their richest compounds are ar-turmerone (21.67%), elemenone (19.41%), curdione (40.23%) and (36.47%), respectively. Moreover, C. kwangsiensis essential oils display the strongest DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity (IC50 , 3.47 µg/ml), much higher than ascorbic acid (6.50 µg/ml). C. phaeocaulis oils show the best antibacterial activities against Escherichia coli (MIC, 235.54 µg/ml), Pseudomonas aeruginosa (391.31 µg/ml) and Staphylococcus aureus (378.36 µg/ml), while C. wenyujin and C. kwangsiensis oils show optimum activities against Candida albicans (208.61 µg/ml) and Saccharomyces cerevisiae (193.27 µg/ml), respectively. C. phaeocaulis (IC50 , 4.63 µg/ml) and C. longa essential oils (73.05 µg/ml) have the best cytotoxicity against LNCaP and HepG2, respectively. C. kwangsiensis oils also exhibit the strongest anti-inflammatory activities by remarkably down-regulating expression of COX-2 and TNF-α. Therefore, due to their different chemical compositions and bioactivities, traditional Chinese Curcuma herbs should be differentially served as natural additives for food, pharmaceutical, and cosmetic.

mTOR inhibition prevents rapid-onset of carcinogen-induced malignancies in a novel inducible HPV-16 E6/E7 mouse model.

The rising incidence of human papillomavirus (HPV)-associated malignancies, especially for oropharyngeal cancers, has highlighted the urgent need to understand how the interplay between high-risk HPV oncogenes and carcinogenic exposure results in squamous cell carcinoma (SCC) development. Here, we describe an inducible mouse model expressing high risk HPV-16 E6/E7 oncoproteins in adults, bypassing the impact of these viral genes during development. HPV-16 E6/E7 genes were targeted to the basal squamous epithelia in transgenic mice using a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA) system. After doxycycline induction, both E6 and E7 were highly expressed, resulting in rapid epidermal hyperplasia with a remarkable expansion of the proliferative cell compartment to the suprabasal layers. Surprisingly, in spite of the massive growth of epithelial cells and their stem cell progenitors, HPV-E6/E7 expression was not sufficient to trigger mTOR activation, a key oncogenic driver in HPV-associated malignancies, and malignant progression to SCC. However, these mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Thus, only few oncogenic hits may be sufficient to induce cancer in E6/E7 expressing cells. All HPV-E6/E7 expressing SCC lesions exhibited increased mTOR activation. Remarkably, rapamycin, an mTOR inhibitor, abolished tumor development when administered to HPV-E6/E7 mice prior to DMBA exposure. Our findings revealed that mTOR inhibition protects HPV-E6/E7 expressing tissues form SCC development upon carcinogen exposure, thus supporting the potential clinical use of mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies.

TLR7-mediated skin inflammation remotely triggers chemokine expression and leukocyte accumulation in the brain.

BACKGROUND: The relationship between the brain and the immune system has become increasingly topical as, although it is immune-specialised, the CNS is not free from the influences of the immune system. Recent data indicate that peripheral immune stimulation can significantly affect the CNS. But the mechanisms underpinning this relationship remain unclear. The standard approach to understanding this relationship has relied on systemic immune activation using bacterial components, finding that immune mediators, such as cytokines, can have a significant effect on brain function and behaviour. More rarely have studies used disease models that are representative of human disorders. METHODS: Here we use a well-characterised animal model of psoriasis-like skin inflammation-imiquimod-to investigate the effects of tissue-specific peripheral inflammation on the brain. We used full genome array, flow cytometry analysis of immune cell infiltration, doublecortin staining for neural precursor cells and a behavioural read-out exploiting natural burrowing behaviour. RESULTS: We found that a number of genes are upregulated in the brain following treatment, amongst which is a subset of inflammatory chemokines (CCL3, CCL5, CCL9, CXCL10, CXCL13, CXCL16 and CCR5). Strikingly, this model induced the infiltration of a number of immune cell subsets into the brain parenchyma, including T cells, NK cells and myeloid cells, along with a reduction in neurogenesis and a suppression of burrowing activity. CONCLUSIONS: These findings demonstrate that cutaneous, peripheral immune stimulation is associated with significant leukocyte infiltration into the brain and suggest that chemokines may be amongst the key mediators driving this response.

Anti-inflammatory and antioxidative effects of six pentacyclic triterpenes isolated from the Mexican copal resin of Bursera copallifera.

BACKGROUND: Bursera copallifera (Burseraceae) releases a resin known as "copal ancho" which has been used, since pre-Colombian times, as ceremonially burned incense and to treat tooth ache, tumors, arthritis, cold, cough, and various inflammatory conditions; however, its anti-inflammatory potential is poorly studied. The aim of the present study was to isolate, quantify, and to investigate the anti-inflammatory activity of triterpene compounds isolated from the copal resin of B. copallifera. METHODS: The constituents present in the total resin of B. copallifera were obtained by successive chromatographic procedures, and quantitative analysis was performed by High Performance Liquid Chromatography (HPLC). Anti-inflammatory effects of the isolated triterpenes were investigated to determine their inhibitory effects on phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema in mice, viability and nitric oxide (NO) production inhibition on lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and inhibition of cyclooxygenase (COX)-1, COX-2 and secretory Phospholipase A2 (sPLA2) activities in vitro. RESULTS: Quantitative phytochemical analysis of the copal resin showed the presence of six pentacyclic triterpenes of which, 3-epilupeol (59.75 % yield) and α-amyrin (21.1 % yield) are the most abundant. Among the isolated triterpenes, 3-epilupeol formiate (Inhibitory Concentration 50 % (IC50) = 0.96 µmol), α.amyrin acetate (IC50 = 1.17 µmol), lupenone (IC50 = 1.05 µmol), and 3-epilupeol (IC50 = 0.83 µmol) showed marked inhibition of the edema induced by TPA in mice. α-amyrin acetate and 3-epilupeol acetate, at 70 µM, also inhibited the activity of COX-2 by 62.85 and 73.28 % respectively, while α-amyrin and 3-epilupeol were the best inhibitors of the production of NO in LPS-activated RAW 264.7 cells with IC50 values of 15.5 and 8.98 µM respectively, and did not affected its viability. All compounds moderately inhibited the activity of PLA2. CONCLUSIONS: This work supports the folk use of B. copallifera and provides the basis for future investigations about the therapeutic use of this resin in treating inflammation.

Microarray analyses demonstrate the involvement of type I interferons in psoriasiform pathology development in D6-deficient mice.

The inflammatory response is normally limited by mechanisms regulating its resolution. In the absence of resolution, inflammatory pathologies can emerge, resulting in substantial morbidity and mortality. We have been studying the D6 chemokine scavenging receptor, which played an indispensable role in the resolution phase of inflammatory responses and does so by facilitating removal of inflammatory CC chemokines. In D6-deficient mice, otherwise innocuous cutaneous inflammatory stimuli induce a grossly exaggerated inflammatory response that bears many similarities to human psoriasis. In the present study, we have used transcriptomic approaches to define the molecular make up of this response. The data presented highlight potential roles for a number of cytokines in initiating and maintaining the psoriasis-like pathology. Most compellingly, we provide data indicating a key role for the type I interferon pathway in the emergence of this pathology. Neutralizing antibodies to type I interferons are able to ameliorate the psoriasis-like pathology, confirming a role in its development. Comparison of transcriptional data generated from this mouse model with equivalent data obtained from human psoriasis further demonstrates the strong similarities between the experimental and clinical systems. As such, the transcriptional data obtained in this preclinical model provide insights into the cytokine network active in exaggerated inflammatory responses and offer an excellent tool to evaluate the efficacy of compounds designed to therapeutically interfere with inflammatory processes.

Detoxification of toxic phorbol esters from Malaysian Jatropha curcas Linn. kernel by Trichoderma spp. and endophytic fungi.

The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%-99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%-92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%-96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.

Occular and dermal toxicity of Jatropha curcas phorbol esters.

Jatropha curcas seeds are a promising feedstock for biodiesel production. However, Jatropha seed oil and other plant parts are toxic due to the presence of phorbol esters (PEs). The ever-increasing cultivation of toxic genotype of J. curcas runs the risk of increased human exposure to Jatropha products. In the present study, effects of J. curcas oil (from both toxic and nontoxic genotypes), purified PEs-rich extract and purified PEs (factors C1, C2, C(3mixture), (C4+C5)) on reconstituted human epithelium (RHE) and human corneal epithelium (HCE) were evaluated in vitro. The PEs were purified from toxic Jatropha oil. In both RHE and HCE, the topical application of PEs containing samples produced severe cellular alterations such as marked oedema, presence of less viable cell layers, necrosis and/or partial tissue disintegration in epithelium and increased inflammatory response (interleukin-1α and prostaglandin E2). When compared to toxic oil, histological alterations and inflammatory response were less evident (P<0.05) in nontoxic oil indicating the severity of toxicity was due to PEs. Conclusively, topical applications of Jatropha PEs are toxic towards RHE and HCE models, which represents dermal and occular toxicity respectively. Data obtained from this study would aid in the development of safety procedures for Jatropha biodiesel industries. It is advised to use protective gloves and glasses when handling PEs containing Jatropha products.

Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product.

Jatropha seed cake contains high amounts of protein and other nutrients, however it has a drawback due to toxic compounds. The aim of this study was to investigate the methods applied to detoxify the main toxin, phorbol esters in jatropha seed cake, to a safe and acceptable level by maintaining the nutritional values. Phorbol esters are tetracyclic diterpenoids-polycyclic compounds that are known as tumor promoters and hence exhibited the toxicity within a broad range of species. Mismanagement of the jatropha waste from jatropha oil industries would lead to contamination of the environment, affecting living organisms and human health through the food chain, so several methods were tested for reducing the toxicity of the seed cake. The results from this investigation showed that heat treatments at either 120°C or 220°C for 1 hour and then mixing with adsorbing bentonite (10%), nanoparticles of zinc oxide (100 µg/g) plus NaHCO3 at 4%, followed by a 4-week incubation period yielded the best final product. The remaining phorbol esters concentration (0.05-0.04 mg/g) from this treatment was less than that reported for the nontoxic jatropha varieties (0.11-0.27 mg/g). Nutritional values of the seed cake after treatment remained at the same levels found in the control group and these values were crude protein (20.47-21.40 + 0.17-0.25%), crude lipid (14.27-14.68 + 0.13-0.14%) and crude fiber (27.33-29.67 + 0.58%). A cytotoxicity test conducted using L929 and normal human dermal fibroblast cell lines confirmed that most of the toxic compounds, especially phorbol esters, were shown as completely eliminated. The results suggested that the detoxification of phorbol esters residues in the jatropha seed cake was possible while it also retained nutritional values. Therefore, the methods to detoxify phorbol esters are necessary to minimize the toxicity of jatropha seed cake. Further, it is essential to reduce the possible environmental impacts that may be generated throughout the jatropha waste-handling process. However additional tests such as digestibility as well as acceptability of the treated jatropha seed cake should be conducted using both in vivo and in vitro studies before recommending the jatropha seed cake as a source of renewable animal feed and other value-added products.

Transient receptor potential vanilloid 4 activated inflammatory signals by intestinal epithelial cells and colitis in mice.

BACKGROUND & AIMS: Ligand-gated calcium channels have been reported to be involved in the pathogenesis of inflammatory bowel disease. One family member, transient receptor potential vanilloid 4 (TRPV4), is activated by arachidonic acid derivatives that might be released on inflammation, yet its role in gastrointestinal inflammation has not been characterized. We investigated whether TRPV4 activation participates in intestinal inflammation and its expression and functions in the gastrointestinal tract. METHODS: TRPV4 expression was studied in human colon samples, human intestinal epithelial cell lines (Caco-2 and T84), and inflamed colons of mice. Calcium mobilization and cytokine release were analyzed in intestinal epithelial cells exposed to the selective TRPV4 agonist 4α-phorbol-12,13-didecanoate (4αPDD). Mice were killed 3, 6, or 24 hours after intracolonic administration of 4αPDD; inflammatory parameters were measured in their colon tissues, and paracellular colonic permeability was measured by the passage of (51)Cr-EDTA from the colon lumen to the blood. RESULTS: High levels of TRPV4 were detected in Caco-2 cells and in epithelial cells of human colon tissue samples; its expression was up-regulated in colons from inflamed mice compared with noninflamed control mice. Administration of 4αPDD to Caco-2 and T84 cells caused a dose-dependent increase in intracellular calcium concentration and chemokine release. In mice, intracolonic administration of 4αPDD caused colitis to develop 3 to 6 hours later; inflammation resolved by 24 hours. Increased colonic permeability was observed in vivo 3 hours after intracolonic administration of 4αPDD. CONCLUSIONS: TRPV4 is expressed and functional in intestinal epithelial cells; its activation in the gastrointestinal tract causes increases in intracellular calcium concentrations, chemokine release, and colitis.

Light induced degradation of phorbol esters.

Jatropha curcas (Jatropha) is a tropical shrub that is gaining popularity as a biofuel feedstock plant. Phorbol esters (PEs) are tetracyclic tiglian diterpenoids that are present in Jatropha seeds and other parts of plant. Epidermal cell irritating and cancer promoting PEs not only reduce commercial values of Jatropha seed cake but also cause some safety and environment concerns on PE leaching to soil. A simple bioassay of PE toxicity was conducted by incubating 48 h old brine shrimp (Artemia salina) nauplii with Jatropha oil for 24 h. 1-4% of Jatropha oil (corresponding to PE concentration of 25-100 mg L(-1)) had mortality rate of 5-95%, with LC50 estimated to be 2.7% of oil or 67 mg L(-1) of PE. Jatropha oil was incubated with clay or black soil (autoclaved or non-autoclaved) in the darkness or under sunlight for different periods of time before oil was re-extracted and tested for PE content by HPLC and for remaining toxicity with the brine shrimp bioassay. Under sunlight, PE decreased to non-detectable level within six days. Toxicity reduced to less than 5% mortality rate that is comparable to rapeseed oil control within the same period. In contrast, PE level and toxicity remained little changed when Jatropha oil was incubated in the darkness. Such PE degradation/detoxification was also found independent of the presence of soil or soil microorganisms. We conclude that sunlight directly degrades and detoxifies PEs and this finding should alleviate the concern on long term environmental impact of PE leaching.

Activities of Jatropha curcas phorbol esters in various bioassays.

Jatropha curcas seeds contain 30-35% oil, which can be converted to high quality biodiesel. However, Jatropha oil is toxic, ascribed to the presence of phorbol esters (PEs). In this study, isolated phorbol ester rich fraction (PEEF) was used to evaluate the activity of PEs using three aquatic species based bioassays (snail (Physa fontinalis), brine shrimp (Artemeia salina), daphnia (Daphnia magna)) and microorganisms. In all the bioassays tested, increase in concentration of PEs increased mortality with an EC(50) (48 h) of 0.33, 26.48 and 0.95 mg L(-1) PEs for snail, artemia and daphnia, respectively. The sensitivity of various microorganisms for PEs was also tested. Among the bacterial species tested, Streptococcus pyogenes and Proteus mirabilis were highly susceptible with a minimum inhibitory concentration (MIC) of 215 mg L(-1) PEs; and Pseudomonas putida were also sensitive with MIC of 251 mg L(-1) PEs. Similarly, Fusarium species of fungi exhibited EC(50) of 58 mg L(-1) PEs, while Aspergillus niger and Curvularia lunata had EC(50) of 70 mg L(-1). The snail bioassay was most sensitive with 100% snail mortality at 1 µg of PEs mL(-1). In conclusion, snail bioassay could be used to monitor PEs in Jatropha derived products such as oil, biodiesel, fatty acid distillate, kernel meal, cake, glycerol or for contamination in soil or other environmental matrices. In addition, PEs with molluscicidal/antimicrobial activities could be utilized for agricultural and pharmaceutical applications.

Localisation of antinutrients and qualitative identification of toxic components in Jatropha curcas seed.

BACKGROUND: Jatropha curcas seed oil is a promising feedstock for biodiesel production. The seeds contain major toxic (phorbol esters, PEs) and antinutritional (phytate and trypsin inhibitor) factors. In the present study the localisation of antinutrients and a rapid qualitative method for detecting the presence of PEs were investigated. RESULTS: Kernels were separated into cotyledon, hypocotyl, kernel coat and endosperm. The majority of phytate (96.5%), trypsin inhibitor (95.3%) and PEs (85.7%) were localised in the endosperm. Based on PEs, a qualitative method was developed to differentiate between toxic and non-toxic Jatropha genotypes. In this method, PEs were easily detected by passing methanol extracts of kernels (Jatropha toxic and non-toxic genotypes) through a solid phase extraction (SPE) column and measuring the absorption of the resulting eluates at 280 nm. For raw kernels, SPE eluates with absorbance ≥ 0.056 were considered as toxic and those with absorbance ≤0.032 as non-toxic. For defatted kernel meals, SPE eluates with absorbance ≥ 0.059 were considered as toxic and those with absorbance ≤0.043 as non-toxic. CONCLUSION: The majority of antinutrients/toxic compounds are localised in the endosperm of the kernel. The qualitative method developed for rapid identification of toxic PEs could be useful in screening the toxicity of Jatropha-based products in the biodiesel industry. Further confirmation of PEs should be established by high-performance liquid chromatography.

Oleanolic acid protects against mast cell-mediated allergic responses by suppressing Akt/NF-κB and STAT1 activation.

BACKGROUND: Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient. PURPOSE: Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders. METHODS: We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock. RESULTS: OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation. CONCLUSION: The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease.

Loss of syndecan-1 is associated with malignant conversion in skin carcinogenesis.

Syndecan-1 (sdc-1) is a cell surface proteoglycan that mediates the interaction of cells with their matrix, influencing attachment, migration, and response to growth factors. In keratinocytes, loss of sdc-1 delays wound healing, reduces migration, and increases Transforming growth factor beta (TGFbeta) 1 expression. In this study we show that sdc-1 expression is significantly reduced in basal cell, squamous cell, and metastatic human skin cancers compared to normal human skin. In experimental mouse skin tumor induction, compared to wildtype (wt) BALB/c mice, papilloma formation in sdc-1 null mice was reduced by 50% and the percent of papillomas converting to squamous cell carcinoma (SCC) was enhanced. sdc-1 expression on wt mouse papillomas decreased as they converted to SCC. Furthermore, papillomas forming on sdc-1 null mice expressed suprabasal alpha3 and beta4 integrins; suprabasal beta4 integrin is a marker of a high risk for progression. While the proliferative response to phorbol-12-myristate-13-acetate (TPA) did not differ among the genotypes, sdc-1 null mice had an enhanced inflammatory response and retained higher levels of total TGFbeta1 within their skin after TPA treatment. sdc-1 null keratinocytes, transduced in vitro by oncogenic ras(Ha), expressed higher levels of beta4 integrin and had enhanced pSmad2 signaling and reduced senescence when compared to wt ras(Ha)-transduced keratinocytes. When ras(Ha)-transduced cells of both genotypes were grafted onto nude mice, null tumors converted to SCC with higher frequency confirming the skin painting experiments. These data indicate that sdc-1 is important both early in the development of skin tumors and in progression of skin cancers suggesting that reduced expression of sdc-1 could be a useful marker for progression in neoplastic skin lesions.

Are Jatropha curcas phorbol esters degraded by rumen microbes?

BACKGROUND: Jatropha curcas, a non-edible oil plant, is being promoted as a biofuel plant in a number of countries in tropical and subtropical regions. The kernel meal left after extraction of the oil is a potentially protein-rich feed ingredient. However, the presence of highly toxic phorbol esters limits its use. Degradation of J. curcas phorbol esters by rumen microbes, using an in vitro rumen fermentation system, has been investigated in this study. RESULTS: The difference between phorbol ester contents in the residues obtained with and without substrates at 0, 24, 48 or 72 h of the incubations was statistically similar. Phorbol esters did not affect either the gas or short chain production in the in vitro rumen fermentation system. CONCLUSIONS: Rumen microbes can not degrade phorbol esters. In addition, the phorbol esters do not adversely affect rumen fermentation. Ruminants are expected to be as prone as monogastric animals to the toxicity of Jatropha seeds.

Traditional detoxification of Jatropha curcas L. seeds.

ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha curcas L. is a plant with high cultural significance for quilombola communities of Oriximiná (Pará State, Brazil). Although the plant is highly toxic, its seeds are used in these communities to treat tuberculosis and related diseases and symptoms. AIM OF THE STUDY: This study was designed to provide a scientific rationale for the traditional detoxification method and use of J. curcas seeds in quilombola communities of Oriximiná. MATERIALS AND METHODS: J. curcas seeds were manually separated into testa, tegmen, endosperm, and embryo, and then methanolic extracts of each sample were prepared. The traditional preparation of J. curcas seeds consists of a water extract of endosperms that is known as "milk of pinhão-branco". The content of phorbol esters (PEs) in the extracts was analyzed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD). The cytotoxic activity was evaluated in human monocytic cell line THP-1 by Resazurin Reduction Assay, and antimycobacterial activity was assessed by determining Minimal Inhibitory Concentration (MIC) values against H37Rv and BCG strains using the Resazurin Microtiter Assay (REMA). RESULTS: The content analysis revealed that the distribution of PEs within the seeds is not homogeneous. High contents were found in tegmens (4.22 ±â€¯0.25-15.52 ±â€¯0.06 mg/g) and endosperms (1.61 ±â€¯0.07-5.00 ±â€¯0.42 mg/g), while concentrations found in testas and embryos were all below 0.5 mg/g. The traditional preparation derived from the endosperm of J. curcas contained significantly less PEs than the endosperms (0.01 ±â€¯0.005 mg/g). Against THP-1 cells, all the parts of the seed showed cytotoxic activity, while the traditional preparation was considered non-cytotoxic. Nevertheless, only the tegmen and endosperm of J. curcas were considered active against M. tuberculosis and M. bovis (MIC = 200 µg/mL). CONCLUSION: The results of this study indicated that the traditional processing performed by the quilombola people from Oriximiná is effective in reducing the toxicity of J. curcas seeds. Although inactive against mycobacteria, the extensive use of the traditional preparation and its low toxicity encourage further studies to investigate other biological activities.

Current Strategies for the Detoxification of Jatropha curcas Seed Cake: A Review.

Jatropha curcas is an important oilseed plant, with considerable potential in the development of biodiesel. Although Jatropha seed cake, the byproduct of oil extraction, is a residue rich in nitrogen, phosphorus, potassium, and carbon, with high protein content suitable for application in animal feed, the presence of toxic phorbol esters limits its application in feed supplements and fertilizers. This review summarizes the current methods available for detoxification of this residue, based upon chemical, physical, biological, or combined processes. The advantages and disadvantages of each process are discussed, and future directions involving genomic and proteomic approaches for advancing our understanding of biodegradation processes involving microorganisms are highlighted.