Rethinking the Roles of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Bearing a dismal 5-year survival rate, pancreatic ductal adenocarcinoma (PDAC) is a challenging disease that features a unique fibroinflammatory tumor microenvironment. As major components of the PDAC tumor microenvironment, cancer-associated fibroblasts are still poorly understood and their contribution to the several hallmarks of PDAC, such as resistance to therapies, immunosuppression, and high incidence of metastasis, is likely underestimated. There have been encouraging advances in the understanding of these fascinating cells, but many controversies remain, leaving the field still actively exploring the full scope of their contributions in PDAC progression. Here we pose several important considerations regarding PDAC cancer-associated fibroblast functions. We posit that transcriptomic analyses be interpreted with caution, when aiming to uncover the functional contributions of these cells. Moreover, we propose that normalizing these functions, rather than eliminating them, will provide the opportunity to enhance therapeutic response. Finally, we propose that cancer-associated fibroblasts should not be studied in isolation, but in conjunction with its extracellular matrix, because their respective functions are coordinated and concordant.

Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling.

The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.

NetrinG1+ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5ß1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5ß1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5ß1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.

In Vitro Models to Study Angiogenesis and Vasculature.

The development of vasculature in vivo is an extremely complex process that requires temporal and spatial coordination between multiple cell types to produce an effective vessel. The formation of vasculature from preexisting blood vessels, known as angiogenesis, plays important roles in several physiological and pathological processes, including wound healing, organ development and growth, ischemia, inflammatory disorders, fibrosis, and cancer. Means to deconstruct these complicated biological systems are necessary to gain mechanistic insight into their development, function, and modulation that can be tested in in vivo models and ultimately the clinic. In this chapter, we will first review the classical in vitro techniques to study angiogenesis. Next, we will explore the exciting recent advances that rely on 3D multicellular systems to more accurately mimic vasculature development in vitro. Finally, we will discuss the applications of in vitro angiogenic methods to study related vasculature phenomena, such as vasculogenic mimicry.

In Vitro Tube Formation Assays in Matrigel.

Vasculature development is a combination of complex processes that require precise coordination of multiple cell types, through time and space, to generate functional blood-carrying vessels. Moreover, vasculature development can be altered when normal physiological conditions are disrupted, such as in cancer, and means to study blood vessels are of great importance. While the gold standard to explore these processes is the use of in vivo animal models, they are costly and time-consuming, and it is often difficult to dissect the molecular mechanisms involved. Thus, there are several ways to deconstruct vasculature development in vitro, in order to produce tunable systems that lead to a better understanding of cellular and molecular communication between different cell types involved, such as endothelial cells and supporting mesenchymal cells. In this method chapter, we will go into detail for one of the most popular ways of studying vasculature development in the context of cancer, which is the application of Matrigel to study tube formation of various cell types involved with vasculature development. We will provide step-by-step instructions to perform mono- and co-cultures of the major cells involved with the production of vasculature, how the results of these assays can be interpreted, and some advice to avoid common mistakes associated with Matrigel tube formation assays.

Antioxidant, antiproliferative and healing properties of araticum (Annona crassiflora Mart.) peel and seed.

Araticum (Annona crassiflora Mart.) is a native fruit from Brazilian Cerrado widely used by folk medicine. Nevertheless, the biological effects of its seeds and peel have not been extensively evaluated. We evaluate herein the antioxidant, antiproliferative and healing potential of araticum peel and seeds extracts. HPLC-ESI-MS/MS analysis showed flavonoids, namely epicatechin and quercetin, as the main compounds in peel and seeds extracts, respectively. These extracts showed high content of phenolic compounds (7254.46 and 97.74 µg/g extract) and, as consequence, high antioxidant capacity. Interesting, the seeds extract was more effective than peel extract against all tested cancer cells, especially on NCI-ADR/RES (multidrug resistant ovary adenocarcinoma) cell line. In the cell migration assay by using HaCaT (keratinocyte), the seeds extract induced migration, while the peel extract showed an inhibitory effect. In this way, phenolic content could be related to antioxidant capacity, but it was not related to antiproliferative and healing effect. The araticum seeds extract showed an interesting response to in vitro biological assay although of its low content of phenolic compounds. Unidentified compounds, such as alkaloids and annonaceous acetogenins could be related to it. Araticum has potential to be used as therapeutic plant especially as antiproliferative and healing drug.

Chemical composition and antiproliferative activity of Croton campestris A.St.-Hil. essential oil.

Herein, the antiproliferative potential of the essential oil obtained from fresh leaves of Croton campestris against human tumour cell lines was investigated for the first time. Furthermore, the essential oil obtained by hydrodistillation had the composition determined by gas chromatography-mass spectrometry (GC/MS). Ten major components were identified that comprised 91.59% of the total content, with 23.8% consisting of (Z)-caryophyllene and 16.08% consisting of γ-elemene as main components. The cytotoxic activity was observed mainly for breast (MCF-7) and colon (HT-29) human tumour cell lines, with GI50 (50% growth inhibition) concentration of 8.61 and 9.94 µg/mL, respectively. The results of this study showed that the essential oil obtained from Croton campestris A.St.-Hil. represents a potential source for the search of new antitumour agents.

Steroidal hormone and morphological responses in the prostate anterior lobe in different cancer grades after Celecoxib and Goniothalamin treatments in TRAMP mice.

Prostate cancer is the second most diagnosed cancer in the world, and alternative methods to prevent and treat different lesion grades need to be evaluated. The objective was to evaluate the morphological, hormonal, and inflammatory responses in the prostate anterior lobe in transgenic adenocarcinoma of the mouse prostate (TRAMP), following Celecoxib and Goniothalamin (GTN) treatments. All animals were treated for 4 weeks, from 8 weeks of age and euthanized either immediately after treatment (12-week-old mice: immediate response) or later (22-week-old mice: late response). The results showed a significant increase of high-grade prostatic intraepithelial neoplasia (HGPIN) and well-differentiated adenocarcinoma (WDA), according to the age in the control groups. Celecoxib treatment decreased the WDA incidence in the late response group. GTN led to a significant healthy tissue increase, and an LGPIN and HGPIN decrease in the immediate response group. In the late response group, GTN led to healthy area increase and there was no occurrence of WDA. AR and ERα immunoexpressions were reduced by both treatments in the immediate response groups. However, only GTN was able to decrease the ERα level in the late response group. Regarding COX-2 immunoreactivity, both treatments reduced the frequency of this enzyme. We can conclude that the prostate anterior lobe is a good model to study prostate cancer, considering its slow progression. Both treatments led to cancer delay in the prostate anterior lobe. However, GTN pointed towards a better treatment spectrum in the signaling pathways in the prostate microenvironment, particularly in ERα.

Thiosemicarbazones and 4-thiazolidinones indole-based derivatives: Synthesis, evaluation of antiproliferative activity, cell death mechanisms and topoisomerase inhibition assay.

In this study, we report the synthesis and structural characterization of a series of thiosemicarbazone and 4-thiazolidinones derivatives, as well as their in vitro antiproliferative activity against eight human tumor cell lines. For the most potent compound further studies were performed evaluating cell death induction, cell cycle profile, ctDNA interaction and topoisomerase IIα inhibition. A synthetic three-step route was established for compounds (2a-e and 3a-d) with yields ranging from 32 to 95%. Regarding antiproliferative activity, compounds 2a-e and 3a-d showed mean GI50 values ranging between 1.1 µM (2b) - 84.65 µM (3d). Compound 2b was the most promising especially against colorectal adenocarcinoma (HT-29) and leukemia (K562) cells (GI50 = 0.01 µM for both cell lines). Mechanism studies demonstrated that 24 h-treatment with compound 2b (5 µM) induced phosphatidylserine residues exposition and G2/M arrest on HT-29 cells. Moreover, 2b (50 µM) was able to interact with ctDNA and inhibited topoisomerase IIα activity. These results demonstrate the importance of thiosemicarbazone, especially the derivative 2b, as a promising candidate for anticancer therapy.

Characterization of phenolic compounds in chia (Salvia hispanica L.) seeds, fiber flour and oil.

The consumption of chia seeds products has increased recently and it has been suggested that the inclusion of this functional food in a daily human diet could contribute to improve consumers' health. However, a better knowledge about the composition of these products is mandatory. In this work, the phenolic compounds from commercial samples of chia seed, fiber flour and oil were extracted using an ultrasound-assisted methodology and were separated and identified by high-performance liquid chromatography coupled to a mass spectrometer. Methanol:water extracts were prepared and submitted to an acidic hydrolysis. Crude and hydrolyzed extracts were analyzed and phenolic compounds found were mainly caffeic acid and danshensu and its derivatives, such as rosmarinic and salvianolic acids. TPC was higher in the hydrolyzed extracts. These results supply new information about the main phenolic compounds presents in chia, which are important dietary sources of natural antioxidants for prevention of diseases caused by oxidative stress.

Goniothalamin and Celecoxib Effects During Aging: Targeting Pro-Inflammatory Mediators in Chemoprevention of Prostatic Disorders.

BACKGROUND: Prostate is highly affected by aging, which lead to inflammatory disorders that can predispose to cancer development. Chemoprevention has emerged as a new therapeutic approach, intensifying studies evaluating the biological properties of new compounds. The aim of this study was to characterize the inflammatory responses in the prostate ventral lobe from senile mice treated with Goniothalamin (GTN), a promising natural compound with anti-inflammatory and antiproliferative properties. Its activity was compared to Celecoxib, an established nonsteroidal anti-inflammatory drug (NSAID). METHODS: The animals were divided into: Control groups; Young (18-week-old FVB), Senile (52-week-old FVB). Treated groups: Senile-Goniothalamin (150 mg/kg orally), Senile-Celecoxib (10 mg/kg orally). The ventral lobe was collected after 4 weeks for light microscopy, immunohistochemistry, ELISA, and Western blotting analysis. RESULTS: Both treatments were efficient in controlling the inflammatory process in the prostate from senile mice, maintaining the glandular morphology integrity. GTN reduced all inflammatory mediators evaluated (TNF-α, COX-2, iNOS) and different from Celecoxib, it also decreased the protein levels of NF-kB and p-NF-kB. CONCLUSIONS: Finally, GTN and Celecoxib controlled inflammation in the prostate, and sensitized the senescent microenvironment to anti-inflammatory stimuli. Thus, both treatments are indicated as potential drugs in the prostatic diseases prevention during senescence. Prostate 77:838-848, 2017. © 2017 Wiley Periodicals, Inc.

Anti-inflammatory natural product goniothalamin reduces colitis-associated and sporadic colorectal tumorigenesis.

The tumor microenvironment offers multiple targets for cancer therapy, including pro-tumorigenic inflammation. Natural compounds represent an enormous source of new anti-inflammatory and anticancer agents. We previously showed that the styryl lactone goniothalamin (GTN) has promising antiproliferative and anti-inflammatory activities. Because inflammation is a major driver of colorectal cancer (CRC), we therefore evaluated the therapeutic and preventive potentials of GTN in colitis, colitis-associated cancer (CAC) and spontaneous CRC. First, in a simplistic model of inflammation in vitro, GTN was able to inhibit cytokine production in bone marrow-derived macrophages induced by lipopolysaccharide. Next, in dextran sulfate sodium (DSS) induced-colitis model, mice treated with GTN displayed restored tissue architecture, increased cell proliferation in the colonic crypts and reduced epithelial damage. Moreover, colon tissue from GTN-treated mice had significantly less expression of the inflammatory genes interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), S100A9, interleukin 23A (IL-23A), IL-22 and IL-17A In the azoxymethane/DSS model of CAC, GTN reduced tumor multiplicity, load and size. Additionally, GTN suppressed production of IL-6, IL-17 and TNF-α in tumor tissue, as well as abrogated stromal immune cell activation and nuclear translocation of NF-κB. Finally, in a tamoxifen inducible model of sporadic CRC, GTN-treated mice had significantly fewer tumors and decreased levels of IL-17A, IL-6, S100A9 and TNF-α protein within the tumors. These results suggest that GTN possesses anti-inflammatory and antitumor activities and represents a preventive and therapeutic agent modulating the inflammatory environment in the colon during colitis as well as CAC and CRC development.

Synthesis and evaluation of novel hybrids ß-carboline-4-thiazolidinones as potential antitumor and antiviral agents.

A series of novel hybrids ß-carboline-4-thiazolidinones were synthesized and evaluated for their in vitro antitumor activity against human cancer cell lines and for antiviral activity towards Herpes simplex virus type-1 (HSV-1). From the N'-(2-ylidene-4-thiazolidinone)-ß-carboline-3-carbohydrazide series (9-11), compounds 9c and 11d were the most active, showing growth inhibition 50% (GI50) values less than 5 µM for all cell lines tested. Compound 9c, bearing the 4-dimethylaminophenyl group at C-1 of ß-carboline was selected for further investigation concerning cell death and cell cycle profile, focusing on the human renal adenocarcinoma cell line 786-0. Treatments with 25 µM of compound 9c induced cell death after 15 h of treatment, characterized by phosphatidylserine exposure and loss of membrane integrity. Moreover, treatment with 12.5 µM promoted a sub-G1 arrest, which indicates cell death. Derivatives of the N-(2-substituted-aryl-4-thiazolidinone)-ß-carboline-3-carboxamide series (18-23) showed a potent activity and high selectivity for glioma (U251) and ovarian (OVCAR-3) cancer cell lines. Also, some ß-carboline-4-thiazolidinone hybrids showed potent antiviral activity against Herpes simplex virus type-1. The N-(2-substituted-aryl-4-thiazolidinone)-carboxamide moiety in 18, 19 and 22 confer a potent anti-HSV-1 activity for these derivatives, which presented EC50 values of 0.80, 2.15 and 2.02 µM, respectively. The assay results showed that the nature of 4-thiazolidinone moiety and of the substituent attached at the 3- and 1- position of ß-carboline nucleus influenced the antitumor and antiviral activities.

Antiproliferative Effect of Synadenium grantii Hook f. stems (Euphorbiaceae) and a Rare Phorbol Diterpene Ester.

Synadenium grantii is frequently used for the treatment of various diseases such as allergies, gastric disorders, and especially cancer. The aim of this study was to evaluate the possible antiproliferative potential of the methanol extract, fractions, and pure compounds from the stems of S grantii Phytochemical analysis was carried out by conventional chromatographic techniques, and the antiproliferative activity was analyzed using the sulforhodamine B assay and an MTT-based assay. Nonpolar fraction and its subfractions from the stems of S grantii exhibited promising cytostatic effect against several human tumor cell lines (glioma, breast, kidney, and lung), with total grown inhibition values ranging from 0.37 to 2.9 µg/mL. One of the active principles of this plant was identified as a rare phorbol diterpene ester, denoted as 3,4,12,13-tetraacetylphorbol-20-phenylacetate. This compound demonstrated antiproliferative activity against glioma, kidney, lung, and triple-negative breast cancer cell lines. These results demonstrate that S grantii stems produce active principles with relevant antiproliferative potential.

Goniothalamin prevents the development of chemically induced and spontaneous colitis in rodents and induces apoptosis in the HT-29 human colon tumor cell line.

Colon cancer is the third most incident type of cancer worldwide. One of the most important risk factors for colon cancer development are inflammatory bowel diseases (IBD), thus therapies focusing on IBD treatment have great potential to be used in cancer prevention. Nature has been a source of new therapeutic and preventive agents and the racemic form of the styryl-lactone goniothalamin (GTN) has been shown to be a promising antiproliferative agent, with gastroprotective, antinociceptive and anti-inflammatory effects. As inflammation is a well-known tumor promoter, the major goal of this study was to evaluate the therapeutic and preventive potentials of GTN on chemically induced and spontaneous colitis, as well as the cytotoxic effects of GTN on a human colon tumor cell line (HT-29). GTN treatments inhibited TNBS-induced acute and chronic colitis development in Wistar rats, reducing myeloperoxidase levels and inflammatory cells infiltration in the mucosa. In spontaneous-colitis using IL-10 deficient mice (C57BL/6 background), GTN prevented colitis development through downregulation of TNF-α, upregulation of SIRT-1 and inhibition of proliferation (PCNA index), without signs of toxicity after three months of treatment. In HT-29 cells, treatment with 10µM of GTN induced apoptosis by increasing BAX/BCL2, p-JNK1/JNK1, p-P38/P38 ratios as well as through ROS generation. Caspase 8, 9 and 3 activation also occurred, suggesting caspase-dependent apoptotic pathway, culminating in PARP-1 cleavage. Together with previous data, these results show the importance of GTN as a pro-apoptotic, preventive and therapeutic agent for IBD and highlight its potential as a chemopreventive agent for colon cancer.

In vitro antiproliferative activity of uncommon xanthones from branches of Garcinia achachairu.

Context Garcinia achachairu Rusby (Clusiaceae) popularly known as 'achachairu' is used in folk medicine to treat rheumatism, inflammation, pain and gastric disorder. Objective The present study investigated the chemical profile and antiproliferative effects of the methanolic extract, fractions and two xanthones, against some carcinoma cell lines in vitro. Materials and methods The compounds were isolated and identified by chromatographic and spectroscopic methods. The extract, fractions and compounds were tested human tumour cell lines of U-251 (glioma), MCF-7 (breast), NCI/ADR-RES (ovary expressing multi-drug resistance phenotype), 786-0 (kidney), NCI-H460 (lung, non-small cells), PC-3 (prostate) and HT-29 (colon), non-tumour cell line HaCat (human keratinocytes) in doses of 0.25-250 µg mL (-) (1) for 48 h. The antiproliferative activity was determined by spectrophotometric quantification (at 540 nm) of the cellular protein content using sulphorhodamine B assay. The prediction of parameters involved in the molecular bioavailability was executed directly by ChemDoodle (version 5.0.1) software (iChemLabs, LLC, Somerset, NJ). Results 3-Demethyl-2-geranyl-4-prenylbellidypholine (1) and 1,5,8-trihydroxy-4',5'-dimethyl-2H-pyrane (2,3:3,2)-4-(3-methylbut-2-enyl) xanthone (2), gartanin (3) and stigmasterol (4) were identified on the basis of spectroscopic techniques. Compounds 1 and 2 exhibited cytocidal activity, especially against breast, prostate and kidney cell lines, with TGI values of 15.8, 4.9, 9.1 and 39.4, 44.7, 40.9 µg/mL, respectively. Discussion and conclusion The presence of two sets of hydrophobic and hydrophilic groups in separate domains in each molecule might play a role in the mediation of tumour-specific action. Our data show that G. achachairu have potent antiproliferative action and should be considered an important source of potent anticancer compounds.

Synthesis of thiophene-thiosemicarbazone derivatives and evaluation of their in vitro and in vivo antitumor activities.

A series of thiophene-2-thiosemicarbazones derivatives (5-14) was synthesized, characterized and evaluated for their antitumor activity. They were tested in vitro against human tumor cell lines through the colorimetric method. The results revealed that compounds 7 and 9 were the most effective in inhibiting 50% of the cell growth after 48 h of treatment. As compound 7 showed a potent antiproliferative profile, it has been chosen for further studies in 786-0 cell line by flow cytometry. Treatments with compound 7 (50 µM) induced early phosphatidylserine exposure after 18 h of exposure and this process progressed phosphatidylserine exposure with loss of cell membrane integrity after 24 h of treatment, suggesting a time-dependent cell death process. Regarding the cell cycle profile, no changes were observed after treatment with compound 7 (25 µM), suggesting a mechanism of cell death independent on the cell cycle. The in vivo studies show that compound 7 possess low acute toxicity, being the doses of 30-300 mgKg(-1) chosen for studies in Ehrlich solid tumor model in mice. All doses were able to inhibit tumor development being the lowest one the most effective. Our findings highlight thiophene-2-thiosemicarbazones as a promising class of compounds for further studies concerning new anticancer therapies.

(-)-Tarchonanthuslactone: Design of New Analogues, Evaluation of their Antiproliferative Activity on Cancer Cell Lines, and Preliminary Mechanistic Studies.

Natural products containing the α,ß-unsaturated δ-lactone skeleton have been shown to possess a variety of biological activities. The natural product (-)-tarchonanthuslactone (1) possessing this privileged scaffold is a popular synthetic target, but its biological activity remains underexplored. Herein, the total syntheses of dihydropyran-2-ones modeled on the structure of 1 were undertaken. These compounds were obtained in overall yields of 17-21 % based on the Keck asymmetric allylation reaction and were evaluated in vitro against eight different cultured human tumor cell lines. We further conducted initial investigation into the mechanism of action of selected analogues. Dihydropyran-2-one 8 [(S,E)-(6-oxo-3,6-dihydro-2H-pyran-2-yl)methyl 3-(3,4-dihydroxyphenyl)acrylate], a simplified analogue of (-)-tarchonanthuslactone (1) bearing an additional electrophilic site and a catechol system, was the most cytotoxic and selective compound against six of the eight cancer cell lines analyzed, including the pancreatic cancer cell line. Preliminary studies on the mechanism of action of compound 8 on pancreatic cancer demonstrated that apoptotic cell death takes place mediated by an increase in the level of reactive oxygen species. It appears as though compound 8, possessing two Michael acceptors and a catechol system, may be a promising scaffold for the selective killing of cancer cells, and thus, it deserves further investigation to determine its potential for cancer therapy.

Anti-inflammatory and antinociceptive effects of racemic goniothalamin, a styryl lactone.

AIMS: The present study aimed to further investigate the anti-inflammatory activity of goniothalamin (GTN), a styryl lactone, as well as its antinociceptive effects. MAIN METHODS: The anti-inflammatory activity was evaluated in models of paw edema induced by different mediators in mice and carrageenan-induced peritonitis. Evaluation of the antinociceptive effect was performed through acetic acid-induced writhing test and formalin test. Activity of GTN on gene expression levels of interleukin-1beta (IL-1ß), induced nitric oxidase synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated in vitro in lipopolysaccharide (LPS)-stimulated macrophage (RAW 264.7), as well as gene expression and protein levels of tumor necrosis factor-alpha (TNF-α). KEY FINDINGS: Pretreatment with GTN (300 mg/kg) significantly reduced paw edema induced by compound 48/80, prostaglandin E2, phospholipase A2 and bradykinin. GTN (10, 30 and 100mg/kg) inhibited leukocyte migration in the peritonitis model and gene expression levels of IL-1ß, iNOS and TNF-α, as well as TNF-α protein levels, in LPS-stimulated macrophages, without affecting COX-2 gene expression levels. GTN inhibited nociception induced by acetic acid in the writhing model and in the formalin test, when both neurogenic and inflammatory phases were inhibited. SIGNIFICANCE: For the first time the acute anti-inflammatory profile of GTN is characterized and its antinociceptive activity reported. The current study shows that GTN inhibits both vascular and cellular phases of inflammation, with bradykinin and PLA2 induced inflammation being the most affected by GTN. Its anti-inflammatory effects also involved the in vitro inhibition of gene expression of alarm cytokines and mediators as IL-1ß, iNOS and TNF-α.

Different cell death responses induced by eupomatenoid-5 in MCF-7 and 786-0 tumor cell lines.

Natural products remain an important source of new drugs, including anticancer drugs. Recently, our group reported the anticancer activity of eupomatenoid-5 (eup-5), a neolignan isolated from Piper regnellii (Miq.) C. DC. var. regnellii leaves. In vitro studies demonstrated that MCF-7 (breast) and 786-0 (kidney) were among the cancer cell lines most sensitive to eup-5 treatment. The current results demonstrate that mitochondrial membrane depolarization and generation of reactive oxygen species are implicated in eup-5-mediated cytotoxic effects on these cancer cells lines. In MCF-7 cells, eup-5 led to phosphatidylserine externalization and caspase activation, whereas the same did not occur in 786-0 cells. Scanning electron microscopy revealed a reduction of microvilli density, as well as cell morphology alterations. Moreover, treated MCF-7 cells exhibited well-characterized apoptosis alterations, while treated 786-0 cells exhibited characteristics of programmed necroptosis process. These findings support the possibility that different mechanisms may be targeted by eup-5 in cell death response.