RESUMO
Malignant mesothelioma (MM) is a currently incurable, aggressive cancer derived from mesothelial cells, most often resulting from asbestos exposure. The current first-line treatment in unresectable MM is cisplatin/pemetrexed, which shows very little long-term effectiveness, necessitating research for novel therapeutic interventions. The existing chemotherapies often act on the cytoskeleton, including actin filaments and microtubules, but recent advances indicate the 'fourth' form consisting of the family of septins, representing a novel target. The septin inhibitor forchlorfenuron (FCF) and FCF analogs inhibit MM cell growth in vitro, but at concentrations which are too high for clinical applications. Based on the reported requirement of the chloride group in the 2-position of the pyridine ring of FCF for MM cell growth inhibition and cytotoxicity, we systematically investigated the importance (cell growth-inhibiting capacity) of the halogen atoms fluorine, chlorine, bromine and iodine in the 2- or 3-position of the pyridine ring. The MM cell lines ZL55, MSTO-211H, and SPC212, and-as a control-immortalized Met-5A mesothelial cells were used. The potency of the various halogen substitutions in FCF was mostly correlated with the atom size (covalent radius); the small fluoride analogs showed the least effect, while the largest one (iodide) most strongly decreased the MTT signals, in particular in MM cells derived from epithelioid MM. In the latter, the strongest effects in vitro were exerted by the 2-iodo and, unexpectedly, the 2-trifluoromethyl (2-CF3) FCF analogs, which were further tested in vivo in mice. However, FCF-2-I and, more strongly, FCF-2-CF3 caused rapidly occurring strong symptoms of systemic toxicity at doses lower than those previously obtained with FCF. Thus, we investigated the effectiveness of FCF (and selected analogs) in vitro in MM cells which were first exposed to cisplatin. The slowly appearing population of cisplatin-resistant cells was still susceptible to the growth-inhibiting/cytotoxic effect of FCF and its analogs, indicating that cisplatin and FCF target non-converging pathways in MM cells. Thus, a combination therapy of cisplatin and FCF (analogs) might represent a new avenue for the treatment of repopulating chemo-resistant MM cells in this currently untreatable cancer.
Assuntos
Antineoplásicos , Mesotelioma Maligno , Mesotelioma , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/metabolismo , Cisplatino/farmacologia , Halogênios/metabolismo , Mesotelioma/tratamento farmacológico , Camundongos , Compostos de Fenilureia/farmacologia , Piridinas , Septinas/metabolismoRESUMO
Human septins comprise a family of 13 genes that encode conserved GTP-binding proteins. They form nonpolar complexes, which assemble into higher-order structures, such as bundles, scaffolding structures, or rings. Septins are counted among the cytoskeletal elements. They interact with the actin and microtubule networks and can bind to membranes. Many cellular functions with septin participation have been described in the literature, including cytokinesis, motility, forming of scaffolding platforms or lateral diffusion barriers, vesicle transport, exocytosis, and recognition of micron-scale curvature. Septin dysfunction has been implicated in diverse human pathologies, including neurodegeneration and tumorigenesis. Moreover, septins are thought to affect the outcome of host-microbe interactions. Implication of septins has been demonstrated in fungal, bacterial, and viral infections. Knowledge on the precise function of a particular septin in the different steps of the virus infection and replication cycle is still limited. Published data for vaccinia virus (VACV), hepatitis C virus (HCV), influenza A virus (H1N1 and H5N1), human herpesvirus 8 (HHV-8), and Zika virus (ZIKV), all of major concern for public health, will be discussed here.
RESUMO
Malignant mesothelioma (MM) is one of the most aggressive cancer types with a patient's life expectancy of typically less than one year upon diagnosis. The urgency of finding novel therapeutic approaches to treat mesothelioma is evident. Here we tested the effect of the plant-growth regulator forchlorfenuron (FCF), an inhibitor of septin function(s) in mammalian cells, on the viability and proliferation of MM cell lines, as well as other tumor cell lines derived from lung, prostate, colon, ovary, cervix and breast. Exposure to FCF strongly inhibited proliferation of human and mouse (most efficiently epithelioid) MM cells and all other tumor cells in a concentration-dependent manner and led to cell cycle arrest and cell death. The role of septin 7 (SEPT7), a presumably essential target of FCF in MM cells was confirmed by an shRNA strategy. FCF was robustly inhibiting tumor cell growth in vitro at low micromolar (IC50: ≈20-60µM) concentrations and more promisingly also in vivo. Initial experiments with FCF analogous revealed the importance of FCF's chloride group for efficient cell growth inhibition. FCF's rather low systemic toxicity might warrant for an extended search for other related and possibly more potent FCF analogues to target MM and putatively other septin-dependent tumors.
RESUMO
The Ca2+-binding protein parvalbumin (PV) and mitochondria play important roles in Ca2+ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.
Assuntos
Células Epiteliais/citologia , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Parvalbuminas/metabolismo , Animais , Sinalização do Cálcio , Tamanho Celular , Cães , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Células Madin Darby de Rim Canino , Mitocôndrias/metabolismo , MitofagiaRESUMO
BACKGROUND: Calretinin is the most widespread positive marker for the immunohistochemical identification of malignant mesothelioma (MM) and was proposed to serve as a blood-based biomarker. Functionally, evidence has accumulated that calretinin might be implicated in MM tumorigenesis. We aimed to identify calretinin (CR; Calb2) in murine MM and reactive mesothelial cells in granuloma from asbestos-exposed NF2+/- mice, a line heterozygous for the tumor suppressor merlin (NF2), used as a mouse MM model. Additionally, we sought to ascertain the presence of calretinin in MM cell lines from other mouse strains. We also intended to investigate the role of calretinin in mesotheliomagenesis by comparing the survival of asbestos-exposed NF2+/- and NF2+/-CR-/- mice. METHODS: NF2+/- and NF2+/-CR-/- mice, both lines on a C57Bl/6J background, were exposed to asbestos following an established protocol. Tumor histology and asbestos-induced mortality were assessed. MM and granuloma from NF2+/- mice were analyzed with immunohistochemical methods for calretinin expression. Levels of Calb2 mRNA and calretinin expression in tumors and MM cell lines of various mouse strains were determined by RT-qPCR and Western blot analysis, respectively. RESULTS: No expression of calretinin at the protein level was detected, neither in MM from NF2+/- mice, NF2+/- MM-derived cell lines nor immortalized mesothelial cells of mouse origin. At the mRNA level we detected Calb2 expression in MM cell lines from different mouse strains. Survival of NF2+/- and NF2+/-CR-/- mice exposed to asbestos showed no significant difference in a log-rank (Kaplan-Meier) comparison. CONCLUSIONS: The concomitant determination of calretinin and mesothelin blood levels has been proposed for early detection of human MM. Mouse MM models based on asbestos exposure are assumed to yield helpful information on the time course of appearance of mesothelin and calretinin in the blood of asbestos-treated mice determining the earliest time point for interventions. However, the observed absence of calretinin in MM from NF2+/- mice and derived cell lines, as well as from MM cells from Balb/c and C3H mice likely precludes the use of calretinin as a biomarker for mouse MM. The results also indicate possible species differences with respect to an involvement of calretinin in the formation of MM.
RESUMO
Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines, and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single-nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naïve mice supporting the role of the environment in tumor progression.
Assuntos
Asbesto Crocidolita/efeitos adversos , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , Mesotelioma/genética , Edição de RNA , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal , Animais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Ativação de Macrófagos , Mesotelioma/induzido quimicamente , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Mutação , Fosfoproteínas , Polimorfismo de Nucleotídeo Único , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Biological (or cellular) noise is the random quantitative variability of proteins and other molecules in individual, genetically identical cells. As the result of biological noise in the levels of some transcription factors that determine a cell's differentiation status, differentiated cells may dedifferentiate to a stem cell state given a sufficiently long time period. Here, to provide direct evidence supporting this hypothesis, we used a live-cell monitoring system based on enhanced green fluorescent protein (eGFP) expression to continuously assess the "stemness" of individual human and murine malignant mesothelioma cells over a period of up to 3 months. Re-expression of the transcription factors, the top hierarchical stemness markers Sox2 (SRY-box 2) and Oct4 (octamer-binding transcription factor), monitored as cell eGFP expression was observed in a subpopulation of differentiated eGFP(-) malignant mesothelioma cells. However, we found that this transition was extremely rare. Of note, when it did occur, neighboring cells that were not direct descendants of a newly emerged eGFP(+) stem cell were more likely than non-neighboring cells to also become an eGFP(+) stem cell. This observation suggested a positional effect and led to a clustered "mosaic" reappearance of eGFP(+) stem cells. Moreover, stem cells reappeared even in cell cultures derived from one single differentiated eGFP(-) cell. On the basis of our experimental in vitro and in vivo findings, we developed a tumor growth model to predict the clustered localization of cancer stem cells within a tumor mass.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Artefatos , Produtos Biológicos/metabolismo , Técnicas de Cultura de Células , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50-500µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50-500µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Mentol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismoRESUMO
Sensory neuron subpopulations as well as breast and prostate cancer cells express functional transient receptor potential vanilloid type 1 (TRPV1) ion channels; however little is known how TRPV1 activation leads to biological responses. Agonist-induced activation of TRPV1 resulted in specific spatiotemporal patterns of cytoplasmic Ca2+ signals in breast and prostate cancer-derived cells. Capsaicin (CAPS; 50µM) evoked intracellular Ca2+ oscillations and/or intercellular Ca2+ waves in all cell lines. As evidenced in prostate cancer Du 145 cells, oscillations were largely dependent on the expression of functional TRPV1 channels in the plasma membrane, phospholipase C activation and on the presence of extracellular Ca2+ ions. Concomitant oscillations of the mitochondrial matrix Ca2+ concentration resulted in mitochondria energization evidenced by increased ATP production. CAPS-induced Ca2+ oscillations also occurred in a subset of sensory neurons, yet already at lower CAPS concentrations (1µM). Stimulation of ectopically expressed TRPV1 channels in CAPS-insensitive NIH-3T3 cells didn't provoke CAPS-triggered Ca2+ oscillations; rather it resulted in low-magnitude, long-lasting elevations of the cytosolic Ca2+ concentration. This indicates that sole TRPV1 activation is not sufficient to generate Ca2+ oscillations. Instead the initial TRPV1-mediated signal leads to the activation of the inositol phospholipid pathway. This in turn suffices to generate a biologically relevant frequency-modulated Ca2+ signal.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/metabolismo , Fosfolipases Tipo C/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Linhagem Celular Tumoral , Diterpenos/farmacologia , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células NIH 3T3 , Cultura Primária de Células , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/genética , Gânglio Trigeminal/citologia , Gânglio Trigeminal/efeitos dos fármacos , Fosfolipases Tipo C/genéticaRESUMO
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit ß (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit ß was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit ß levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.
Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Parvalbuminas/metabolismo , Animais , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Citosol/metabolismo , Cães , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Canais Iônicos/metabolismo , Células Madin Darby de Rim Canino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Tamanho Mitocondrial/fisiologia , Células de Purkinje/metabolismo , Proteína Desacopladora 2RESUMO
The short-chain fatty acid butyrate plays an essential role in colonic mucosa homeostasis through the capacity to block the cell cycle, regulate differentiation and to induce apoptosis. The beneficial effect of dietary fibers on preventing colon cancer is essentially mediated through butyrate, derived from luminal fermentation of fibers by intestinal bacteria. In epithelial cells of the colon, both in normal and colon cancer cells, the expression of several genes is positively or negatively regulated by butyrate likely through modulation of histone acetylation and thereby affecting the transcriptional activity of genes. Calretinin (CALB2) is a member of the EF-hand family of Ca(2+)-binding proteins and is expressed in a majority of poorly differentiated colon carcinoma and additionally in mesothelioma of the epithelioid and mixed type. Since CALB2 is one of the genes negatively regulated by butyrate in colon cancer cells and butyrate decreases calretinin protein expression levels in those cells, we investigated whether expression is regulated via putative butyrate-responsive elements (BRE) in the human CALB2 promoter. We identified two elements that act as butyrate-sensitive repressors in all colon cancer cell lines tested (CaCo-2, HT-29, Co-115/3). In contrast, in cells of mesothelial origin, MeT-5A and ZL34, the same two elements do not operate as butyrate-sensitive repressors and calretinin expression levels are insensitive to butyrate indicative of cell type-specific regulation of the CALB2 promoter. Calretinin expression in colon cancer cells is negatively regulated by butyrate via a bipartite BRE flanking the TATA box and this may be linked to butyrate's chemopreventive activity.
Assuntos
Butiratos/farmacologia , Carcinoma/genética , Neoplasias do Colo/genética , Mesotelioma/genética , Regiões Promotoras Genéticas/genética , Elementos de Resposta , Proteína G de Ligação ao Cálcio S100/genética , Sequência de Bases , Células CACO-2 , Calbindina 2 , Carcinoma/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Primers do DNA/química , Células HT29 , Humanos , Mesotelioma/metabolismo , Dados de Sequência Molecular , Proteína G de Ligação ao Cálcio S100/metabolismo , TATA BoxRESUMO
The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.