RESUMO
Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3--cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays-with some discrepancies-link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and-in some molecular subtypes-improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 µM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3--dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by â¼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.
Assuntos
Neoplasias da Mama , Proliferação de Células , Guanidinas , Receptor ErbB-2 , Trocador 1 de Sódio-Hidrogênio , Sulfonas , Guanidinas/farmacologia , Feminino , Animais , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/genética , Camundongos , Humanos , Sulfonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Linhagem Celular Tumoral , Concentração de Íons de HidrogênioRESUMO
AIM: To assess the impact of endurance training on skeletal muscle release of H+ and K+. METHODS: Nine participants performed one-legged knee extension endurance training at moderate and high intensities (70%-85% of Wpeak), three to four sessions·week-1 for 6 weeks. Post-training, the trained and untrained (control) leg performed two-legged knee extension at low, moderate, and high intensities (40%, 62%, and 83% of Wpeak) in normoxia and hypoxia (~4000 m). The legs were exercised simultaneously to ensure identical arterial inflow concentrations of ions and metabolites, and identical power output was controlled by visual feedback. Leg blood flow was measured (ultrasound Doppler), and acid-base variables, lactate- and K+ concentrations were assessed in arterial and femoral venous blood to study K+ and H+ release. Ion transporter abundances were assessed in muscle biopsies. RESULTS: Lactate-dependent H+ release was similar in hypoxia to normoxia (p = 0.168) and was lower in the trained than the control leg at low-moderate intensities (p = 0.060-0.006) but similar during high-intensity exercise. Lactate-independent and total H+ releases were higher in hypoxia (p < 0.05) and increased more with power output in the trained leg (leg-by-power output interactions: p = 0.02). K+ release was similar at low intensity but lower in the trained leg during high-intensity exercise in normoxia (p = 0.024) and hypoxia (p = 0.007). The trained leg had higher abundances of Na+/H+ exchanger 1 (p = 0.047) and Na+/K+ pump subunit α (p = 0.036). CONCLUSION: Moderate- to high-intensity endurance training increases lactate-independent H+ release and reduces K+ release during high-intensity exercise, coinciding with increased Na+/H+ exchanger 1 and Na+/K+ pump subunit α muscle abundances.
Assuntos
Treino Aeróbico , Hipóxia , Ácido Láctico , Perna (Membro) , Músculo Esquelético , Potássio , Humanos , Potássio/metabolismo , Potássio/sangue , Hipóxia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/irrigação sanguínea , Perna (Membro)/irrigação sanguínea , Adulto , Ácido Láctico/sangue , Adulto Jovem , Prótons , Fluxo Sanguíneo Regional , ATPase Trocadora de Sódio-Potássio/metabolismo , Exercício Físico/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismoRESUMO
AIMS: Heart failure with preserved ejection fraction (HFpEF) causes substantial morbidity and mortality. Importantly, atrial remodelling and atrial fibrillation are frequently observed in HFpEF. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have recently been shown to improve clinical outcomes in HFpEF, and post-hoc analyses suggest atrial anti-arrhythmic effects. We tested if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx, which is known to cause atrial arrhythmias, and if that is responsive to treatment with the SGTL2i empagliflozin. METHODS AND RESULTS: Cardiomyocytes were isolated from atrial biopsies of 124 patients (82 with HFpEF) undergoing elective cardiac surgery. Na influx was measured with the Na-dye Asante Natrium Green-2 AM (ANG-2). Compared to patients without heart failure (NF), Na influx was doubled in HFpEF patients (NF vs. HFpEF: 0.21 ± 0.02 vs. 0.38 ± 0.04â mmol/L/min (N = 7 vs. 18); P = 0.0078). Moreover, late INa (measured via whole-cell patch clamp) was significantly increased in HFpEF compared to NF. Western blot and HDAC4 pulldown assay indicated a significant increase in CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation in HFpEF compared to NF, whereas NaV1.5 protein and mRNA abundance remained unchanged. Consistently, increased Na influx was significantly reduced by treatment not only with the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), late INa inhibitor tetrodotoxin (TTX) but also with sodium/hydrogen exchanger 1 (NHE1) inhibitor cariporide. Importantly, empagliflozin abolished both increased Na influx and late INa in HFpEF. Multivariate linear regression analysis, adjusting for important clinical confounders, revealed HFpEF to be an independent predictor for changes in Na handling in atrial cardiomyocytes. CONCLUSION: We show for the first time increased Na influx in human atrial cardiomyocytes from HFpEF patients, partly due to increased late INa and enhanced NHE1-mediated Na influx. Empagliflozin inhibits Na influx and late INa, which could contribute to anti-arrhythmic effects in patients with HFpEF.
Assuntos
Potenciais de Ação , Compostos Benzidrílicos , Glucosídeos , Insuficiência Cardíaca , Miócitos Cardíacos , Inibidores do Transportador 2 de Sódio-Glicose , Volume Sistólico , Humanos , Glucosídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/enzimologia , Masculino , Feminino , Idoso , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/patologia , Pessoa de Meia-Idade , Compostos Benzidrílicos/farmacologia , Volume Sistólico/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Sódio/metabolismo , Átrios do Coração/metabolismo , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/fisiopatologia , Átrios do Coração/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Células Cultivadas , Função do Átrio Esquerdo/efeitos dos fármacos , Trocador 1 de Sódio-HidrogênioRESUMO
OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.
Assuntos
Aminoácido Oxirredutases , Neoplasias de Cabeça e Pescoço , Trocador 1 de Sódio-Hidrogênio , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias da Língua , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Humanos , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Neoplasias da Língua/metabolismo , Microambiente Tumoral , Técnicas de Silenciamento de Genes , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Carcinogênese/genética , Colágeno/metabolismo , Células Matadoras Naturais , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genéticaRESUMO
Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.
Assuntos
Encéfalo , Microglia , Trocador 1 de Sódio-Hidrogênio , Animais , Camundongos , Encéfalo/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Oligodendroglia/metabolismo , Transdução de Sinais/genética , Trocador 1 de Sódio-Hidrogênio/metabolismoRESUMO
Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1, which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain.
Assuntos
Neuralgia , Neuroblastoma , Ratos , Humanos , Animais , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Glicóis , Preparações de Ação Retardada , RNA Interferente Pequeno/genéticaRESUMO
Tumour cell detachment from the primary tumour is an early and crucial step of the metastatic cascade. At the single cell level, it was already shown that migrating melanoma cells establish both intra- and extracellular pH gradients and that the Na+ /H+ exchanger NHE1 accumulates at the leading edges to strengthen cell-matrix interactions. However, less is known about the role of NHE1 in collective cell migration and the specific pH microenvironment at tumour cell-cell contacts. We used MV3 melanoma cells transfected with a NHE1-expressing vector or a control vector. NHE1 localization at cell-cell contacts was assessed via immunofluorescence imaging. Collective migration was analysed by live-cell imaging. The NHE1 activity and the perimembranous pH were measured both intra- and extracellularly by ratiometric fluorescence microscopy. NHE1 clearly localizes at cell-cell contacts. Its overexpression further increases migratory speed and translocation in multidirectional pathway analyses. NHE1 overexpressing MV3 cells also move further away from their neighbouring cells during wound closure assays. pH measurements revealed that the NHE1 is highly active at cell-cell contacts of melanoma cells. NHE1-mediated pH dynamics at such contact sites are more prominent in NHE1-overexpressing melanoma cells. Our findings highlight the contribution of the NHE1 towards modulation and plasticity of melanoma cell-cell contacts. We propose that its localization and functional activity at cell-cell contacts promotes evasion of single melanoma cells from the primary tumour.
Assuntos
Melanoma , Humanos , Trocador 1 de Sódio-Hidrogênio/metabolismo , Melanoma/metabolismo , Linhagem Celular Tumoral , Trocadores de Sódio-Hidrogênio/metabolismo , Comunicação Celular , Concentração de Íons de Hidrogênio , Microambiente TumoralRESUMO
Intrinsic or acquired radioresistance often limits the efficacy of radiation therapy (RT), thereby leading to local control failure. Cancerous cells have abnormal pH dynamics due to high metabolic demands, but it is unclear how pH dynamics contribute to radioresistance. In this study, we investigated the role of Na-H exchange 1 (NHE1), the major intracellular pH (pHi) regulator, in RT response. We observed that RT increased NHE1 expression and modulated pHi in MDA-MB-231 human breast cancer cells. When combined with RT, pharmacological NHE1 inhibition by 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) reduced pHi and clonogenic survival. EIPA attenuated radiation-damaged DNA repair, increasing G2/M cell cycle arrest. The combination of EIPA and RT increased apoptotic cell death while decreasing phosphorylation of NF-κB p65. Similarly, the knockdown of NHE1 increased radiosensitivity with lower pHi and increased apoptosis. Consistent with in vitro data, the EIPA plus RT inhibited the growth of MDA-MB-231 xenograft tumors in mice to a greater extent than either EIPA or RT alone. EIPA abrogated the RT-induced increase in NHE1 and phospho-NF-κB p65 expression in tumor tissues. Such coincidence of increased NHE1 level, pHi, and NF-κB activation was also found in radioresistant MDA-MB-231 cells, which were reversed by EIPA treatment. Bioinformatics analysis of RNA sequencing data revealed that inhibiting NHE1 reversed three core gene networks that were up-regulated in radioresistant cells and correlated with high NHE1 expression in patient samples: NF-κB, senescence, and extracellular matrix. Taken together, our findings suggest that NHE1 contributes to RT resistance via NF-κB-mediated signaling networks, and NHE1 may be a promising target for improving RT outcomes.
Assuntos
Neoplasias da Mama , NF-kappa B , Humanos , Camundongos , Animais , Feminino , NF-kappa B/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Amilorida/farmacologia , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Concentração de Íons de HidrogênioRESUMO
As a previously discovered target of DNA damage, Na+/H+ exchanger 1 (NHE1) plays a role in regulation of intracellular pH (pHi) through the extrusion of intracellular proton (H+) in exchange for extracellular sodium (Na+). Its abnormal expression and dysfunction have been reported in solid tumor and hematopoietic malignancies. Here, we reported that suppression of NHE1 in BCR-ABL+ hematopoietic malignancies' K562 cells treated with Etoposide was manipulated by miR-19 and c-MYC. Inhibition of miR-19 or c-MYC enhanced the expression of NHE1 and sensitized K562 cells to Etoposide in vitro. The in vivo nude mouse transplantation model was also performed to confirm the enhanced sensitivity of K562 cells to Etoposide by inhibiting the miR-19 or c-MYC pathway. TCGA analysis conferred a negative correlation between miR-19 level and leukemia patients' survival. Thus, our results provided a potential management by which the c-MYC-miRNA 19 pathway might have a crucial impact on sensitizing K562 cells to Etoposide in the therapeutic approaches.
Assuntos
Neoplasias Hematológicas , Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Regulação Leucêmica da Expressão Gênica , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: Sodium-glucose cotransporter-2 (SGLT2) inhibitors exhibit cardioprotective properties in patients with diabetes. However, SGLT2 is not expressed in the heart, and the underlying molecular mechanisms are not fully understood. We investigated whether the SGLT2 inhibitor luseogliflozin exerts beneficial effects on high glucose-exposed cardiomyocytes via the suppression of sodium-hydrogen exchanger-1 (NHE-1) activity. METHODS: Mouse cardiomyocytes were incubated under normal or high glucose conditions with vehicle, luseogliflozin, or the NHE-1 inhibitor cariporide. NHE-1 activity and gene expression were evaluated by the SNARF assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Six-week-old male db/db mice were treated with vehicle or luseogliflozin for 6 weeks, and the hearts were collected for histological, RT-PCR, and western blot analyses. RESULTS: High glucose increased NHE-1 activity and transforming growth factor (Tgf)-ß2 mRNA levels in cardiomyocytes, both of which were inhibited by luseogliflozin or cariporide, whereas their combination showed no additive suppression of Tgf-ß2 mRNA levels. Luseogliflozin attenuated cardiac hypertrophy and fibrosis in db/db mice in association with decreased mRNA and protein levels of TGF-ß2. CONCLUSIONS: Luseogliflozin may suppress cardiac hypertrophy in diabetes by reducing Tgf-ß2 expression in cardiomyocytes via the suppression of NHE-1 activity.
Assuntos
Diabetes Mellitus , Miócitos Cardíacos , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Cardiomegalia/patologia , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/farmacologia , Sorbitol/análogos & derivados , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
This review summarizes and describes the current evidence addressing how sodium-glucose cotransporter 2 (SGLT2) inhibitors alter the function of sodium-hydrogen exchanger 1 (NHE-1), in association with their protective effects against adverse cardiovascular events. In the heart, SGLT2 inhibitors modulate the function of NHE-1 (either by direct inhibition or indirect attenuation of protein expression), which promotes cardiac contraction and an enhanced energy supply, in association with improved mitochondrial function, reduced inflammation/oxidative/endoplasmic reticulum stress, and attenuated fibrosis and apoptotic/autophagic cell death. The vasodilating effect of SGLT2 inhibitors has also been proposed due to NHE-1 inhibition. Moreover, platelet-expressed NHE-1 might serve as a target for SGLT2 inhibitors, since these drugs and selective NHE-1 inhibitors produce comparable activity against adenosine diphosphate-stimulated platelet activation. Overall, it is promising that the modulation of the functions of NHE-1 on the heart, blood vessels, and platelets may act as a contributing pathway for the cardiovascular benefits of SGLT2 inhibitors in diabetes and heart failure.
Assuntos
Diabetes Mellitus Tipo 2 , Inibidores do Transportador 2 de Sódio-Glicose , Difosfato de Adenosina , Compostos Benzidrílicos/farmacologia , Diabetes Mellitus Tipo 2/complicações , Glucose , Glucosídeos/farmacologia , Humanos , Sódio/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Trocador 1 de Sódio-HidrogênioRESUMO
Breast cancer metastasis is the leading cause of cancer-related deaths. Hypoxia in the tumor mass is believed to trigger cell migration, which is involved in a crucial process of breast cancer metastasis. However, the molecular mechanisms underlying aggressive behavior under hypoxic conditions have not been fully elucidated. Here, we demonstrate the significant motility of MDA-MB-231 cells cultured under hypoxic conditions compared to that of cells cultured under normoxic conditions. MDA-MB-231 cells under hypoxic conditions showed a significant increase in Na+/H+ exchanger isoform 1 (NHE1) expression level, which was observed to co-locate in lamellipodia formation. Inhibition of NHE1 significantly suppressed the intracellular pH and the expression of mesenchymal markers, thereby blocking the high migration activity in hypoxia. Moreover, treatment with ciglitazone, a potent and selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, modulated hypoxia-enhanced motion in cells via the repression of NHE1. These findings highlight that NHE1 is required for migratory activity through the enhancement of epithelial-mesenchymal transition (EMT) in MDA-MB-231 cells under hypoxic conditions, and we propose new drug repurposing strategies targeting hypoxia based on NHE1 suppression by effective usage of PPARγ agonists.
Assuntos
Neoplasias da Mama/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Humanos , Modelos Biológicos , PPAR gama/agonistas , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Hipóxia Tumoral/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
In breast cancer, it is the resulting metastasis that is the primary cause of fatality. pH regulatory proteins and the tumor microenvironment play an important role in metastasis of cancer cells and acid-extruding proteins are critical in this process. There are several types of breast cancer and triple-negative breast cancer tends to be more metastatic and invasive and is itself is composed of several types. MDA-MB-468 are a triple-negative breast cancer cell line and are classified as basal-like and basal tumors account for up to 15% of breast cancers. Here we examined the effect of removal of the acid-extruding protein, the Na+/H+ exchanger isoform one, from MDA-MB-468 cells. NHE1 was deleted from these cells using the CRISPR/Cas9 system. Western blotting and measurement of activity confirmed the absence of the protein. In wounding/cell migration experiments, deletion of NHE1 reduced the rate of cell migration in the presence of low- or high-serum concentrations. Anchorage-dependent colony formation was also greatly reduced by deletion of the NHE1 protein. Cell proliferation was not affected by knockout of NHE1. The results demonstrate that NHE1 has an important role in migration and invasion of basal-like triple-negative breast cancer cells.
Assuntos
Movimento Celular , Proteínas de Neoplasias/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+ T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates.
Assuntos
Amilorida/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/uso terapêutico , Esomeprazol/uso terapêutico , Inibidores da Bomba de Prótons/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Amilorida/uso terapêutico , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Trocador 1 de Sódio-Hidrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Macrófagos Associados a Tumor/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation V600E (BRAFV600E) is involved in glioblastoma multiforme (GBM). Na/H exchanger 1 (NHE1), a main pH regulator affecting cell microenvironment, is hyper-expressed in GBM. However, the relationship between BRAFV600E signal pathway and NHE1 in GMB cells remains unclear. This study found that NHE1 was a downstream target of BRAFV600E and an upstream factor of extracellular signal-regulated kinase (ERK). In addition, there was a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAFV600E mutation contributing to the proliferation and invasion of GBM cells. Moreover, the proliferation and invasion abilities of BRAFV600E-mutant and BRAF wild type GBM cells were all suppressed by the NHE1 inhibitor, BRAFV600E inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors was better than each single drug both in vitro and in vivo. Combination of BRAFV600E and NHE1 inhibitors could be considered as a new therapeutic regimen for GBM, especially for GBM with BRAFV600E.
Assuntos
Carcinogênese/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica , Glioblastoma/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioblastoma/patologia , Humanos , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidoresRESUMO
Prostate cancer is the fourth most commonly diagnosed cancer, and although it is often a slow-growing malignancy, it is the second leading cause of cancer-associated deaths in men and the first in Europe and North America. In many forms of cancer, when the disease is a solid tumor confined to one organ, it is often readily treated. However, when the cancer becomes an invasive metastatic carcinoma, it is more often fatal. It is therefore of great interest to identify mechanisms that contribute to the invasion of cells to identify possible targets for therapy. During prostate cancer progression, the epithelial cells undergo epithelial-mesenchymal transition that is characterized by morphological changes, a loss of cell-cell adhesion, and invasiveness. Dysregulation of pH has emerged as a hallmark of cancer with a reversed pH gradient and with a constitutively increased intracellular pH that is elevated above the extracellular pH. This phenomenon has been referred to as "a perfect storm" for cancer progression. Acid-extruding ion transporters include the Na+/H+ exchanger NHE1 (SLC9A1), the Na+HCO3- cotransporter NBCn1 (SLC4A7), anion exchangers, vacuolar-type adenosine triphosphatases, and the lactate-H+ cotransporters of the monocarboxylate family (MCT1 and MCT4 (SLC16A1 and 3)). Additionally, carbonic anhydrases contribute to acid transport. Of these, several have been shown to be upregulated in different human cancers including the NBCn1, MCTs, and NHE1. Here the role and contribution of acid-extruding transporters in prostate cancer growth and metastasis were examined. These proteins make significant contributions to prostate cancer progression.
Assuntos
Carcinoma , Neoplasias da Próstata , Carcinoma/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na+/H+ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1-alone, or with inhibitors combining NHE1 or uPA inhibition-generally did not prevent prostate cancer cell migration. However, uPA inhibition-but not NHE1 inhibition-prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.
Assuntos
Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Neoplasias da Próstata/genética , Microambiente Tumoral , Regulação para CimaRESUMO
Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett's esophagus, and this is presumably a compensatory mechanism against this toxic agent.
Assuntos
Esôfago de Barrett/genética , Proliferação de Células/genética , Esôfago/metabolismo , Interferência de RNA , Fumaça , Trocador 1 de Sódio-Hidrogênio/genética , Animais , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Linhagem Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Esôfago/patologia , Expressão Gênica , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Fumar , Trocador 1 de Sódio-Hidrogênio/metabolismo , Nicotiana/químicaRESUMO
Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3- cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3- cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3- cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3- cotransport associates with severe breast cancer: enlarged CO2/HCO3--dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3--dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3- cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Idoso , Animais , Bicarbonatos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Pessoa de Meia-Idade , Organoides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/genética , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio , TranscriptomaRESUMO
SGLT-2i's exert direct anti-inflammatory and anti-oxidative effects on resting endothelial cells. However, endothelial cells are constantly exposed to mechanical forces such as cyclic stretch. Enhanced stretch increases the production of reactive oxygen species (ROS) and thereby impairs endothelial barrier function. We hypothesized that the SGLT-2i's empagliflozin (EMPA), dapagliflozin (DAPA) and canagliflozin (CANA) exert an anti-oxidative effect and alleviate cyclic stretch-induced endothelial permeability in human coronary artery endothelial cells (HCAECs). HCAECs were pre-incubated with one of the SGLT-2i's (1 µM EMPA, 1 µM DAPA and 3 µM CANA) for 2 h, followed by 10% stretch for 24 h. HCAECs exposed to 5% stretch were considered as control. Involvement of ROS was measured using N-acetyl-l-cysteine (NAC). The sodium-hydrogen exchanger 1 (NHE1) and NADPH oxidases (NOXs) were inhibited by cariporide, or GKT136901, respectively. Cell permeability and ROS were investigated by fluorescence intensity imaging. Cell permeability and ROS production were increased by 10% stretch; EMPA, DAPA and CANA decreased this effect significantly. Cariporide and GKT136901 inhibited stretch-induced ROS production but neither of them further reduced ROS production when combined with EMPA. SGLT-2i's improve the barrier dysfunction of HCAECs under enhanced stretch and this effect might be mediated through scavenging of ROS. Anti-oxidative effect of SGLT-2i's might be partially mediated by inhibition of NHE1 and NOXs.