RESUMO
The nonphysiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels affect therapeutic response by performing drug screening in human plasma-like medium. We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that recently failed in phase III clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response and how incorporation of human-specific physiological nutrient medium might help identify compounds whose efficacy could be influenced in humans.
Assuntos
Glicina , Sulfonas , Ácido Úrico , Humanos , Ácido Úrico/metabolismo , Glicina/farmacologia , Glicina/análogos & derivados , Sulfonas/farmacologia , Meios de Cultura , Avaliação Pré-Clínica de Medicamentos/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.
RESUMO
Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.
Assuntos
Ácido Aspártico , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Glicólise , Malatos/metabolismo , Camundongos , Neurônios/fisiologia , Piruvatos/metabolismoRESUMO
The human microbiome is essential for the correct functioning of many host physiological processes, including metabolic regulation and immune responses. Increasing evidence indicates that the microbiome may also influence cancer development, progression, and the response to therapy. Although most studies have focused on the effect of the gut microbiome, many other organs such as the skin, vagina, and lungs harbor their own microbiomes that are different from the gut. Tumor development has been associated with dysbiosis not only in the gut but also in the tissue from which the tumor originated. Furthermore, the intratumoral microbiota has a distinct signature in each tumor type. Here, we review the mechanisms by which the organ-specific microbiome can contribute to carcinogenesis: release of toxins that cause DNA damage and barrier failure; alteration of immune responses to create a local inflammatory or immunosuppressive environment; and regulation of nutrient levels in the tumor microenvironment through metabolite production and consumption. Solving the puzzle of how the microbiome influences the carcinogenesis process and treatment response requires an understanding of the two ways the microbiome can interact with cancer cells and the tumor microenvironment: through systemic effects exerted by the gut microbiota and local effects of the intratumoral microbiota. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Microbiota , Neoplasias , Animais , HumanosRESUMO
BACKGROUND: d-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown. METHODS: To study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery. RESULTS: We found that PHGDH depletion is well tolerated, and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown and impaired serine synthesis, liver and pancreatic functions were normal. Interestingly, diminished PHGDH expression reduced liver serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain, polyunsaturated tails upon PHGDH knockdown. CONCLUSIONS: These results suggest that dietary serine is adequate to support the function of healthy, adult murine tissues, but PHGDH-derived serine supports liver ceramide synthesis and sustains general lipid homeostasis.
RESUMO
The pathology of Charcot-Marie-Tooth (CMT), a disease arising from mutations in different genes, has been associated with an impairment of mitochondrial dynamics and axonal biology of mitochondria. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause several forms of CMT neuropathy, but the pathogenic mechanisms involved remain unclear. GDAP1 is an outer mitochondrial membrane protein highly expressed in neurons. It has been proposed to play a role in different aspects of mitochondrial physiology, including mitochondrial dynamics, oxidative stress processes, and mitochondrial transport along the axons. Disruption of the mitochondrial network in a neuroblastoma model of GDAP1-related CMT has been shown to decrease Ca2+ entry through the store-operated calcium entry (SOCE), which caused a failure in stimulation of mitochondrial respiration. In this review, we summarize the different functions proposed for GDAP1 and focus on the consequences for Ca2+ homeostasis and mitochondrial energy production linked to CMT disease caused by different GDAP1 mutations.
Assuntos
Cálcio/metabolismo , Doença de Charcot-Marie-Tooth/etiologia , Doença de Charcot-Marie-Tooth/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Transporte Biológico , Doença de Charcot-Marie-Tooth/patologia , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Mutação , Neurônios/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
Mitochondria undergo frequent morphological changes to control their function. We show here that T-cell intracellular antigens (TIA1b/TIARb) and Hu antigen R (HuR) have antagonistic roles in mitochondrial function by modulating the expression of mitochondrial shaping proteins. Expression of TIA1b/TIARb alters the mitochondrial dynamic network by enhancing fission and clustering, which is accompanied by a decrease in respiration. In contrast, HuR expression promotes fusion and cristae remodeling and increases respiratory activity. Mechanistically, TIA proteins downregulate the expression of optic atrophy 1 (OPA1) protein via switching of the splicing patterns of OPA1 to facilitate the production of OPA1 variant 5 (OPA1v5). Conversely, HuR enhances the expression of OPA1 mRNA isoforms through increasing steady-state levels and targeting translational efficiency at the 3' untranslated region. Knockdown of TIA1/TIAR or HuR partially reversed the expression profile of OPA1, whereas knockdown of OPA1 or overexpression of OPA1v5 provoked mitochondrial clustering. Middle-term expression of TIA1b/TIARb triggers reactive oxygen species production and mitochondrial DNA damage, which is accompanied by mitophagy, autophagy, and apoptosis. In contrast, HuR expression promotes mitochondrion-dependent cell proliferation. Collectively, these results provide molecular insights into the antagonistic functions of TIA1b/TIARb and HuR in mitochondrial activity dynamics and suggest that their balance might contribute to mitochondrial physiopathology.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Expressão Gênica/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proliferação de Células , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/genética , Antígeno-1 Intracelular de Células TRESUMO
GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca2+ levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca2+ release from de ER and Ca2+ inflow through SOCE in neuroblastoma cells result in a Ca2+-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca2+ levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca2+ mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Linhagem Celular Tumoral , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/- mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Assuntos
Antiporters/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antiporters/genética , Canais de Cálcio/genética , Respiração Celular/efeitos dos fármacos , Expressão Gênica , Ácido Glutâmico/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca(2+)-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade.
Assuntos
Trifosfato de Adenosina/metabolismo , Antiporters/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Potenciais de Ação , Animais , Antiporters/genética , Cálcio/metabolismo , Respiração Celular , Células Cultivadas , Córtex Cerebral/citologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Convulsões/metabolismoRESUMO
Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca(2+) signal or to Ca(2+) itself. [Na(+)]i, [Ca(2+)]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca(2+) clearly showed that Ca(2+)-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca(2+) may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca(2+) entry in mitochondria through the calcium uniporter (MCU). Two Ca(2+)-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca(2+) of 300nM and 3.4µM, respectively. The lack of SCaMC-3 results in a smaller Ca(2+)-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca(2+)-dependent responses to all workloads: a reduction of about 65-70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K(+)-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K(+)-depolarization, this occurs in spite of the presence of large [Ca(2+)]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca(2+)-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca(2+) is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.
Assuntos
Cálcio/fisiologia , Mitocôndrias/fisiologia , Neurônios/fisiologia , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Citosol/metabolismo , Humanos , Transporte de Íons , Mitocôndrias/metabolismo , Neurônios/metabolismo , Ácido Pirúvico/metabolismoRESUMO
GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot-Marie-Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca(2+) stores. GDAP1 silencing does not affect mitochondrial Ca(2+) uptake, ER-Ca(2+), or Ca(2+) flow from ER to mitochondria, but reduces Ca(2+) inflow through store-operated Ca(2+) entry (SOCE) following mobilization of ER-Ca(2+) and SOCE-driven Ca(2+) entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca(2+) uptake in mitochondria.