RESUMEN
BACKGROUND: The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca2+. METHODS: A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2',7'-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. RESULTS: CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. CONCLUSION: Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca2+ signaling and CaMKK2 pathways.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Carcinoma de Células Escamosas , Neoplasias de la Boca , Especies Reactivas de Oxígeno , Humo , Productos de Tabaco , Humanos , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Línea Celular Tumoral , Humo/efectos adversos , Carcinoma de Células Escamosas/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Productos de Tabaco/efectos adversos , Apoptosis/efectos de los fármacos , Nicotiana/química , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
Application of physical forces, ranging from ultrasound to electric fields, is recommended in various clinical practice guidelines, including those for treating cancers and bone fractures. However, the mechanistic details of such treatments are often inadequately understood, primarily due to the absence of comprehensive study models. In this study, we demonstrate that an alternating magnetic field (AMF) inherently possesses a direct anti-cancer effect by enhancing oxidative phosphorylation (OXPHOS) and thereby inducing metabolic reprogramming. We observed that the proliferation of human glioblastoma multiforme (GBM) cells (U87 and LN229) was inhibited upon exposure to AMF within a specific narrow frequency range, including around 227 kHz. In contrast, this exposure did not affect normal human astrocytes (NHA). Additionally, in mouse models implanted with human GBM cells in the brain, daily exposure to AMF for 30 min over 21 days significantly suppressed tumor growth and prolonged overall survival. This effect was associated with heightened reactive oxygen species (ROS) production and increased manganese superoxide dismutase (MnSOD) expression. The anti-cancer efficacy of AMF was diminished by either a mitochondrial complex IV inhibitor or a ROS scavenger. Along with these observations, there was a decrease in the extracellular acidification rate (ECAR) and an increase in the oxygen consumption rate (OCR). This suggests that AMF-induced metabolic reprogramming occurs in GBM cells but not in normal cells. Our results suggest that AMF exposure may offer a straightforward strategy to inhibit cancer cell growth by leveraging oxidative stress through metabolic reprogramming.
Asunto(s)
Neoplasias Encefálicas , Proliferación Celular , Glioblastoma , Magnetoterapia , Reprogramación Metabólica , Fosforilación Oxidativa , Especies Reactivas de Oxígeno , Animales , Humanos , Ratones , Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patología , Magnetoterapia/métodos , Campos Magnéticos , Reprogramación Metabólica/efectos de la radiación , Mitocondrias/metabolismo , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.
Asunto(s)
Proteínas de Unión al Calcio , Carcinoma de Células Escamosas , Movimiento Celular , Mitocondrias , Neoplasias de la Boca , Subtipo EP4 de Receptores de Prostaglandina E , Animales , Humanos , Ratones , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Calmodulina/metabolismo , Calmodulina/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Mitocondrias/metabolismo , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismoRESUMEN
Intracellular calcium (Ca2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca2+ entry (SOCE) is a major mechanism by which Ca2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca2+ entry (SOCE) is also a receptor-regulated Ca2+ entry pathway that maintains Ca2+ homeostasis by sensing reduced Ca2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors located in the ER membrane. The, IP3 receptors in the ER membrane trigger a rapid and transient release of Ca2+ from the ER store. The resulting depletion of ER Ca2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca2+ channel subunits (also known as CRACM1) on the PM, leading to Ca2+ influx from the extracellular space to increase intracellular Ca2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca2+ release-activated Ca2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Humanos , Señalización del Calcio/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismoRESUMEN
BACKGROUND: The influence of neurological or balance dysfunction on cognitive impairment has not been well studied. We compared the results of the balance test, measured by either head or foot sway to consider whole body sway, with those of the cognitive impairment test. METHODS: Individuals of either gender, aged over 60 years, underwent a 30 s balance test. We measured sway while standing on one-leg or two-legs. Sway was evaluated by the distance or area of movement of the head or foot pressure. We also evaluated the effect of visual condition: eyes-open (EO) or -closed (EC). The Mini-Mental State Examination (MMSE) was used to evaluate the degree of cognitive impairment. RESULTS: The head sway area standing on one leg was significantly correlated to MMSE score with EO (correlation r = -0.462). In standing on two legs, no sway test results showed a significant correlation to MMSE scores with EO. With EC, the magnitude of sway became greater, and was significantly correlated to MMSE scores in the head distance. CONCLUSION: Although the correlation between head sway and MMSE was not strong, head sway showed a stronger correlation than did foot pressure sway. Standing on one leg, as measured by head sway area, may thus predict cognitive impairment.
RESUMEN
Despite exhibiting cardiotoxicity, doxorubicin (DOX) is widely used for cancer treatments. Cardiac fibroblasts (CFs) are important in the pathogenesis of heart failure. This necessitates the study of the effect of DOX on CFs. The impairment of calcium (Ca2+) homeostasis is a common mechanism of heart failure. Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2⺠entry pathway that maintains calcium balance by sensing reduced calcium stores in the endoplasmic reticulum. ORAI1, a calcium channel protein and the most important component of SOCE, is highly expressed in human cardiac fibroblasts (HCFs). It is upregulated in CFs from failing ventricles. However, whether ORAI1 in HCFs is increased and/or plays a role in DOX-induced cardiotoxicity remains unknown. In this study, we aimed to elucidate the relationship between ORAI1/SOCE and DOX-induced heart failure. Induction of apoptosis by DOX was characterized in HCFs. Apoptosis and cell cycle analyses were performed by fluorescence-activated cell sorting (FACS). Reactive oxygen species (ROS) production was measured using fluorescence. YM-58483 was used as an ORAI1/SOCE inhibitor. ORAI1-knockdown cells were established by RNA interference. In vivo experiments were performed by intraperitoneally injecting YM-58483 and DOX into mice. We first demonstrated that DOX significantly increased the protein expression level of p53 in HCFs by western blotting. FACS analysis revealed that DOX increased early apoptosis and induced cell cycle arrest in the G2 phase in fibroblasts. DOX also increased ROS production. DOX significantly increased the expression level of ORAI1 in CFs. Both YM-58483 and ORAI1 gene knockdown attenuated DOX-induced apoptosis. Similarly, YM-58483 attenuated cell cycle arrest in the G2 phase, and ORAI1 knockdown attenuated DOX-induced ROS production in HCFs. In the animal experiment, YM-58483 attenuated DOX-induced apoptosis. In HCFs, ORAI1/SOCE regulates p53 expression and plays an important role in DOX-induced cardiotoxicity. ORAI1 may serve as a new target for preventing DOX-induced heart failure.
Asunto(s)
Calcio , Insuficiencia Cardíaca , Humanos , Animales , Ratones , Proteína p53 Supresora de Tumor , Doxorrubicina/toxicidad , Apoptosis , Insuficiencia Cardíaca/inducido químicamente , Proteína ORAI1/genéticaRESUMEN
Doxorubicin (DOX)-induced cardiomyopathy has a poor prognosis. No early detection or effective treatment methods are available in clinical. The mechanisms of cardiotoxicity were considered as oxidative stress and apoptosis in cardiomyocytes. However, the effect of DOX on cardiac fibroblasts remains to be developed. We investigated the direct effect of DOX on the function of human cardiac fibroblasts (HCFs) independently of cell death pathway. Animal study showed that lower dose of DOX (4â mg/kg/week for 3 weeks, i.p.) than a toxic cumulate dose, induced perivascular fibrosis without cell death in hear of mice. DOX increased the protein expression of α-SMA (a marker of trans-differentiation) in HCFs culture cells, indicating that DOX promoted the trans-differentiation of HCFs into myofibroblast. DOX also increased the mRNA and protein expression of matrix metalloproteinase (MMP)-1 in less than 0.1â µM which did not induce cell apoptosis of HCFs cells via PI3K/Akt pathway in HCFs. DOX increased Interleukin-6 (IL-6) via transforming growth factor (TGF)-ß/Smad pathway. In addition, DOX induced the mitochondrial damage and increased the expression of Interleukin-1 (IL-1) via stress-activated protein kinases (SAPK)/ c-Jun NH-2termial kinase (JNK). A peroxisome proliferator-activated receptor gamma (PPARγ) agonist, pioglitazone hydrochloride attenuated the expression of fibrotic marker such as α-SMA and galectin-3 and collagen1 via SAPK/JNK signaling. Pioglitazone also suppressed DOX-induced early fibrotic response in vivo. In conclusion, these findings suggested that low dose DOX induced reactive fibrotic change of cardiac fibroblasts via cell death-independent pathway. There may be potentially new mechanisms of DOX induced cardiotoxicity in clinical usage.
Asunto(s)
Doxorrubicina , Fosfatidilinositol 3-Quinasas , Animales , Apoptosis , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , Fibroblastos , Fibrosis , Ratones , Miocitos Cardíacos , Estrés Oxidativo , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group (n = 30) and an HSMM sheet group (n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.
Asunto(s)
Anastomosis Quirúrgica/métodos , Colon/cirugía , Mioblastos Esqueléticos/fisiología , Animales , Colon/patología , Humanos , Masculino , Ratones Desnudos , RatasRESUMEN
OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.
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Proteínas de Unión al Calcio/metabolismo , Conducto Arterioso Permeable/metabolismo , Conducto Arterial/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Conducto Arterial/anomalías , Conducto Arterioso Permeable/genética , Conducto Arterioso Permeable/patología , Células Endoteliales/patología , Matriz Extracelular/genética , Matriz Extracelular/patología , Humanos , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/patología , FN-kappa B/metabolismo , Técnicas de Cultivo de Órganos , Proteína Quinasa C/metabolismo , Ratas Wistar , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
Ubiquitination is a representative, reversible biological process of the post-translational modification of various proteins with multiple catalytic reaction sequences, including ubiquitin itself, in addition to E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, deubiquitinating enzymes, and proteasome degradation. The ubiquitin-proteasome system is known to play a pivotal role in various molecular life phenomena, including the cell cycle, protein quality, and cell surface expressions of ion-transporters. As such, the failure of this system can lead to cancer, neurodegenerative diseases, cardiovascular diseases, and hypertension. This review article discusses Nedd4-2/NEDD4L, an E3-ubiquitin ligase involved in salt-sensitive hypertension, drawing from detailed genetic dissection analysis and the development of genetically engineered mice model. Based on our analyses, targeting therapeutic regulations of ubiquitination in the fields of cardio-vascular medicine might be a promising strategy in future. Although the clinical applications of this strategy are limited, compared to those of kinase systems, many compounds with a high pharmacological activity were identified at the basic research level. Therefore, future development could be expected.
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Canales Epiteliales de Sodio/metabolismo , Hipertensión/metabolismo , Túbulos Renales Distales/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Aldosterona/metabolismo , Animales , Canales Epiteliales de Sodio/genética , Humanos , Ubiquitina-Proteína Ligasas Nedd4/genética , Cloruro de Sodio/metabolismoRESUMEN
OBJECTIVE: Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 (prostaglandin E receptor 4) in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-ß-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. CONCLUSIONS: Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.
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Aneurisma de la Aorta Abdominal/etiología , Inflamación/etiología , Músculo Liso Vascular/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/fisiología , Transducción de Señal/fisiología , Angiotensina II/administración & dosificación , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta Abdominal/patología , Cloruro de Calcio/administración & dosificación , Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Interleucina-6/genética , Macrófagos/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoE , Ratones Transgénicos , Monocitos/patología , Músculo Liso Vascular/química , Miocitos del Músculo Liso/metabolismo , Proteína-Lisina 6-Oxidasa/análisis , Proteína-Lisina 6-Oxidasa/genética , Receptores de Citocinas/genética , Subtipo EP4 de Receptores de Prostaglandina E/genéticaRESUMEN
AIMS: Doxorubicin (DOX)-induced heart failure has a poor prognosis, and effective treatments have not been established. Because DOX shows cumulative cardiotoxicity, we hypothesized that minimal cardiac remodelling occurred at the initial stage in activating cardiac fibroblasts. Our aim was to investigate the initial pathophysiology of DOX-exposed cardiac fibroblasts and propose prophylaxis. METHODS AND RESULTS: An animal study was performed using a lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulative dose. Histological analysis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, picrosirius red staining, and immunohistochemical staining. The mechanism was analysed in vitro with a low dose of DOX, which did not induce cell apoptosis. Microarray analysis was performed. Differentially expressed genes were confirmed by enrichment analysis. Mitochondrial damage was assessed by mitochondrial membrane potential. The production of inflammatory cytokines and fibrosis markers was assessed by western blot, quantitative polymerase chain reaction, and ELISA. A phosphokinase antibody array was performed to detect related signalling pathways. Low-dose DOX did not induced cell death, and fibrosis was localized to the perivascular area in mice. Microarray analysis suggested that DOX induced genes associated with the innate immune system and inflammatory reactions, resulting in cardiac remodelling. DOX induced mitochondrial damage and increased the expression of interleukin-1. DOX also promoted the expression of fibrotic markers, such as alpha smooth muscle actin and galectin-3. These responses were induced through stress-activated protein kinase/c-Jun NH2-terminal kinase signalling. A peroxisome proliferator-activated receptor (PPARγ) agonist attenuated the expression of fibrotic markers through suppressing stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, this molecule also suppressed DOX-induced early fibrotic responses in vivo. CONCLUSIONS: Low-dose DOX provoked reactive fibrosis through sterile inflammation evoked by the damaged mitochondria.
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Insuficiencia Cardíaca , Miocitos Cardíacos , Animales , Doxorrubicina , Fibrosis , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/patología , Inflamación , Ratones , Miocitos Cardíacos/patologíaRESUMEN
The EP4 prostanoid receptors are one of four receptor subtypes for prostaglandin E2 (PGE2 ). Therefore, EP4 may play an important role in cancer progression. However, little information is available regarding their function per se, including migration and the cellular signaling pathway of EP4 in oral cancer. First, we found that mRNA and protein expression of EP4 was abundantly expressed in human-derived tongue squamous cell carcinoma cell lines HSC-3 and OSC-19. The EP4 agonist (ONO-AE1-437) significantly promoted cell migration in HSC-3 cells. In contrast, knockdown of EP4 reduced cell migration. Furthermore, we confirmed that knockdown of EP4 suppressed metastasis of oral cancer cells in the lungs of mice in vivo. Therefore, we focused on the mechanism of migration/metastasis in EP4 signaling. Interestingly, EP4 agonist significantly induced intracellular Ca2+ elevation not in only oral cancer cells but also in other cells, including normal cells. Furthermore, we found that EP4 activated PI3K and induced Ca2+ influx through Orai1 without activation of store depletion and stromal interaction molecule 1 (STIM1). Immunoprecipitation showed that EP4 formed complexes with Orai1 and TRPC1, but not with STIM. Moreover, the EP4 agonist ONO-AE1-437 phosphorylated ERK and activated MMP-2 and MMP-9. Knockdown of Orai1 negated EP4 agonist-induced ERK phosphorylation. Taken together, our data suggested that EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration. Migration is regulated by EP4/PI3K/Orai1 signaling in oral cancer.
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Movimiento Celular/fisiología , Proteína ORAI1/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Animales , Calcio/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Humanos , Células MCF-7 , Fosforilación/fisiología , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Neoplasias de la Lengua/metabolismoRESUMEN
Although doxorubicin (DOX)-induced cardiomyopathy causes lethal heart failure (HF), no early detection or effective treatment methods are available. The principal mechanisms of cardiotoxicity are considered to involve oxidative stress and apoptosis of cardiomyocytes. However, the effect of DOX on cardiac fibroblasts at non-lethal concentrations remains unknown. The aim of this study was to investigate the direct effect of doxorubicin on the activation of cardiac fibroblasts independent of cell death pathways. We first found that DOX induced α-SMA expression (marker of trans-differentiation) at a low concentration range, which did not inhibit cell viability. DOX also increased MMP1, IL-6, TGF-ß and collagen expression in human cardiac fibroblasts (HCFs). In addition, DOX promoted Akt and Smad phosphorylation. A Smad inhibitor prevented DOX-induced α-SMA and IL-6 protein expression. An PI3K inhibitor also prevented MMP1 mRNA expression in HCFs. These findings suggest that DOX directly induces fibrotic changes in HCFs via cell death-independent pathways. Furthermore, we confirmed that these responses are organ- and species-specific for HCFs based on experiments using different types of human and murine fibroblast cell lines. These results suggest potentially new mechanisms of DOX-induced cardiotoxicity from the viewpoint of fibrotic changes in cardiac fibroblasts.
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Doxorrubicina/farmacología , Fibroblastos/citología , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/genética , Miocitos Cardíacos/citología , Actinas/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos , Transducción de Señal/efectos de los fármacos , Especificidad de la EspecieRESUMEN
Prevention of cardiomyocyte death is an important therapeutic strategy for heart failure. In this study, we focused on translationally controlled tumor protein (TCTP), a highly conserved protein that is expressed ubiquitously in mammalian tissues, including heart. TCTP plays pivotal roles in survival of certain cell types, but its function in cardiomyocytes has not been examined. We aimed to clarify the role of TCTP in cardiomyocyte survival and the underlying mechanism. Here, we demonstrated that downregulation of TCTP with siRNA induced cell death of cardiomyocytes with apoptotic and autophagic features, accompanied with mitochondrial permeability transition pore (mPTP) opening. TCTP loss did not induce cell death of cardiac fibroblasts. Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) was found to mediate the TCTP-loss-induced cardiomyocyte death. In exploring the clinical significance of the TCTP expression in the heart, we found that DOX treatment markedly downregulated the protein expression of TCTP in cultured cardiomyocytes and in mouse heart tissue. Exogenous rescue of TCTP expression attenuated DOX-induced cardiomyocyte death. In mice, cardiomyocyte-specific overexpression of TCTP resulted in decreased susceptibility to DOX-induced cardiac dysfunction, accompanied with attenuated induction of Bnip3. Dihydroartemisinin, a pharmacological TCTP inhibitor, induced development of heart failure and cardiomyocyte death in control mice, but not in mice with cardiomyocyte-specific TCTP overexpression. Our findings revealed TCTP has a pivotal role in cardiomyocyte survival, at least in part through a Bnip3-dependent mechanism. TCTP could be considered as a candidate therapeutic target to prevent DOX-induced heart failure.
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Biomarcadores de Tumor/metabolismo , Supervivencia Celular/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Células Cultivadas , Doxorrubicina/toxicidad , Insuficiencia Cardíaca/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Proteínas Mitocondriales/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
The article Epac activation inhibits IL-6-induced cardiac myocyte dysfunction.
RESUMEN
BACKGROUND: It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of ß-adrenergic receptor (ß-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. MethodsâandâResults: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. CONCLUSIONS: These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.
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Fibrilación Atrial/prevención & control , AMP Cíclico/antagonistas & inhibidores , Fibrilación Ventricular/prevención & control , Animales , Fibrilación Atrial/etiología , Calcio/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/fisiología , Ratones , Ratones Noqueados , Quinolinas/uso terapéutico , Retículo Sarcoplasmático/metabolismo , Fibrilación Ventricular/etiologíaRESUMEN
Vascular smooth muscle cell (VSMC) migration and the subsequent intimal thickening play roles in vascular restenosis. We previously reported that an exchange protein activated by cAMP 1 (Epac1) promotes platelet-derived growth factor (PDGF)-induced VSMC migration and intimal thickening. Because basic fibroblast growth factor (bFGF) also plays a pivotal role in restenosis, we examined whether Epac1 was involved in bFGF-mediated VSMC migration. bFGF-induced lamellipodia formation and migration were significantly decreased in VSMCs obtained from Epac1-/- mice compared to those in Epac1+/+-VSMCs. The bFGF-induced phosphorylation of Akt and glycogen synthase kinase 3ß (GSK3ß), which play a role in bFGF-induced cell migration, was attenuated in Epac1-/--VSMCs. Intimal thickening induced by the insertion of a large wire was attenuated in Epac1-/- mice, and was accompanied by the decreased phosphorylation of GSK3ß. These data suggest that Epac1 deficiency attenuates bFGF-induced VSMC migration, possibly via Akt/GSK3ß pathways.
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Movimiento Celular/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/deficiencia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Animales , Células Cultivadas , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/fisiopatología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
We previously identified a novel nanomagnetic particle, N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)]. Fe(Salen) not only shows antitumor effects but also magnetic properties. We found that Fe(Salen) can be used for magnet-guided drug delivery and visualization of accumulated drug by magnetic resonance imaging (MRI) because of its magnetism. In addition, Fe(Salen) can generate heat by itself when exposed to an alternating current magnetic field (AMF), resulting in a hyperthermia effect. Herein, we partly elucidated the antitumor mechanism of Fe(Salen) and carried out an i.v. repeated dose toxicity study to decide the therapeutic amount. Furthermore, we evaluated the antitumor effect of selective intra-arterial injection or i.v. injection of Fe(Salen) by catheter and the hyperthermia effect of Fe(Salen) when exposed to AMF in vivo. We used a rabbit model grafted with VX2 cells (rabbit squamous cell carcinoma) on the right leg. Intra-arterial injection of Fe(Salen) showed a greater antitumor effect than did i.v. injection. The combination of Fe(Salen) intra-arterial injection and AMF exposure showed a greater antitumor effect than did either Fe(Salen) or methotrexate (MTX) without AMF exposure, suggesting that AMF exposure greatly enhanced the antitumor effect of Fe(Salen) by arterial injection by catheter. This is the first report that the effectiveness of Fe(Salen) was evaluated in the point of administration route; that is, selective intra-arterial injection by catheter. Taken together, these results indicate a new administration route; that is, selective arterial injection of Fe(Salen) by catheter, and the development of a new strategy of simultaneous hyperthermia-chemotherapy in the future.