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He, we show that combined use of the EZH2 inhibitor GSK126 and the CDK4/6 inhibitor abemaciclib synergistically enhances antitumoral effects in preclinical GBM models. Dual blockade led to HIF1α upregulation and CalR translocation, accompanied by massive impairment of mitochondrial function. Basal oxygen consumption rate, ATP synthesis, and maximal mitochondrial respiration decreased, confirming disrupted endoplasmic reticulum-mitochondrial homeostasis. This was paralleled by mitochondrial depolarization and upregulation of the UPR sensors PERK, ATF6α, and IRE1α. Notably, dual EZH2/CDK4/6 blockade also reduced 3D-spheroid invasion, partially inhibited tumor growth in ovo, and led to impaired viability of patient-derived organoids. Mechanistically, this was due to transcriptional changes in genes involved in mitotic aberrations/spindle assembly (Rb, PLK1, RRM2, PRC1, CENPF, TPX2), histone modification (HIST1H1B, HIST1H3G), DNA damage/replication stress events (TOP2A, ATF4), immuno-oncology (DEPDC1), EMT-counterregulation (PCDH1) and a shift in the stemness profile towards a more differentiated state. We propose a dual EZH2/CDK4/6 blockade for further investigation.
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Preclinical studies rarely test the efficacy of therapies in both sexes. The field of oncology is no exception in this regard. In a model of syngeneic, orthotopic, metastasized pancreatic ductal adenocarcinoma we evaluated the impact of sex on pathological features of this disease as well as on the efficacy and possible adverse side effects of a novel, small molecule-based therapy inhibiting KRAS:SOS1, MEK1/2 and PI3K signaling in male and female C57BL/6J mice. Male mice had less tumor infiltration of CD8-positive cells, developed bigger tumors, had more lung metastasis and a lower probability of survival compared to female mice. These more severe pathological features in male animals were accompanied by higher distress at the end of the experiment. The evaluated inhibitors BI-3406, trametinib and BKM120 showed synergistic effects in vitro. This combinatorial therapy reduced tumor weight more efficiently in male animals, although the drug concentrations were similar in the tumors of both sexes. These results underline the importance of sex-specific preclinical research and at the same time provide a solid basis for future studies with the tested compounds.
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The major risk factor for chronic disease is chronological age, and age-related chronic diseases account for the majority of deaths worldwide. Targeting senescent cells that accumulate in disease-related tissues presents a strategy to reduce disease burden and to increase healthspan. The senolytic combination of the tyrosine-kinase inhibitor dasatinib and the flavonol quercetin is frequently used in clinical trials aiming to eliminate senescent cells. Here, our goal was to computationally identify natural senotherapeutic repurposing candidates that may substitute dasatinib based on their similarity in gene expression effects. The natural senolytic piperlongumine (a compound found in long pepper), and the natural senomorphics parthenolide, phloretin and curcumin (found in various edible plants) were identified as potential substitutes of dasatinib. The gene expression changes underlying the repositioning highlight apoptosis-related genes and pathways. The four compounds, and in particular the top-runner piperlongumine, may be combined with quercetin to obtain natural formulas emulating the dasatinib + quercetin formula.
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Quercetina , Senoterapia , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Quercetina/farmacologia , Quercetina/uso terapêutico , Senescência Celular , Expressão GênicaRESUMO
ß-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.
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Neoplasias Pulmonares , Sesquiterpenos Policíclicos , Fator A de Crescimento do Endotélio Vascular , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Regulação para Baixo , Secretoma , Fatores de Crescimento do Endotélio Vascular , Hipóxia , Meios de Cultivo CondicionadosRESUMO
INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.
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Dermatopatias , Neoplasias Cutâneas , Animais , Camundongos , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Histonas , Oncologia , Terapia Combinada , Modelos Animais de DoençasRESUMO
The monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2), is involved in the formation, progression, and destabilization of atheromatous plaques. Flavonoids, found in fruits and vegetables, have been associated with various health-promoting properties, including antioxidant, anti-inflammatory, and cardioprotective effects. In the present study, the flavonoids quercetin, kaempferol, and luteolin, but not cannflavin A, were shown to substantially inhibit interleukin (IL)-1ß-induced MCP-1 mRNA and protein expression in human coronary artery endothelial cells (HCAEC). At the functional level, conditioned medium (CM) from IL-1ß-stimulated HCAEC caused an increase in the migration of THP-1 monocytes compared with CM from unstimulated HCAEC. However, this induction was suppressed when IL-1ß-treated HCAEC were coincubated with quercetin, kaempferol, or luteolin. The functional importance of MCP-1 in IL-1ß-induced monocyte migration was supported by experiments showing that neutralization of MCP-1 in the CM of IL-1ß-treated HCAEC led to a significant inhibition of migration. In addition, a concentration-dependent induction of monocyte migration in the presence of recombinant MCP-1 was demonstrated. Collectively, the flavonoids quercetin, kaempferol, and luteolin were found to exert potential antiatherogenic effects in HCAEC, challenging further studies with these compounds.
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Quimiocina CCL2 , Monócitos , Humanos , Quimiocina CCL2/metabolismo , Monócitos/metabolismo , Quempferóis/farmacologia , Quempferóis/metabolismo , Flavonoides/farmacologia , Flavonoides/metabolismo , Quercetina/farmacologia , Quercetina/metabolismo , Células Endoteliais/metabolismo , Vasos Coronários/metabolismo , Luteolina/farmacologia , Células Cultivadas , Fatores Imunológicos/farmacologiaRESUMO
JZL184, an inhibitor of monoacylglycerol lipase (MAGL) and thus of the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), mediates various anticancer effects in preclinical studies. However, studies on the effect of this or other MAGL inhibitors under hypoxia, an important factor in tumor biology and response to cancer therapy, have not yet been performed in cancer cells. In the present study, the impact of the conditioned media (CM) of A549 and H358 lung cancer cells incubated with JZL184 under hypoxic conditions on the angiogenic properties of human umbilical vein endothelial cells (HUVECs) was investigated. Treatment of HUVECs with CM derived from cancer cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation compared with unconditioned medium, which was inhibited when cancer cells were incubated with JZL184. In this process, JZL184 led to a significant increase in 2-AG levels in both cell lines. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. Antiangiogenic and VEGF-lowering effects were also demonstrated for the MAGL inhibitor MJN110. Receptor antagonist experiments suggest partial involvement of the cannabinoid receptors CB1 and CB2 in the antiangiogenic and VEGF-lowering effects induced by JZL184. The functional importance of VEGF for angiogenesis in the selected system is supported by observations showing inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs by CM from hypoxic cancer cells treated with JZL184 or when hypoxic cancer cell-derived CM was spiked with a neutralizing VEGF antibody. On the other hand, JZL184 did not exert a direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. In conclusion, these results reveal a novel mechanism of antiangiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.
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Neoplasias Pulmonares , Fator A de Crescimento do Endotélio Vascular , Humanos , Células Endoteliais , Neoplasias Pulmonares/tratamento farmacológico , HipóxiaRESUMO
Atherosclerosis is associated with vascular smooth muscle cell proliferation, chronic vascular inflammation, and leukocyte adhesion. In view of the cardioprotective effects of cannabinoids described in recent years, the present study investigated the impact of the non-psychoactive phytocannabinoids cannabidiol (CBD) and tetrahydrocannabivarin (THCV) on proliferation and migration of human coronary artery smooth muscle cells (HCASMC) and on inflammatory markers in human coronary artery endothelial cells (HCAEC). In HCASMC, CBD and THCV at nontoxic concentrations exhibited inhibitory effects on platelet-derived growth factor-triggered proliferation (CBD) and migration (CBD, THCV). When interleukin (IL)-1ß- and lipopolysaccharide (LPS)-stimulated HCAEC were examined, both cannabinoids showed a concentration-dependent decrease in the expression of vascular cell adhesion molecule-1 (VCAM-1), which was mediated independently of classical cannabinoid receptors and was not accompanied by a comparable inhibition of intercellular adhesion molecule-1. Further inhibitor experiments demonstrated that reactive oxygen species, p38 mitogen-activated protein kinase activation, histone deacetylase, and nuclear factor κB (NF-κB) underlie IL-1ß- and LPS-induced expression of VCAM-1. In this context, CBD and THCV were shown to inhibit phosphorylation of NF-κB regulators in LPS- but not IL-1ß-stimulated HCAEC. Stimulation of HCAEC with IL-1ß and LPS was associated with increased adhesion of monocytes, which, however, could not be significantly abolished by CBD and THCV. In summary, the results highlight the potential of the non-psychoactive cannabinoids CBD and THCV to regulate inflammation-related changes in HCASMC and HCAEC. Considering their effect on both cell types studied, further preclinical studies could address the use of CBD and THCV in drug-eluting stents for coronary interventions.
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Canabidiol , Canabinoides , Humanos , Vasos Coronários/metabolismo , Lipopolissacarídeos/farmacologia , Células Endoteliais/metabolismo , Molécula 1 de Adesão de Célula Vascular , NF-kappa B/metabolismo , Canabinoides/farmacologia , Canabidiol/farmacologia , Inflamação , Músculo Liso/metabolismoRESUMO
Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.
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Carcinoma de Células Escamosas , Melanoma , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Carcinoma de Células Escamosas/patologia , Glicosídeos/farmacologia , Apoptose , Linhagem Celular Tumoral , Melanoma/patologia , Mitocôndrias/metabolismo , Complexo I de Transporte de Elétrons/metabolismoRESUMO
Despite the well-described anticarcinogenic effects of endocannabinoids, the influence of the endocannabinoid system on tumor angiogenesis is still debated. In the present study, conditioned medium (CM) from A549 and H358 lung cancer cells treated with ascending concentrations of the monoacylglycerol lipase (MAGL) inhibitor JZL184 and 2-arachidonoylglycerol (2-AG), a prominent MAGL substrate, caused a concentration-dependent reduction in human umbilical vein endothelial cell (HUVEC) migration and tube formation compared with CM from vehicle-treated cancer cells. Comparative experiments with MAGL inhibitors JW651 and MJN110 showed the same results. On the other hand, the angiogenic properties of HUVECs were not significantly altered by direct stimulation with JZL184 or 2-AG or by exposure to CM of JZL184- or 2-AG-treated non-cancerous bronchial epithelial cells (BEAS-2B). Inhibition of HUVEC migration and tube formation by CM of JZL184- and 2-AG-treated A549 cells was abolished in the presence of the CB1 antagonist AM-251. Increased release of tissue inhibitor of metalloproteinase-1 (TIMP-1) from JZL184- or 2-AG-stimulated A549 or H358 cells was shown to exert an antiangiogenic effect on HUVECs, as confirmed by siRNA experiments. In addition, JZL184 caused a dose-dependent regression of A549 tumor xenografts in athymic nude mice, which was associated with a decreased number of CD31-positive cells and upregulation of TIMP-1-positive cells in xenograft tissue. In conclusion, our data suggest that elevation of 2-AG by MAGL inhibition leads to increased release of TIMP-1 from lung cancer cells, which mediates an antiangiogenic effect on endothelial cells.
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Neoplasias Pulmonares , Inibidor Tecidual de Metaloproteinase-1 , Camundongos , Animais , Humanos , Monoacilglicerol Lipases , Células Endoteliais , Camundongos Nus , Monoglicerídeos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
BACKGROUND: Recent studies suggest that low-dose acetylsalicylic acid (ASA) can lower pregnancy-associated morbidity. METHODS: This review is based on pertinent publications that were retrieved by a selective search in PubMed, with special attention to systematic reviews, metaanalyses, and randomized controlled trials. RESULTS: Current meta-analyses document a reduction of the risk of the occurrence of pre-eclampsia (RR 0.85, NNT 50), as well as beneficial effects on the rates of preterm birth (RR 0.80, NNT 37), fetal growth restriction (RR 0.82, NNT 77), and perinatal death (RR 0.79, NNT 167). Moreover, there is evidence that ASA raises the rate of live births after a prior spontaneous abortion, while also lowering the rate of spontaneous preterm births (RR 0.89, NNT 67). The prerequisites for therapeutic success are an adequate ASA dose, early initiation of ASA, and the identification of women at risk of pregnancy-associated morbidity. Side effects of treatment with ASA in this patient group are rare and mainly involve bleeding in connection with the pregnancy (RR 0.87, NNH 200). CONCLUSION: ASA use during pregnancy has benefits beyond reducing the risk of pre-eclampsia. The indications for taking ASA during pregnancy may be extended at some point in the future; at present, in view of the available evidence, it is still restricted to high-risk pregnancies.
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Pré-Eclâmpsia , Nascimento Prematuro , Gravidez , Recém-Nascido , Feminino , Humanos , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/prevenção & controle , Aspirina/uso terapêutico , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/prevenção & controle , Retardo do Crescimento Fetal/tratamento farmacológico , Retardo do Crescimento Fetal/prevenção & controleRESUMO
The endocannabinoid system has been shown to be involved in various skin functions, such as melanogenesis and the maintenance of redox balance in skin cells exposed to UV radiation, as well as barrier functions, sebaceous gland activity, wound healing and the skin's immune response. In addition to the potential use of cannabinoids in the treatment and prevention of skin cancer, cannabinoid compounds and derivatives are of interest as potential systemic and topical applications for the treatment of various inflammatory, fibrotic and pruritic skin conditions. In this context, cannabinoid compounds have been successfully tested as a therapeutic option for the treatment of androgenetic alopecia, atopic and seborrhoeic dermatitis, dermatomyositis, asteatotic and atopic eczema, uraemic pruritis, scalp psoriasis, systemic sclerosis and venous leg ulcers. This review provides an insight into the current literature on cannabinoid compounds as potential medicines for the treatment of skin diseases.
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Canabinoides , Dermatopatias , Neoplasias Cutâneas , Humanos , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Dermatopatias/tratamento farmacológico , Pele , Prurido , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.
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Therapeutic drug monitoring of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) is based on a complex procedure and is therefore not possible in most laboratories, especially in emergency cases. This work addresses the question of whether therapeutic drug monitoring of nabiximols can be performed using an immunological urine-based test system for cannabinoid abuse. Seventeen patients with multiple sclerosis were included in this study. Administered doses of nabiximols were correlated with immunologically determined urine concentrations of cannabinoids using the DRITM Cannabinoid (THC) Assay. Significant correlations with the administered nabiximols doses were found for creatinine-normalized urine concentrations of cannabinoids without (r = 0.675; p = 0.0015) and after (r = 0.650; p = 0.0044) hydrolysis, as well as for gas-chromatography-coupled mass spectrometry (GC/MS)-measured concentrations of the THC metabolite 11-nor-9-carboxy-Δ9-THC (THC-COOH) in urine samples (r = 0.571; p = 0.0084) by Pearson's correlation. In addition, doses were significantly correlated with plasma THC-COOH concentrations (r = 0.667; p = 0.0017) measured by GC/MS. Simple immunological cannabinoid measurements in urine samples could provide an estimate of nabiximols dosage, although the correlations obtained here were weak because of the small number of patients observed. Longitudinal monitoring of individual patients is expected to exhibit good results of therapeutic drug monitoring of nabiximols.
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Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.
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Depending on their composition, plastics have a cytotoxic potential that needs to be evaluated before they are used in dentistry, e.g., as orthodontic removable appliances. Relevant guidelines set out requirements that a potential new resin in the medical field must meet, with a wide scope for experimental design. In the present study, test specimens of different geometries consisting of varying polymers (Orthocryl®, Orthocryl® LC, Loctite® EA 9483, Polypropylene) were soaked for different periods of time, then transferred to cell culture medium for 24 h, which was subsequently used for 24-h cultivation of A549 cells, followed by cytotoxicity assays (WST-1, Annexin V-FITC-propidium iodide (PI) flow cytometry). In this context, a reduction in the cytotoxic effect of the eluates of test specimens prepared from Orthocryl® LC and Loctite® EA 9483 was particularly evident in the Annexin V-FITC-PI assay when the soaking time was extended to 48 h and 168 h, respectively. Consistent with this, a reduced release of potentially toxic monomers into the cell culture medium, as measured by gas chromatography-mass spectrometry, was observed when the prior soaking time of test specimens of all geometries was extended. Remarkably, a significant increase in cytotoxic effect was observed in the WST-1 assay, which was accompanied by a higher release of monomers when the thickness of the test sample was increased from 0.5 to 1.0 mm, although an elution volume adapted to the surface area was used. However, further increasing the thickness to 3.0 mm did not lead to an increase in the observed cytotoxicity or monomer release. Test specimens made of polypropylene showed no toxicity under all test specimen sizes and soaking time conditions. Overall, it is recommended to perform toxicity studies of test specimens using different geometries and soaking times. Thereby, the influence of the different specimen thicknesses should also be considered. Finally, an extension of the test protocols proposed in ISO 10993-5:2009 should be considered, e.g., by flow cytometry or monomer analysis as well as fixed soaking times.
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Polipropilenos , Água , Teste de Materiais/métodos , Metacrilatos , Metilmetacrilatos/químicaRESUMO
Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and ß-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.
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Glioblastoma , Arginina/metabolismo , Autofagia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes , Glioblastoma/genética , HumanosRESUMO
Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.
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Antineoplásicos , Glicosídeos , Animais , Antineoplásicos/farmacocinética , Técnicas de Cultura de Células , Cromatografia Líquida/métodos , Humanos , Indóis , Camundongos , Oximas , Oxindóis , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
Drugs targeting the endocannabinoid system are of interest as potential systemic chemotherapeutic treatments and for palliative care in cancer. In this context, cannabinoid compounds have been successfully tested as a systemic therapeutic option in preclinical models over the past decades. Recent findings have suggested an essential function of the endocannabinoid system in the homeostasis of various skin functions and indicated that cannabinoids could also be considered for the treatment and prophylaxis of tumour diseases of the skin. Cannabinoids have been shown to exert their anticarcinogenic effects at different levels of skin cancer progression, such as inhibition of tumour growth, proliferation, invasion and angiogenesis, as well as inducing apoptosis and autophagy. This review provides an insight into the current literature on cannabinoid compounds as potential pharmaceuticals for the treatment of melanoma and squamous cell carcinoma.
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Silmitasertib (CX-4945) is currently being investigated in clinical trials against various types of cancer. The U.S. Food and Drug Administration (FDA) has already granted orphan drug designation to the compound for the treatment of advanced cholangiocarcinoma, medulloblastoma, and biliary tract cancer. Silmitasertib inhibits the serine/threonine protein kinase CK2, which exerts a proliferation-promoting and anti-apoptotic effect on cancer cells. In view of current and future applications, the measurement of silmitasertib levels in plasma is expected to play an important role in the evaluation of therapeutic and toxic concentrations in cancer patients. In the present work, we therefore present an LC-MS/MS method for the quantification of silmitasertib in human plasma. Using a simple liquid-liquid extraction with ethyl acetate and a mixture of n-hexane and ethyl acetate, this method can be performed in any laboratory with mass spectrometry. The validation was carried out according to the FDA guideline.