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Ten previously undescribed metabolites were isolated from Peperomia incana (Haw.) A. Dietr. (Piperaceae), among which four contained a chromene moiety, two were identified as meroterpene lactones, and four were cannabinoid-like compounds. While the chemical structures of the compounds were assigned based on HRESIMS and 1D and 2D-NMR spectra analyses, the relative and absolute configurations were assigned from NOE correlations and a combination of ECD data and X-ray single crystal analyses, respectively. In a cytotoxic assay against a panel of seven human cancer cell lines (A549, MDA-MB-231, HeLa, DU 145, 5637, Hep G2, and MIA PaCa-2, which represent non-small cell lung cancer, as well as breast, cervical, prostate, bladder, liver, and pancreas carcinomas, respectively) most of the isolated compounds showed promising cytotoxic activities. The incanachromenes B, and incanabinoids A and C exhibited the highest cytotoxicity toward all tested cancer cell lines with IC50 values in the range of 5.0-10.0 µM, whereas incanolides A, B, and incanabinoid B showed the lowest cytotoxic activity. In addition, incanachromene C and incanabinoid C produced a significant antibacterial effect toward planktonic cells and biofilms of multidrug-resistant Staphylococcus aureus strains.
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Antineoplásicos , Canabinoides , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Staphylococcus aureus Resistente à Meticilina , Peperomia , Humanos , Peperomia/química , Antineoplásicos/farmacologia , Estrutura MolecularRESUMO
Evidence for clinical efficacy of a semisynthetic derivative of arglabin in anticancer treatment prompted us to examine molecular mechanisms and cellular targets of arglabin. Arglabin, a sesquiterpene lactone isolated from Artemisia glabella was cytotoxic to different human cancer cell lines including those derived from advanced triple-negative breast, lung, androgen-dependent and androgen-independent prostate carcinomas. Noteworthy, arglabin was less toxic to non-neoplastic prostate epithelial cells indicating selectivity for cancer cells. At the molecular level, prior to any biochemical signs of cellular toxicity, arglabin reduced levels of cell-surface sulphanyl groups and inhibited phosphorylation of the redox-sensitive receptor tyrosine kinase EGFR, the only active RTK in PC-3 prostate cancer cells among 49 TRKs analyzed by the assay. Henceforth, arglabin inhibited the EGFR downstream signaling pathways mTORC1 and mTORC2. Accordingly, arglabin induced autophagosome formation and autophagic flux, inhibited phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and impeded cell cycle progression and proliferation of PC-3 cells. In agreement with inhibition of the mTORC2 pathway, arglabin induced sustained actin polymerization, inhibited cell migration, and triggered apoptosis in vitro in 2D cell culture and colony formation assay and in vivo in prostate cancer xenografts grown on chick chorioallantoic membranes. Under physiological conditions, arglabin rapidly formed adducts with reduced glutathione (GSH). Moreover, thiol-based antioxidants GSH and ß-mercaptoethanol abolished arglabin-induced cancer cell toxicity, whereas the non-thiol antioxidant trolox was ineffective pointing to a crucial role of interaction with cell-surface sulphanyl groups for arglabin cytotoxic activity against cancer cells.
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Próstata , Neoplasias da Próstata , Humanos , Masculino , Próstata/patologia , Androgênios/metabolismo , Apoptose , Neoplasias da Próstata/patologia , Fosforilação , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proliferação de Células , Inibidores de Proteínas Quinases/farmacologia , Receptores ErbB/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Complementary medicine (CM) is frequently used by patients, but little is known about the usage of CM in surgical patients. The study aimed to elucidate the relevance of CM in surgery. METHODS: This cross-sectional, multi-center survey utilized a paper-based questionnaire consisting of 21 questions to capture CM usage and interest as well as CM communication in visceral and thoracic surgical patients being hospitalized at the corresponding departments of surgery at the University Medical Centers in Freiburg, Heidelberg und Ulm, Germany. RESULTS: Overall, 151 patients consented to the survey. On average, current CM usage was stated by 44% of patients. Most frequently used CM approaches were physical exercise (63%), nutritional supplements (59%) and herbal medicine (56%). Strong interest in CM counselling was stated by 51% of patients. Almost 80% of patients wanted to be treated in a holistic manner and desired for reliable information about CM as well as CM informed physicians. Only 12% of patients communicated CM usage and interest with their attending physician. Review of literature revealed similar results showing an overall CM usage of 43%, preferring nutritional supplements and herbal medicine. CONCLUSION: The results of our cross-sectional study indicate a high percentage of CM users and a strong interest in CM among surgical patients. Indeed, the current communication about CM between patients and surgeons is poor. With respect to safety and quality reasons, but also to pay attention to patients' demands, physicians should be aware of patients' CM usage in surgery. TRIAL REGISTRATION: German Clinical Trial register (DRKS00015445).
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Terapias Complementares , Comunicação , Estudos Transversais , Alemanha , Humanos , Inquéritos e QuestionáriosRESUMO
Oleogum resins of the genus Commiphora have been used in traditional medicines for centuries. More than 200 Commiphora species exhibit highly variable phytochemical compositions. A novel highly selective, sensitive, accurate HPLC-MS/MS method was developed and validated to quantify five characteristic phytosteroids and furanosesquiterpenoids, namely (E)-guggulsterone, (Z)-guggulsterone, curzerenone, furanoeudesma-1,3-diene, and myrrhone. The resulting contents and additionally GC analysis were used to classify and differentiate Commiphora oleogum resins of the species C. myrrha, C. erythraea, C. mukul, C. holtziana, C. confusa, and C. kua, as well as unspecified resins. Interestingly, a Commiphora sample from Ogaden, Ethiopia, comprised 446 ng/mg guggulsterones presumed to be unique to C. mukul from the Indian subcontinent. However, Commiphora from Ogaden differed considerably from C. mukul in respect to guggulsterones isomer's ratio. Moreover, the cytotoxicity of Commiphora extracts, essential oils, botanical drugs containing Commiphora, and pure compounds against the epidermoid carcinoma A431, malignant melanoma RPMI-7951 and SK-MEL-28 cells was investigated in vitro. Thereby, especially C. mukul extract and C. myrrha essential oil exhibited high cytotoxicity against skin cancer cells with IC50 of 2.9-10.9 µg/mL, but were less toxic to normal keratinocytes. In summary, Commiphora oleogum resins and its phytochemicals warrant further investigation aiming at chemotaxonomical classification as well as application in skin cancer treatment.
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Óleos Voláteis , Neoplasias Cutâneas , Commiphora/química , Humanos , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Resinas Vegetais/química , Resinas Vegetais/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Espectrometria de Massas em TandemRESUMO
PURPOSE: Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. METHODS: Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. RESULTS: We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis in vitro and in vivo. CONCLUSION: In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.
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Sarcoma Sinovial , Animais , Apoptose , Carcinogênese , Linhagem Celular Tumoral , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma Sinovial/tratamento farmacológico , Sarcoma Sinovial/genética , Sarcoma Sinovial/metabolismoRESUMO
In vitro analysis of human macrophages is generally hampered by the necessity to differentiate them from peripheral blood monocytes. We have analyzed to which extent noncancerous SC monocytes could be used as an in vitro macrophage model. Macrophages differentiated from peripheral monocytes using standard CSF1 and CSF2 protocols for M2 and M1 precursors, respectively, were compared with THP-1-derived macrophages treated with PMA and with SC-derived macrophages differentiated either by CSF1, CSF2, or PMA according to different protocols. The optimal condition for generation of SC macrophages was treatment with PMA for 3 days, followed by 5-days culture without PMA and 24-h polarization with LPS/IFN-γ or IL-4/IL-13. Similar to THP-1, SC cells do not express the monocyte marker CD14 and differentiation to macrophages results neither in CD68 nor in CD14 expression, both of which were expressed by monocyte-derived macrophages. Similar to THP-1-macrophages, a proportion of SC macrophages can be polarized to the M1-like subtype that is characterized by higher expression of CD38, CD86, CD80, TNF-α, and IL-1ra, whereas treatment with IL4/IL13 did not lead to expression of the M2-associated receptors CD163, CD206, and only slightly increased the CD200R expression. Still, SC-M1 express much lower levels of the M1-associated markers compared with monocyte-derived M1 and no IL-1ß. The data demonstrate that SC-derived macrophages differ from monocyte-derived macrophages in respect of their morphology, expression of important macrophage markers, phagocytosis. Yet, polarized SC-M1-like cells may with restrictions serve as a model for M1 macrophages, though this model does not provide significant advantages over already well-described THP-1-M1-like cells.
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Interleucina-13 , Monócitos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. From the leaves of Callistemon citrinus, we have isolated a novel phloroglucinol dimer, calcitrinone A, and analyzed its potential anticancer activity using the triple-negative breast cancer cell line MDA-MB-231. Calcitrinone A decreased the total intracellular ATP levels, inhibited proliferation, and induced apoptosis in MDA-MB-231 cells, but was less toxic to peripheral blood mononuclear cells. The antiproliferative and apoptosis-inducing effects of calcitrinone A were confirmed in vivo using breast cancer xenografts grown on chick chorioallantoic membranes. Mechanistic analysis showed mitochondrial membrane-potential dissipation and interference with energy-yielding processes resulting in cell accumulation in the S phase of the cell cycle. Seahorse assay analysis revealed an early inhibition of mitochondrial oxidative phosphorylation (OXPHOS). At the molecular level, calcitrinone A inhibited activity of the succinate-coenzyme Q reductase (SQR) (mitochondrial complex II). In silico docking identified the coenzyme Q binding pocket as a possible high affinity binding site for calcitrinone A in SQR. Inhibition of complex II was accompanied by strong elevation of mitochondrial superoxide and cytoplasmic ROS. Calcitrinone A might be a promising anticancer lead compound acting through the interference with the mitochondrial complex II activity.
RESUMO
A series of aryl-substituted 3-amino-1-aryl-8-methoxy-1H-benzo[f]chromene-2-carbonitriles (4a-4q) were designed and synthesized via reaction of 6-methoxy-2-naphthol with a mixture of appropriate aromatic aldehydes and malononitrile under microwave conditions. The structures of the novel compounds 4b, 4c, 4f, 4g, 4i, 4l, 4m, and 4o-4q were established according to IR, 1H-NMR, 13C-NMR/13C-NMR-DEPT, and MS. The benzochromene derivative 4c with a single chlorine at the meta position of the phenyl ring and, to a lesser extent, other benzochromenes with monohalogenated phenyl ring (4a, 4c-4f) exhibited the highest cytotoxicity against six human cancer cell lines MDA-MB-231, A549, HeLa, MIA PaCa-2, 5,637, and Hep G2. The mechanisms of the cytotoxic activities of benzochromenes with monohalogenated phenyl ring (4a, 4c-4f) were further analyzed using triple-negative breast cancer cell line MDA-MB-231. Cell cycle analysis showed accumulation of the treated cells in S phase for 4a, 4d-4f, and S-G2/M phases for 4c. In vivo, 4a and 4c-4f inhibited growth, proliferation, and triggered apoptosis in preestablished breast cancer xenografts grown on the chick chorioallantoic membranes while exhibiting low systemic toxicity. Compounds 4a and 4c-4f increased levels of mitochondrial superoxide and decreased mitochondrial membrane potential resulting in initiation of apoptosis as demonstrated by caspase 3/7 activation. In addition, 4c induced general oxidative stress in cancer cells. The SAR study confirmed that halogens of moderate size at meta or para positions of the pendant phenyl ring enhance the cytotoxic activity of 3-amino-1-aryl-8-methoxy-1H-benzo[f]chromene-2-carbonitriles, and these compounds could serve as leads for the development of novel anticancer therapies.
RESUMO
BACKGROUND: The results of recent surveys indicate that more than 50% of the German population has experience with complementary and alternative medicine (CAM) or uses CAM regularly. This study investigated the CAM usage and CAM-related needs of hospitalized patients at university medical centres in the state of Baden-Württemberg, Germany. METHODS: A multi-centre, paper-based, pseudonymous survey was carried out by the members of the Academic Centre for Complementary and Integrative Medicine. Patients of all ages, regardless of sex, diagnosis and treatment, who were hospitalized in the Department of Cardiology, Gastroenterology, Oncology, Gynaecology or Surgery at the university medical centres in Freiburg, Heidelberg, Tübingen and Ulm were eligible for inclusion. RESULTS: Of the 1275 eligible patients, 67% (n = 854) consented to participate in the survey. Forty-eight percent of the study participants stated that they were currently using CAM. The most frequently used therapies were exercise (63%), herbal medicine (54%) and dietary supplements (53%). Only 16% of the patients discussed CAM usage with their attending physician. Half of the patients (48%) were interested in CAM consultations. More than 80% of the patients desired reliable CAM information and stated that physicians should be better informed about CAM. CONCLUSIONS: The frequency of CAM usage and the need for CAM counselling among hospitalized patients at university medical centres in Baden-Württemberg are high. To better meet patients' needs, CAM research and physician education should be intensified. TRIAL REGISTRATION: German Clinical Trial register ( DRKS00015445 ).
Assuntos
Centros Médicos Acadêmicos , Terapias Complementares/estatística & dados numéricos , Hospitalização , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários , Adulto JovemRESUMO
Oleanolic acid is a widely distributed natural product, which possesses promising antitumor, antiviral, antihyperlipidemic, and anti-inflammatory activities. A heterodimeric complex formed by integrin αM (CD11b) and integrin ß2 (CD18) is highly expressed on monocytes and macrophages. In the current study, we demonstrate that the I domain of αM (αM-I domain) might present a potential cellular target for oleanolic acid. In vitro data show that oleanolic acid induces clustering of αM on macrophages and reduces their non-directional migration. In accordance with experimental data, molecular docking revealed that oleanolic acid binds to the αM-I domain in its extended-open form, the dominant conformation found in αM clusters. Molecular dynamics simulation revealed that oleanolic acid can increase the flexibility of the α7 helix and promote its movement away from the N-terminus, indicating that oleanolic acid may facilitate the conversion of the αM-I domain from the extended-closed to the extended-open conformation. As demonstrated by metadynamics simulation, oleanolic acid can destabilize the local minimum of the αM-I domain in the open conformation partially through disturbance of the interactions between α1 and α7 helices. In summary, we demonstrate that oleanolic acid might function as an allosteric agonist inducing clustering of αM on macrophages by shifting the balance from the closed to the extended-open conformation. The molecular target identified in this study might hold potential for a purposeful use of oleanolic acid to modulate chronic inflammatory responses.
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Herein, a series of aryl-substituted derivatives of 3-amino-1-aryl-9-methoxy-1H-benzo[f]chromene-2-carbonitriles (4a-4q) were designed and synthesized via reaction of 7-methoxy-2-naphthol with a mixture of appropriate aromatic aldehydes and malononitrile under microwave conditions. Among the tested benzochromene, the known compound 4e and four novel compounds 4f, 4j, 4k, 4m exhibited the highest cytotoxicity towards a panel of six human cancer cell lines MDA-MB-231, A549, HeLa, MIA PaCa-2, RPMI 7951, and PC-3. Compound 4j with 2,4-dichloro substitution on the pendant phenyl ring exhibited the highest broad-spectrum cytotoxicity towards all tested cancer cell lines. Compounds 4e, 4f, 4j, 4k, 4m were further selected to study the mechanism of cellular toxicity using the triple-negative breast cancer cells MDA-MB-231. Compounds 4e, 4f, 4j, 4k, 4m induced accumulation of the treated MDA-MB-231 cells in the S phase and 4k additionally in the G2/M phase of the cell cycle. Compounds 4e, 4f, 4j, 4k, 4m induced dissipation of mitochondrial transmembrane potential and activation of caspase 3/7 in MDA-MB-231 cells with 4j being one of the most active. In an in vivo model, compound 4j and less efficiently 4e and 4f inhibited growth and proliferation and triggered DNA fragmentation in MDA-MB-231 xenografts grown on chick chorioallantoic membranes. SAR study confirmed that the 2,4-dichloro substitution pattern on the pendant phenyl ring enhanced the cytotoxic activity of benzochromene.
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Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzopiranos/síntese química , Benzopiranos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
AA amyloidosis belongs to the group of amyloid diseases which can follow chronic inflammatory conditions of various origin. The disease is characterized by the deposition of insoluble amyloid fibrils formed by serum amyloid A1 (SAA1) leading eventually to organ failure. Macrophages are intimately involved in the fibrillogenesis as well as in the clearance of amyloid fibrils. In vivo, macrophages may occur as classically (M1) or alternatively activated (M2) macrophages. We investigate here how SAA1 might affect the macrophage phenotype and function. Gene microarray analysis revealed upregulation of 64 M1-associated genes by SAA1. M1-like polarization was further confirmed by the expression of the M1-marker MARCO, activation of the NF-κB transcription factor, and secretion of the M1-cytokines TNF-α, IL-6, and MCP-1. Additionally, we demonstrate here that M1-polarized macrophages exhibit enhanced fibrillogenic activity towards SAA1. Based on our data, we propose reconsideration of the currently used cellular amyloidosis models towards an in vitro model employing M1-polarized macrophages. Furthermore, the data suggest macrophage repolarization as potential intervention strategy in AA amyloidosis.
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Macrófagos/efeitos dos fármacos , Proteína Amiloide A Sérica/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas Recombinantes/farmacologia , Proteína Amiloide A Sérica/genéticaRESUMO
BACKGROUND: Gremlin-1 is a cystine knot protein and is expressed in organs developing fibrosis. Transient ischaemia leads to myocardial fibrosis, a major determinant of impaired myocardial function. MATERIALS AND METHODS: Expression of Gremlin-1 was investigated in infarcted myocardium by real-time PCR, Western blot analysis, histological and immunohistochemistry staining. We further elaborated the colocalization of Gremlin-1 and TGF-ß proteins by confocal microscopy and co-immunoprecipitation experiments. The interaction between Gremlin-1 and TGF-ß was analysed by photon correlation spectroscopy. Gremlin-1 modulation of the TGF-ß-dependent collagen I synthesis in fibroblasts was investigated using ELISA and immunohistochemistry experiments. The effect of prolonged administration of recombinant Gremlin-1 on myocardial function following ischaemia/reperfusion was accessed by echocardiography and immunohistochemistry. RESULTS: Gremlin-1 is expressed in myocardial tissue and infiltrating cells after transient myocardial ischaemia (P < .05). Gremlin-1 colocalizes with the pro-fibrotic cytokine transforming growth factor-ß (TGF-ß) expressed in fibroblasts and inflammatory cell infiltrates (P < .05). Gremlin-1 reduces TGF-ß-induced collagen production of myocardial fibroblasts by approximately 20% (P < .05). We found that Gremlin-1 binds with high affinity to TGF-ß (KD = 54 nmol/L) as evidenced by photon correlation spectroscopy and co-immunoprecipitation. intravenous administration of m Gremlin-1-Fc, but not of equivalent amount of Fc control, significantly reduced infarct size by approximately 20%. In the m Gremlin-1-Fc group, infarct area was reduced by up to 30% in comparison with mice treated with Fc control (I/LV: 4.8 ± 1.2% vs 6.0 ± 1.2% P < .05; I/AaR: 15.2 ± 1.5% vs 21.1 ± 5%, P < .05). CONCLUSIONS: The present data disclose Gremlin-1 as an antagonist of TGF-ß and presume a role for Gremlin-1/TGF-ß interaction in myocardial remodelling following myocardial ischaemia.
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Fibroblastos/metabolismo , Coração/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Infarto do Miocárdio/genética , Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/metabolismo , Ecocardiografia , Células Endoteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Coração/diagnóstico por imagem , Coração/efeitos dos fármacos , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Microscopia Confocal , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Proteínas Recombinantes , Fator de Crescimento Transformador beta/efeitos dos fármacos , Remodelação Ventricular/genéticaRESUMO
Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 µM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Citocromo P-450 CYP2E1/metabolismo , Dodecanol , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Camundongos Nus , Estresse OxidativoRESUMO
Boswellic acids, and particularly 11-keto-boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and potential antitumor efficacy. Although boswellic acids generally occur as α-isomers (oleanane type) and ß-isomers (ursane type), 11-keto-boswellic acid (KBA) was found only as the ß-isomer, ß-KBA. Here, the existence and natural occurrence of the respective α-isomer, 11-keto-α-boswellic acid (α-KBA), is demonstrated for the first time. Initially, α-KBA was synthesized and characterized by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy, and a highly selective, sensitive, and accurate high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed by Design of Experiments (DoE) using a pentafluorophenyl stationary phase. This method allowed the selective quantification of individual 11-keto-boswellic acids and provided evidence for α-KBA in Boswellia spp. oleogum resins. The contents of α-KBA as well as further boswellic acids and the composition of essential oils were used to chemotaxonomically classify 41 Boswellia oleogum resins from 9 different species. Moreover, α-KBA exhibited cytotoxicity against three treatment-resistant triple-negative breast cancer (TNBC) cell lines in vitro and also induced apoptosis in MDA-MB-231 xenografts in vivo. The respective ß-isomer and the acetylated form demonstrate higher cytotoxic efficacies against TNBC cells. This provides further insights into the structure-activity relationship of boswellic acids and could support future developments of potential anti-inflammatory and antitumor drugs.
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Antineoplásicos/farmacologia , Boswellia/química , Neoplasias de Mama Triplo Negativas/patologia , Triterpenos/farmacologia , Animais , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Galinhas , Humanos , Isomerismo , Triterpenos/síntese química , Triterpenos/química , Triterpenos/isolamento & purificaçãoRESUMO
BACKGROUND AND AIMS: Alcoholic steatohepatitis (ASH)-the inflammation of fatty liver-is caused by chronic alcohol consumption and represents one of the leading chronic liver diseases in Western Countries. ASH can lead to organ dysfunction or progress to hepatocellular carcinoma (HCC). Long-term alcohol abstinence reduces this probability and is the prerequisite for liver transplantation-the only effective therapy option at present. Elevated enzymatic activity of cytochrome P450 2E1 (CYP2E1) is known to be critically responsible for the development of ASH due to excessively high levels of reactive oxygen species (ROS) during metabolization of ethanol. Up to now, no rational drug discovery process was successfully initiated to target CYP2E1 for the treatment of ASH. METHODS: In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. RESULTS: A new class of drug candidates was generated successfully. Two of the most promising small compounds named 12-Imidazolyl-1-dodecanol (abbr.: I-ol) and 1-Imidazolyldodecane (abbr.: I-an) were selected at the end of this process of drug discovery and developability. These new ω-imidazolyl-alkyl derivatives act as strong chimeric CYP2E1 inhibitors at a nanomolar range. They restore redox balance, reduce inflammation process as well as the fat content in the liver and rescue the physiological liver architecture of rats consuming continuously a high amount of alcohol. CONCLUSIONS: Due to its oral application and therapeutic superiority over an off-label use of the hepatoprotector ursodeoxycholic acid (UDCA), this new class of inhibitors marks the first rational, pharmaceutical concept in long-term treatment of ASH.
Assuntos
Inibidores do Citocromo P-450 CYP2E1/uso terapêutico , Citocromo P-450 CYP2E1/metabolismo , Fígado Gorduroso Alcoólico/tratamento farmacológico , Alcanos/síntese química , Alcanos/farmacologia , Alcanos/uso terapêutico , Animais , Citocromo P-450 CYP2E1/química , Inibidores do Citocromo P-450 CYP2E1/síntese química , Inibidores do Citocromo P-450 CYP2E1/farmacologia , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Feminino , Células Hep G2 , Humanos , Lipoproteínas VLDL/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Triglicerídeos/sangue , Ácido Ursodesoxicólico/análiseRESUMO
Triple negative human breast cancer (TNBC) is an aggressive cancer subtype with poor prognosis. Besides the better-known artemisinin, Artemisia annua L. contains numerous active compounds not well-studied yet. High-performance liquid chromatography coupled with diode-array and mass spectrometric detection (HPLC-DAD-MS) was used for the analysis of the most abundant compounds of an Artemisia annua extract exhibiting toxicity to MDA-MB-231 TNBC cells. Artemisinin, 6,7-dimethoxycoumarin, arteannuic acid were not toxic to any of the cancer cell lines tested. The flavonols chrysosplenol d and casticin selectively inhibited the viability of the TNBC cell lines, MDA-MB-231, CAL-51, CAL-148, as well as MCF7, A549, MIA PaCa-2, and PC-3. PC-3 prostate cancer cells exhibiting high basal protein kinase B (AKT) and no ERK1/2 activation were relatively resistant, whereas MDA-MB-231 cells with high basal ERK1/2 and low AKT activity were more sensitive to chrysosplenol d treatment. In vivo, chrysosplenol d and casticin inhibited MDA-MB-231 tumor growth on chick chorioallantoic membranes. Both compounds induced mitochondrial membrane potential loss and apoptosis. Chrysosplenol d activated ERK1/2, but not other kinases tested, increased cytosolic reactive oxygen species (ROS) and induced autophagy in MDA-MB-231 cells. Lysosomal aberrations and toxicity could be antagonized by ERK1/2 inhibition. The Artemisia annua flavonols chrysosplenol d and casticin merit exploration as potential anticancer therapeutics.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Artemisia annua/química , Flavonas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Flavonas/química , Flavonoides/química , Flavonoides/farmacologia , Flavonóis/química , Flavonóis/farmacologia , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias de Mama Triplo Negativas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Photodynamic therapy (PDT) is a non-invasive treatment strategy that includes the combination of three components-a photosensitizer, a light source, and tissue oxygen. PDT can be used for the treatment of skin diseases such as squamous cell carcinoma. The photosensitizer used in this study is the naturally derived chlorophyll derivative chlorin e6 (Ce6), which was encapsulated in ultradeformable ethosomes. Singlet oxygen production by Ce6 upon laser light irradiation was not significantly affected by encapsulation into ethosomes. PDT of squamous cell carcinoma cells treated with Ce6 ethosomes triggered increased mitochondrial superoxide levels and increased caspase 3/7 activity, resulting in concentration- and light-dose-dependent cytotoxicity. Ce6 ethosomes showed good penetration into 3D squamous cell carcinoma spheroids, which upon laser light irradiation exhibited reduced size, proliferation, and viability. The PDT effect of Ce6 ethosomes was specific and showed higher cytotoxicity against squamous cell carcinoma spheroids compared to normal skin fibroblast spheroids. In addition, PDT treatment of squamous cell carcinoma xenografts grown on chorioallantoic membranes of chick eggs (CAM) exhibited reduced expression of Ki-67 proliferation marker and increased terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, indicating reduced proliferation and activation of apoptosis, respectively. The results demonstrate that Ce6-loaded ethosomes represent a convenient formulation for photodynamic treatment of squamous cell carcinoma.
RESUMO
In this study, a series of novel N-feruloyl dipeptides (10-17) have been synthesized through the coupling of N-feruloyl amino acids (6-9) with glycine/alanine methyl ester hydrochloride. Structures of the peptides were assigned using 1D and 2D NMR and HRESIMS. According to initial in vitro cytotoxic screening against the cervix carcinoma cell line KB-3-1, aromatic dipeptides (12, 13, 16, 17) were the most potent ones among all tested feruloyl dipeptides. Accordingly, these peptides were further intensively investigated as potential anticancer agents against a panel of ten cancer cell lines from different tissue origin. Based on that, compound 17 showed the strongest cytotoxic efficiency towards the whole panel of tested cell lines with IC50 values from 2.1 to 7.9 µM. By contrast, the dipeptides 12, 13 and 16 showed moderate to weak cytotoxicity (IC50 16.1-28.3 or >30, 5.7-21.9 and 3.9-21.2 or ≥30 µM, respectively). Mechanistically, compound 17 induced a strong dissipation of the mitochondrial transmembrane potential and an early activation of caspase 3/7 in the triple-negative MDA-MB-231 breast cancer cell line. In an in vivo model, compound 17 inhibited growth, proliferation and induced apoptosis in MDA-MB-231 xenografted onto the chick chorioallantoic membrane. All the synthesized compounds were also tested against a set of pathogenic bacterial strains, displaying no potential activity.