RESUMO
A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen-Schmidt condensation sequence. Structure-activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues. Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.
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Antineoplásicos/farmacologia , Curcumina/farmacologia , Bases de Mannich/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/análogos & derivados , Curcumina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Bases de Mannich/química , Camundongos , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Relação Estrutura-AtividadeRESUMO
Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-ß1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.
Assuntos
Matriz Extracelular/metabolismo , Proteínas Matrilinas/metabolismo , Músculos/fisiologia , Mioblastos/fisiologia , Necrose/terapia , Animais , Apoptose/genética , Linhagem Celular , Proliferação de Células/genética , Venenos Elapídicos/administração & dosagem , Humanos , Proteínas Matrilinas/genética , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculos/patologia , Necrose/induzido quimicamente , Ratos , Ratos Wistar , Regeneração/genética , Fatores de TempoRESUMO
BACKGROUND: Reperfusion of ischemic myocardium may contribute to substantial cardiac tissue damage, but the addition of iron chelators, zinc or zinc complexes has been shown to prevent heart from reperfusion injury. We investigated the possible beneficial effects of an iron-chelating and zinc-complexing agent, Q50, in rat models of ischemia/reperfusion (I/R)-induced myocardial infarction and on global reversible myocardial I/R injury after heart transplantation. METHODS AND RESULTS: Rats underwent 45-min myocardial ischemia by left anterior descending coronary artery ligation followed by 24h reperfusion. Vehicle or Q50 (10 mg/kg, IV) were given 5 min before reperfusion. In a heart transplantation model, donor rats received vehicle or Q50 (30 mg/kg, IV) 1h before the onset of ischemia. In myocardial infarcted rats, increased left ventricular end-systolic and end-diastolic volumes were significantly decreased by Q50 post treatment as compared with the sham group. Moreover, in I/R rat hearts, the decreased dP/dtmax and load-independent contractility parameters were significantly increased after Q50. However, Q50 treatment did not reduce infarct size or have any effect on increased plasma cardiac troponin-T-levels. In the rat model of heart transplantation, 1h after reperfusion, decreased left ventricular systolic pressure, dP/dt(max), dP/dt(min) and myocardial ATP content were significantly increased and myocardial protein expression of superoxide dismutase-1 was upregulated after Q50 treatment. CONCLUSIONS: In 2 experimental models of I/R, administration of Q50 improved myocardial function. Its mechanisms of action implicate in part the restoration of myocardial high-energy phosphates and upregulation of antioxidant enzymes.
Assuntos
Quelantes de Ferro/farmacologia , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Zinco , Animais , Modelos Animais de Doenças , Masculino , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Fatores de Tempo , Troponina T/sangueRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. RESULTS: In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. CONCLUSION: These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Talidomida/análogos & derivados , Talidomida/farmacologia , Animais , Antineoplásicos/farmacocinética , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chaperonina 60/genética , Chaperonina 60/metabolismo , Dietilnitrosamina , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipídeos/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Talidomida/farmacocinética , Carga Tumoral/efeitos dos fármacosRESUMO
Metabolic reprogramming is one of the main hallmarks of cancer [...].
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BACKGROUND & AIMS: The effects of trypsin on pancreatic ductal epithelial cells (PDECs) vary among species and depend on the localization of proteinase-activated receptor 2 (PAR-2). We compared PAR-2 localization in human and guinea-pig PDECs, and used isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on bicarbonate secretion. METHODS: PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functionally, guinea pig PDECs were studied by microperfusion of isolated ducts, measurements of intracellular pH and intracellular Ca(2+) concentration, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. RESULTS: PAR-2 localized to the apical membrane of human and guinea pig PDECs. Trypsin increased intracellular Ca(2+) concentration and intracellular pH and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were markedly reduced. CONCLUSIONS: Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2-dependent inhibition of the apical anion exchanger and the CFTR Cl(-) channel. This could contribute to the development of chronic pancreatitis by decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts.
Assuntos
Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Ductos Pancreáticos/metabolismo , Pancreatite Crônica/enzimologia , Receptor PAR-2/metabolismo , Tripsina/fisiologia , Animais , Resinas de Troca Aniônica/metabolismo , Ativação Enzimática , Células Epiteliais/metabolismo , Cobaias , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ductos Pancreáticos/citologia , Pancreatite Crônica/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor PAR-2/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsinogênio/metabolismoRESUMO
Recently, we presented evidence that high mitochondrial ATP production is a new therapeutic target for cancer treatment. Using ATP as a biomarker, we isolated the "metabolically fittest" cancer cells from the total cell population. Importantly, ATP-high cancer cells were phenotypically the most aggressive, with enhanced stem-like properties, showing multi-drug resistance and an increased capacity for cell migration, invasion and spontaneous metastasis. In support of these observations, ATP-high cells demonstrated the up-regulation of both mitochondrial proteins and other protein biomarkers, specifically associated with stemness and metastasis. Therefore, we propose that the "energetically fittest" cancer cells would be better able to resist the selection pressure provided by i) a hostile micro-environment and/or ii) conventional chemotherapy, allowing them to be naturally-selected for survival, based on their high ATP content, ultimately driving tumor recurrence and distant metastasis. In accordance with this energetic hypothesis, ATP-high MDA-MB-231 breast cancer cells showed a dramatic increase in their ability to metastasize in a pre-clinical model in vivo. Conversely, metastasis was largely prevented by treatment with an FDA-approved drug (Bedaquiline), which binds to and inhibits the mitochondrial ATP-synthase, leading to ATP depletion. Clinically, these new therapeutic approaches could have important implications for preventing treatment failure and avoiding cancer cell dormancy, by employing ATP-depletion therapy, to target even the fittest cancer cells.
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BACKGROUND: Cytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) with potent anticancer activities were synthesized. RESULTS: Cytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs in vitro, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis. CONCLUSIONS: Because of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.
Assuntos
Ácidos Graxos Insaturados/farmacologia , Neoplasias/patologia , Estresse Oxidativo/efeitos dos fármacos , Talidomida/farmacologia , Animais , Linhagem Celular Tumoral , Sinergismo Farmacológico , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Homeostase , Humanos , Neoplasias/metabolismo , Talidomida/análogos & derivados , Peixe-ZebraRESUMO
Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence, distant metastasis and drug-resistance, in the vast majority of cancer patients. Therefore, there is an urgent need to identify new drugs that can target and eradicate CSCs. To identify new molecular targets that are unique to CSCs, we previously compared MCF7 2D-monolayers with 3D-mammospheres, which are enriched in CSCs. We observed that 25 mitochondrial-related proteins were >100-fold over-expressed in 3D-mammospheres. Here, we used these 25 proteins to derive short gene signatures to predict distant metastasis (in N=1,395 patients) and tumor recurrence (in N=3,082 patients), by employing a large collection of transcriptional profiling data from ER(+) breast cancer patients. This analysis resulted in a 4-gene signature for predicting distant metastasis, with a hazard ratio of 1.91-fold (P=2.2e-08). This provides clinical evidence to support a role for CSC mitochondria in metastatic dissemination. Next, we employed a panel of mitochondrial inhibitors, previously shown to target mitochondria and selectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells. Remarkably, these five mitochondrial inhibitors had only minor effects or no effect on MDA-MB-231 tumor formation, but preferentially and selectively inhibited tumor cell metastasis, without causing significant toxicity. Mechanistically, all five mitochondrial inhibitors have been previously shown to induce ATP-depletion in cancer cells. Since 3 of these 5 inhibitors were designed to target the large mitochondrial ribosome, we next interrogated whether genes encoding the large mitochondrial ribosomal proteins (MRPL) also show prognostic value in the prediction of distant metastasis in both ER(+) and ER(-) breast cancer patients. Interestingly, gene signatures composed of 6 to 9 MRPL mRNA-transcripts were indeed sufficient to predict distant metastasis, tumor recurrence and Tamoxifen resistance. These gene signatures could be useful as companion diagnostics to assess which patients may benefit most from anti-mito-ribosome therapy. Overall, our studies provide the necessary proof-of-concept, and in vivo functional evidence, that mitochondrial inhibitors can successfully and selectively target the biological process of cancer cell metastasis. Ultimately, we envision that mitochondrial inhibitors could be employed to develop new treatment protocols, for clinically providing metastasis prophylaxis, to help prevent poor clinical outcomes in cancer patients.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Mitocôndrias/efeitos dos fármacos , Ribossomos Mitocondriais/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/genética , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Compostos Organofosforados/farmacologia , Compostos Organofosforados/uso terapêutico , Prognóstico , Estudo de Prova de Conceito , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Esferoides CelularesRESUMO
Here, we describe the chemical synthesis and biological activity of a new Doxycycline derivative, designed specifically to more effectively target cancer stem cells (CSCs). In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. First, we determined the IC50 of Doxy-Myr using the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show antibiotic activity, against Escherichia coli and Staphylococcus aureus. Interestingly, other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently inhibits tumor cell metastasis in vivo, with little or no toxicity. In summary, by using 9-amino-Doxycycline as a scaffold, here we have designed new chemical entities for their further development as anti-cancer agents. These compounds selectively target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic resistance. Taken together, our current studies provide proof-of-principle, that existing FDA-approved drugs can be further modified and optimized, to successfully target the anchorage-independent growth of CSCs and to prevent the process of spontaneous tumor cell metastasis.
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Using proteomics analysis, we previously compared MCF7 breast cancer cells grown as 3D tumor spheres, with the same cell line grown as monolayers. Our results indicated that during 3D anchorage-independent growth, the cellular machinery associated with i) mitochondrial biogenesis and ii) ribosomal biogenesis, were both significantly increased. Here, for simplicity, we refer to these two new oncogenic hallmarks as "mito-stemness" and "ribo-stemness" features. We have now applied this same type of strategy to begin to understand how fibroblasts and MCF7 breast cancer cells change their molecular phenotype, when they are co-cultured together. We have previously shown that MCF7-fibroblast co-cultures are a valuable model of resistance to apoptosis induced by hormonal therapies, such as Tamoxifen and Fulvestrant. Here, we directly show that these mixed co-cultures demonstrate the induction of mito-stemness and ribo-stemness features, likely reflecting a mechanism for cancer cells to increase their capacity for accumulating biomass. In accordance with the onset of a stem-like phenotype, KRT19 (keratin 19) was induced by ~6-fold during co-culture. KRT19 is a well-established epithelial CSC marker that is used clinically to identify metastatic breast cancer cells in sentinel lymph node biopsies. The potential molecular therapeutic targets that we identified by label-free proteomics of MCF7-fibroblast co-cultures were then independently validated using a bioinformatics approach. More specifically, we employed publically-available transcriptional profiling data derived from primary tumor samples from breast cancer patients, which were previously subjected to laser-capture micro-dissection, to physically separate breast cancer cells from adjacent tumor stroma. This allowed us to directly validate that the proteins up-regulated in this co-culture model were also transcriptionally elevated in patient-derived breast cancer cells in vivo. This powerful approach for target identification and translational validation, including the use of patient outcome data, can now be applied to other tumor types as well, to validate new therapeutic targets that are more clinically relevant, for patient benefit. Moreover, we discuss the therapeutic implications of these findings for new drug development, drug repurposing and Tamoxifen-resistance, to effectively target mito-stemness and ribo-stemness features in breast cancer patients. We also discuss the broad implications of this "organelle biogenesis" approach to cancer therapy.
Assuntos
Neoplasias da Mama , Reprogramação Celular/fisiologia , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/fisiologia , Ribossomos/metabolismo , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Fibroblastos/metabolismo , Humanos , Células MCF-7 , Biogênese de Organelas , Fenótipo , ProteômicaRESUMO
Elevated mitochondrial biogenesis and/or metabolism are distinguishing features of cancer cells, as well as Cancer Stem Cells (CSCs), which are involved in tumor initiation, metastatic dissemination, and therapy resistance. In fact, mitochondria-impairing agents can be used to hamper CSCs maintenance and propagation, toward better control of neoplastic disease. Tri-Phenyl-Phosphonium (TPP)-based mitochondrially-targeted compounds are small non-toxic and biologically active molecules that are delivered to and accumulated within the mitochondria of living cells. Therefore, TPP-derivatives may represent potentially "powerful" candidates to block CSCs. Here, we evaluate the metabolic and biological effects induced by the TPP-derivative, termed Dodecyl-TPP (d-TPP) on breast cancer cells. By employing the 3D mammosphere assay in MCF-7 cells, we demonstrate that treatment with d-TPP dose-dependently inhibits the propagation of breast CSCs in suspension. Also, d-TPP targets adherent "bulk" cancer cells, by decreasing MCF-7 cell viability. The analysis of metabolic flux using Seahorse Xfe96 revealed that d-TPP potently inhibits the mitochondrial oxygen consumption rate (OCR), while simultaneously shifting cell metabolism toward glycolysis. Thereafter, we exploited this ATP depletion phenotype and strict metabolic dependency on glycolysis to eradicate the residual glycolytic CSC population, by using additional metabolic stressors. More specifically, we applied a combination strategy based on treatment with d-TPP, in the presence of a selected panel of natural and synthetic compounds, some of which are FDA-approved, that are known to behave as glycolysis (Vitamin C, 2-Deoxy-Glucose) and OXPHOS (Doxycyline, Niclosamide, Berberine) inhibitors. This two-hit scheme effectively decreased CSC propagation, at concentrations of d-TPP toxic only for cancer cells, but not for normal cells, as evidenced using normal human fibroblasts (hTERT-BJ1) as a reference point. Taken together, d-TPP halts CSCs propagation and targets "bulk" cancer cells, without eliciting the relevant undesirable off-target effects in normal cells. These observations pave the way for further exploring the potential of TPP-based derivatives in cancer therapy. Moreover, TPP-based compounds should be investigated for their potential to discriminate between "normal" and "malignant" mitochondria, suggesting that distinct biochemical, and metabolic changes in these organelles could precede specific normal or pathological phenotypes. Lastly, our data validate the manipulation of the energetic machinery as useful tool to eradicate CSCs.
RESUMO
Tri-phenyl-phosphonium (TPP) is a non-toxic chemical moiety that functionally behaves as a mitochondrial targeting signal (MTS) in living cells. Here, we explored the hypothesis that TPP-related compounds could be utilized to inhibit mitochondria in cancer stem cells (CSCs). We randomly selected 9 TPP-related compounds for screening, using an ATP depletion assay. Based on this approach, five compounds were identified as "positive hits"; two had no detectable effect on ATP production. Remarkably, this represents a >50% hit rate. We validated that the five positive hit compounds all inhibited oxygen consumption rates (OCR), using the Seahorse XFe96 metabolic flux analyzer. Interestingly, these TPP-related compounds were non-toxic and had little or no effect on ATP production in normal human fibroblasts, but selectively targeted adherent "bulk" cancer cells. Finally, these positive hit compounds also inhibited the propagation of CSCs in suspension, as measured functionally using the 3D mammosphere assay. Therefore, these TPP-related compounds successfully inhibited anchorage-independent growth, which is normally associated with a metastatic phenotype. Interestingly, the most effective molecule that we identified contained two TPP moieties (i.e., bis-TPP). More specifically, 2-butene-1,4-bis-TPP potently and selectively inhibited CSC propagation, with an IC-50 < 500 nM. Thus, we conclude that the use of bis-TPP, a "dimeric" mitochondrial targeting signal, may be a promising new approach for the chemical eradication of CSCs. Future studies on the efficacy of 2-butene-1,4-bis-TPP and its derivatives are warranted. In summary, we show that TPP-related compounds provide a novel chemical strategy for effectively killing both i) "bulk" cancer cells and ii) CSCs, while specifically minimizing or avoiding off-target side-effects in normal cells. These results provide the necessary evidence that "normal" mitochondria and "malignant" mitochondria are truly biochemically distinct, removing a significant barrier to therapeutically targeting cancer metabolism.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Trifosfato de Adenosina/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacosRESUMO
Here, we employed a "senolytic" assay system as a screening tool, with the goal of identifying and repurposing FDA-approved antibiotics, for the targeting of the senescent cell population. Briefly, we used two established human fibroblast cell lines (MRC-5 and/or BJ) as model systems to induce senescence, via chronic treatment with a DNA-damaging agent, namely BrdU (at a concentration of 100 µM for 8 days). Cell viability was then monitored by using the SRB assay, to measure protein content. As a consequence of this streamlined screening strategy, we identified Azithromycin and Roxithromycin as two novel clinically-approved senolytic drugs. However, Erythromycin - the very closely-related parent compound - did not show any senolytic activity, highlighting the dramatic specificity of these interactions. Interestingly, we also show that Azithromycin treatment of human fibroblasts was indeed sufficient to strongly induce both aerobic glycolysis and autophagy. However, the effects of Azithromycin on mitochondrial oxygen consumption rates (OCR) were bi-phasic, showing inhibitory activity at 50 µM and stimulatory activity at 100 µM. These autophagic/metabolic changes induced by Azithromycin could mechanistically explain its senolytic activity. We also independently validated our findings using the xCELLigence real-time assay system, which measures electrical impedance. Using this approach, we see that Azithromycin preferentially targets senescent cells, removing approximately 97% of them with great efficiency. This represents a near 25-fold reduction in senescent cells. Finally, we also discuss our current results in the context of previous clinical findings that specifically document the anti-inflammatory activity of Azithromycin in patients with cystic fibrosis - a genetic lung disorder that results in protein mis-folding mutations that cause protein aggregation.
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Azitromicina/farmacologia , Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Roxitromicina/farmacologia , Antibacterianos/farmacologia , Autofagia , Bioensaio , Linhagem Celular , Fibroblastos/fisiologia , HumanosRESUMO
Here, we wish to propose a new systematic approach to cancer therapy, based on the targeting of mitochondrial metabolism, especially in cancer stem cells (CSCs). In the future, we envision that anti-mitochondrial therapy would ultimately be practiced as an add-on to more conventional therapy, largely for the prevention of tumor recurrence and cancer metastasis. This mitochondrial based oncology platform would require a panel of FDA-approved therapeutics (e.g. Doxycycline) that can safely be used to inhibit mitochondrial OXPHOS and/or biogenesis in CSCs. In addition, new therapeutics that target mitochondria could also be developed, to optimize their ability to eradicate CSCs. Finally, in this context, mitochondrial-based biomarkers (i.e. "Mito-signatures") could be utilized as companion diagnostics, to identify high-risk cancer patients at diagnosis, facilitating the early detection of tumor recurrence and the prevention of treatment failure. In summary, we suggest that new clinical trials are warranted to test and possibly implement this emerging treatment strategy, in a variety of human cancer types. This general approach, using FDA-approved antibiotics to target mitochondria, was effective in killing CSCs originating from many different cancer types, including DCIS, breast (ER(+) and ER(-)), prostate, ovarian, lung and pancreatic cancers, as well as melanoma and glioblastoma, among others. Thus, we propose the term MITO-ONC-RX, to describe this anti-mitochondrial platform for targeting CSCs. The use of re-purposed FDA-approved drugs will undoubtedly help to accelerate the clinical evaluation of this approach, as these drugs can move directly into Phase II clinical trials, saving considerable amounts of time (10-15 y) and billions in financial resources.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Mitomicina/farmacologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológicoRESUMO
PURPOSE: The main function of the lacrimal gland is to produce the most aqueous component of the tear film covering the surfaces of the cornea and the conjunctiva. Studies have been conducted that characterize the mixed fluid and protein secretion of isolated acini, but no methods have been developed to characterize lacrimal gland ductal cell (LGDC) secretion. Secretory mechanisms of ductal epithelia may play physiological roles in the maintenance of the standard environments for the cornea and the conjunctiva. METHODS: In this study, the authors developed a rapid method to isolate large quantities of intact lacrimal ducts. The preparation of isolated intact lacrimal gland ducts for the first time enabled the performance of real-time functional experiments on cleaned ducts. Electron microscopy and fluorescence measurements were used to evaluate the viability of lacrimal ducts. RESULTS: Fluorescence measurements showed that LGDCs express functionally active Na(+)/H(+) exchanger (NHE) and Cl(-)/HCO(3)(-) exchanger (AE). Parasympathomimetic stimulation by carbachol stimulated NHE and AE through the elevation of intracellular calcium concentration. This mechanism can play a role in the regulation of ion and water secretion by LGDCs. CONCLUSIONS: The authors have described a lacrimal gland duct isolation technique in which the intact ducts remain viable and the role of duct cells in tear film secretion can be characterized. These data combined with the novel isolation facilitated understanding of the regulation mechanisms of ductal cell secretion at cellular and molecular levels under normal and pathologic conditions.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Aparelho Lacrimal/metabolismo , Técnicas de Cultura de Órgãos/métodos , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Ácidos/metabolismo , Álcalis/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Carbacol/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/ultraestrutura , Concentração de Íons de Hidrogênio , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Parassimpatomiméticos/farmacologia , CoelhosRESUMO
Here, we used MCF7 cells as a model system to interrogate how MYC/RAS co-operativity contributes to metabolic flux and stemness in breast cancer cells. We compared the behavior of isogenic MCF7 cell lines transduced with c-Myc or H-Ras (G12V), either individually or in combination. Cancer stem cell (CSC) activity was measured using the mammosphere assay. c-Myc augmented both mammosphere formation and mitochondrial respiration, without any effects on glycolytic flux. In contrast, H-Ras (G12V) synergistically augmented both mammosphere formation and glycolysis, but only in combination with c-Myc, directly demonstrating MYC/RAS co-operativity. As c-Myc is known to exert its effects, in part, by stimulating mitochondrial biogenesis, we next examined the effects of another stimulus known to affect mitochondrial biogenesis, i.e. ROS production. To pharmacologically induce oxidative stress, we used Rotenone (a mitochondrial inhibitor) to target mitochondrial complex I. Treatment with Rotenone showed bi-phasic effects; low-dose Rotenone (1 to 2.5 nM) elevated mammosphere formation, while higher doses (10 to 100 nM) were inhibitory. Importantly, the stimulatory effects of Rotenone on CSC propagation were blocked using a mitochondrial-specific anti-oxidant, namely Mito-tempo. Thus, "mild" mitochondrial oxidative stress, originating at Complex I, was sufficient to pheno-copy the effects of c-Myc, effectively promoting CSC propagation. To validate the idea that mitochondrial biogenesis is required to stimulate CSC propagation, we employed Doxycycline, a well-established inhibitor of mitochondrial protein translation. Treatment with Doxycycline was indeed sufficient to block the stimulatory effects of H-Ras (G12V), c-Myc, and Rotenone on CSC propagation. As such, Doxycycline provides a strong rationale for designing new therapeutics to target mitochondrial biogenesis, suggesting a new "mutation-independent" approach to cancer therapy. In support of this notion, most currently successful anti-cancer agents therapeutically target "cell phenotypes", such as increased cell proliferation, rather than specific genetic mutations. Remarkably, we demonstrated that Doxycycline inhibits the effects of diverse oncogenic stimuli, of both i) genetic (MYC/RAS) and ii) environmental (Rotenone) origins. Finally, we discuss the advantages of our "Proteomics-to-Genomics (PTG)" approach for in silico validation of new biomarkers and novel drug targets. In this context, we developed a new Myc-based Mito-Signature consisting of 3 mitochondrial genes (HSPD1; COX5B; TIMM44) for effectively predicting tumor recurrence (HR=4.69; p=2.4e-08) and distant metastasis (HR=4.94; p=2.8e-07), in ER(+) in breast cancer patients. This gene signature could serve as a new companion diagnostic for the early prediction of treatment failure in patients receiving hormonal therapy.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Biogênese de Organelas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas ras/metabolismo , Neoplasias da Mama/metabolismo , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Transcriptoma , Proteínas ras/genéticaRESUMO
Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or "suspended animation".In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial "power" (OXPHOS) and ii) elevated CSC activity.Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules "Mitoflavoscins".For example, DPI is ~30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.