RESUMEN
Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.
Asunto(s)
Artemisininas/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Modelos Animales de Enfermedad , Receptores de GABA-A/metabolismo , Transducción de Señal , Animales , Arteméter , Artemisininas/administración & dosificación , Proteínas Portadoras/metabolismo , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus Tipo 1/patología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Estabilidad Proteica/efectos de los fármacos , Ratas , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Pez Cebra , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The clinical development of anticancer metallodrugs is often hindered by the elusive nature of their molecular targets. To identify the molecular targets of an antimetastatic ruthenium organometallic complex based on 1,3,5-triaza-7-phosphaadamantane (RAPTA), we employed a chemical proteomic approach. The approach combines the design of an affinity probe featuring the pharmacophore with mass-spectrometry-based analysis of interacting proteins found in cancer cell lysates. The comparison of data sets obtained for cell lysates from cancer cells before and after treatment with a competitive binder suggests that RAPTA interacts with a number of cancer-related proteins, which may be responsible for the antiangiogenic and antimetastatic activity of RAPTA complexes. Notably, the proteins identified include the cytokines midkine, pleiotrophin and fibroblast growth factor-binding protein 3. We also detected guanine nucleotide-binding protein-like 3 and FAM32A, which is in line with the hypothesis that the antiproliferative activity of RAPTA compounds is due to induction of a G2/M arrest and histone proteins identified earlier as potential targets.
RESUMEN
A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. While these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib), and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune, and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes, and signaling pathways that are simultaneously engaged by multitargeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments.
Asunto(s)
Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Transducción de Señal , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit its therapeutic applications. Here, we used a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anticancer compound YM155 on solute carrier family member 35 F2 (SLC35F2), an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug-importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines, and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo. These findings suggest a new route to targeted DNA damage by exploiting tumor and patient-specific import of YM155.
Asunto(s)
Daño del ADN/efectos de los fármacos , Imidazoles/farmacología , Sustancias Intercalantes/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Naftoquinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular , Clonación Molecular , Ensayo Cometa , Genoma Humano/efectos de los fármacos , Genoma Humano/genética , Haploidia , Humanos , Imidazoles/metabolismo , Inmunohistoquímica , Ratones , Ratones SCID , Naftoquinonas/metabolismo , ARN Neoplásico/química , ARN Neoplásico/genéticaRESUMEN
Deregulation of protein tyrosine kinase signaling has been linked to many diseases, most notably cancer. As a consequence, small molecule inhibitors of protein tyrosine kinases may provide powerful strategies for treatment. Following the successful introduction of imatinib in the treatment of chronic myelogenous leukemia, such drugs are also now evaluated for other types of cancer. However, many developed kinase inhibitors are not very target-specific and therefore may induce side effects. The importance of such side effects is certainly cell-proteome dependent. Understanding the all-inclusive action of a tyrosine kinase inhibitor on each individual cell-type entails the identification of potential targets, combined with monitoring the downstream effects revealing the signaling networks involved. Here, we explored a multilevel quantitative mass spectrometry-based proteomic strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in epidermoid carcinoma cells, as a model system for skin-cancer. More than 25 tyrosine kinases showed affinity to the drugs, with imatinib and nilotinib displaying a high specificity, especially when compared to dasatinib and bosutinib. Consequently, the latter two drugs showed a larger effect on downstream phosphotyrosine signaling. Many of the proteins affected are key regulators in cell adhesion and invasion. Our data represents a multiplexed view on the promiscuous action of certain tyrosine kinase inhibitors that needs to be taking into consideration prior to the application of these drugs in the treatment of different forms of cancer.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/enzimología , Línea Celular Tumoral , Humanos , Espectrometría de Masas , Fosforilación , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteómica , Piel/efectos de los fármacos , Piel/enzimologíaRESUMEN
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Aminoquinolinas/farmacología , Animales , Antineoplásicos/química , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Crizotinib , Cristalización , Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/química , Modelos Animales de Enfermedad , Femenino , Homeostasis/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/química , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Proteómica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Pirazoles/química , Piridinas/química , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genéticaRESUMEN
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Modelos Biológicos , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Biología de Sistemas , Proliferación Celular/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Mapas de Interacción de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.
Asunto(s)
Compuestos de Anilina/química , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Neoplasias Pulmonares/enzimología , Nitrilos/química , Proteínas Tirosina Quinasas/aislamiento & purificación , Pirimidinas/química , Quinolinas/química , Tiazoles/química , Animales , Biopsia , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Cromatografía de Afinidad , Dasatinib , Humanos , Células K562 , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Anotación de Secuencia Molecular , Terapia Molecular Dirigida , Trasplante de Neoplasias , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , ProteómicaRESUMEN
Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABL(T315I). To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABL(T315I) CML cells on c-Myc through nonobvious off targets.
Asunto(s)
Compuestos de Anilina/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Nitrilos/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Proteómica , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-Actividad , Biología de SistemasRESUMEN
Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.
Asunto(s)
Cromatografía de Afinidad/métodos , Composición de Medicamentos/métodos , Preparaciones Farmacéuticas/aislamiento & purificación , Animales , Extractos Celulares , Células Cultivadas , Cromatografía Liquida , Dasatinib , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Espectrometría de Masas , Proteínas/análisis , Pirimidinas/química , Pirimidinas/farmacología , Tinción con Nitrato de Plata , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
The water extract of the lettuce Lactuca sativa, but not the ethyl acetate extract, inhibited the growth of HL-60 leukaemia cells and MCF-7 breast cancer cells. This correlated with the activation of checkpoint kinase 2 (Chk2), the induction of the tumour suppressor p21, and the severe downregulation of the proto-oncogene cyclin D1. The ethyl acetate extract, but not the water extract, induced HL-60 cell death, which correlated with the acetylation of alpha-tubulin. The acetylation of alpha-tubulin is indicative for microtubuli stabilisation such as induced by taxol. The calculated amount for human intake would require approximately 3 kg lettuce to reach the required concentration shown to inhibit 50% HL-60 proliferation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas de Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Lactuca/química , Extractos Vegetales/farmacología , Acetilación , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Ciclina D1/efectos de los fármacos , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Humanos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proto-Oncogenes Mas , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Estrogenic procarcinogenic effects of piceatannol (PIC) contrast reports about anticarcinogenic activities of PIC. To explain this contradiction, we investigated PIC in estrogen-dependent MCF-7 breast cancer cells and elucidated those cellular mechanisms that correlated with the observed cell effects induced by PIC. Low PIC concentrations (50 nM) induced c-Myc that depended on progesterone receptor (PR) and estrogen receptor (ER). PR-mediated c-Myc induction by PIC was independent of nuclear PR activity but depended on mitogen-activated protein kinase (MAPK) signaling and was associated with an acceleration of cancer cell proliferation. In contrast, 25 µM PIC inhibited deoxynucleotide triphosphate synthesis, activated Chk2 and p38-MAPK and this was accompanied by an attenuation of cancer cell growth. Apoptosis was most probably inhibited due to activation of Akt; however, high PIC concentrations (>100 µM) permitted apoptosis-like cell death in consequence to disruption of orchestrated mitotic signaling. The presented results show for the first time that nanomolar PIC concentrations signal through PR and Erk1/2 and provide a mechanistic explanation why moderate wine consumption-but not other alcoholic beverages-increases the breast cancer risk in women. In contrast, higher PIC concentrations in the micromolar range are considered for adjuvant anticancer therapeutic concepts.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/prevención & control , Carcinógenos/toxicidad , Estilbenos/farmacología , Estilbenos/toxicidad , Animales , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Genes myc , Humanos , Receptores de Estrógenos/fisiología , Receptores de Progesterona/fisiología , VinoRESUMEN
AIM OF THIS STUDY: Within the genus Scutellaria various species are used in different folk medicines throughout Asia. Traditional Chinese Medicine (TCM) uses S. baicalensis (Labiatae) to treat various inflammatory conditions. The root shows strong anticancer properties in vitro and was suggested for clinical trials against multiple myeloma. Further, S. barbata was successfully tested against metastatic breast cancer in a phase I/II trial. Therefore, we investigated the anti-cancer properties of S. orientalis L. ssp. carica Edmondson, an endemic subspecies from the traditional medicinal plant S. orientalis L. in Turkey, which is used to promote wound healing and to stop haemorrhage. MATERIALS AND METHODS: Freeze-dried plant material was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, and by investigating protein expression profiles specific for cell cycle arrest and apoptosis. RESULTS: The strongest anti-leukemic activity was shown by the methanol extract, which contained apigenin, baicalein, chrysin, luteolin and wogonin, with an IpC50 of 43 microg/ml (corresponding to 1.3mg/ml of dried plant material) which correlated with cyclin D1- and Cdc25A suppression and p21 induction. At 132 microg/ml (=4 mg/ml of the drug) this extract caused genotoxic stress indicated by substantial phosphorylation of the core histone H2AX (gamma-H2AX) followed by activation of caspase 3 and signature-type cleavage of PARP resulting in a 55% apoptosis rate after 48 hours of treatment. CONCLUSIONS: Here, we report for the first time that S. orientalis L. ssp. carica Edmondson exhibited potent anti-leukaemic properties likely through the anti-proliferative effect of baicalein and the genotoxic property of wogonin.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Scutellaria/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Cromatografía Líquida de Alta Presión , Ciclina D1/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HL-60 , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Fosforilación , Extractos Vegetales/química , Extractos Vegetales/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Turquía , Fosfatasas cdc25/antagonistas & inhibidoresRESUMEN
Hypericum perforatum (St. John's wort) is well-established for its antidepressant activity throughout the world and also various other species within this genus are used in different folk medicines. Hyperforin of St. John's wort inhibited growth of cancer cell lines and the use of hypericin (another compound of H. perforatum) in cancer photodynamic therapy is proposed. Therefore, we investigated the anti-cancer properties of H. adenotrichum Spach (Guttiferae), an endemic species in Turkey called 'kantaron', which is used for wound healing and antiseptic effects. Freeze-dried plant was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, by investigating protein expression profiles specific for cell cycle arrest and apoptosis as well as composition by HPLC. The strongest anti-proliferative activity was determined for the petroleum ether extract with an IpC50 of approximately 5.8 microg/ml medium (referring to 1 mg dried plant) which correlated with cyclin D1 suppression and p21 induction. This extract also induced phosphorylation of H2AX, and activated caspase-3 followed by signature-type cleavage of PARP resulting in approximately 50% apoptosis at 23.2 microg/ml after 24 h of treatment. Neither hyperforin, hypericin, or amentoflavone contributed to these properties. To the best of our knowledge, we report for the first time that the endemic plant H. adenotrichum Spach exhibits potent p53-independent anti-neoplastic properties due to yet unexplored Hypericum constituents.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Hypericum , Leucemia/tratamiento farmacológico , Fitoterapia/métodos , Extractos Vegetales/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Humanos , Hypericum/química , Técnicas In Vitro , TurquíaRESUMEN
The Aracea Anthurium schlechtendalii and Syngonium podophyllum are traditional remedies for the treatment of severe and chronic inflammatory conditions. We cross-examined these plants regarding their anti-neoplastic properties, because several anti-inflammatory molecular targets are common for both pathologic conditions due to similar signalling pathways. Two malignant cell lines, HL-60 and MCF-7, were treated with increasing concentrations of plant extracts of increasing polarity. The potential of the extracts to inhibit the cell cycle and to induce cell death was investigated, because these are relevant endpoints to assess the anti-cancer potential in vitro and the protein expression and cell cycle distribution upon exposure to the strongest extract was analysed. Extracts from S. podophyllum were rather ineffective, but the freeze-dried (but not air-dried) roots of A. schlechtendalii exhibited strong growth inhibitory and apoptosis-inducing properties. In HL-60 cells 50% proliferation inhibition was achieved by 1.7 microg dichloromethane extract/ml medium and correlated with the activation of Chk2, down-regulation of Cdc25A, suppression of cyclin D1 level, and transient induction of p21. This extract efficiently triggered apoptosis, which was confirmed by caspase 3 activation. The polymerisation of alpha-tubulin and its subsequent degradation that depleted the cells from the G2/M contributed to apoptosis induction, because proper spindle-formation during mitosis is mandatory for survival. In conclusion, we demonstrated that A. schlechtendalii root extract specifically targeted carcinogenic mechanisms, because Cdc25A and cyclin D1 are oncogenes that are frequently overexpressed in a variety of cancer entities and further, this extract affected microtubule function reminiscent of taxol.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Araceae/química , Extractos Vegetales/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Ciclina D1/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Extractos Vegetales/química , Proteínas Serina-Treonina Quinasas/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
The effects of heat shock (HS; 42 degrees C) on the cell cycle and underlying molecular mechanisms are astonishingly unexplored. Here, we show that HS caused rapid Cdc25A degradation and a reduction of cell cycle progression. Cdc25A degradation depended on Ser75-Cdc25A phosphorylation caused by p38MAPK and Chk2, which phosphorylated Ser177-Cdc25A that is specific for 14.3.3 binding. Upon HS, Cdc25A rapidly co-localized with 14.3.3 in the perinuclear space that was accompanied with a decrease of nuclear Cdc25A protein levels. Consistently, a 14.3.3 binding-deficient Cdc25A double mutant (Ser177/Ala-Tyr507/Ala) was not degraded in response to HS and there was no evidence for an increased co-localization of Cdc25A with 14.3.3 in the cytosol. Therefore, upon HS, p38, Chk2 and 14.3.3 were antagonists of Cdc25A stability. On the other hand, Cdc25A was protected by Hsp90 in HEK293 cells because the specific inhibition of Hsp90 with Geldanamycin caused Cdc25A degradation in HEK293 implicating that Cdc25A is an Hsp90 client. Specific inhibition of Hsp90 together with HS caused and accelerated degradation of Cdc25A and was highly cytotoxic. The results presented here show for the first time that Cdc25A is degraded by moderate heat shock and protected by Hsp90. We describe the mechanisms explaining HS-induced cell cycle retardation and provide a rationale for a targeted hyperthermia cancer therapy.
Asunto(s)
Proteínas 14-3-3/metabolismo , Respuesta al Choque Térmico , Proteínas Serina-Treonina Quinasas/metabolismo , Fosfatasas cdc25/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas 14-3-3/genética , Línea Celular , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Quinasa de Punto de Control 2 , Citosol/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Fosfatasas cdc25/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Many traditional healing plants successfully passed several hundred years of empirical testing against specific diseases and thereby demonstrating that they are well tolerated in humans. Although quite a few ethno-pharmacological plants are applied against a variety of conditions there are still numerous plants that have not been cross-tested in diseases apart from the traditional applications. Herein we demonstrate the anti-neoplastic potential of two healing plants used by the Maya of the Guatemala/Belize area against severe inflammatory conditions such as neuritis, rheumatism, arthritis, coughs, bruises and tumours. Phlebodium decumanum and Pluchea odorata were collected, dried and freeze dried, and extracted with five solvents of increasing polarity. We tested HL-60 and MCF-7 cells, the inhibition of proliferation and the induction of cell death were investigated as hallmark endpoints to measure the efficiency of anti-cancer drugs. Western blot and FACS analyses elucidated the underlying mechanisms. While extracts of P. decumanum showed only moderate anti-cancer activity and were therefore not further analysed, particularly the dichloromethane extract of P. odorata inhibited the cell cycle in G2-M which correlated with the activation of checkpoint kinase 2, and down-regulation of Cdc25A and cyclin D1 as well as inactivation of Erk1/2. In HL-60 and MCF-7 cells this extract was a very strong inducer of cell death activating caspase-3 followed by PARP signature type cleavage. The initiating death trigger was likely the stabilization of microtubules monitored by the rapid acetylation of alpha-tubulin, which was even more pronounced than that triggered by taxol. The dichloromethane extract of P. odorata contains apolar constituents which inhibit inflammatory responses and exhibit anti-cancer activity. The strong proapoptotic potential warrants further bioassay-guided fractionation to discover and test the active principle(s).