RESUMEN
Targeting the DNA damage response (DDR) is a promising strategy in oncotherapy, as most tumor cells are sensitive to excess damage due to their repair defects. Ataxia telangiectasia mutated and RAD3-related protein (ATR) is a damage response signal transduction sensor, and its therapeutic potential in tumor cells needs to be precisely investigated. Herein, we identified a new axis that could be targeted by ATR inhibitors to decrease the DNA-dependent protein kinase catalytic subunit (DNAPKcs), downregulate the expression of the retinoblastoma (RB), and drive G1/S-phase transition. Four-way DNA Holliday junctions (FJs) assembled in this process could trigger S-phase arrest and induce lethal chromosome damage in RB-positive triple-negative breast cancer (TNBC) cells. Furthermore, these unrepaired junctions also exerted toxic effects to RB-deficient TNBC cells when the homologous recombination repair (HRR) was inhibited. This study proposes a precise strategy for treating TNBC by targeting the DDR and extends our understanding of ATR and HJ in tumor treatment.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , ADN Cruciforme , Neoplasias de la Mama Triple Negativas , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Humanos , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Línea Celular Tumoral , ADN Cruciforme/metabolismo , ADN Cruciforme/genética , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Femenino , Fase S/efectos de los fármacos , Fase S/fisiología , Animales , Antineoplásicos/farmacología , Daño del ADN/fisiología , Daño del ADN/efectos de los fármacosRESUMEN
BACKGROUND: Severe acute pancreatitis (SAP) is a potential fatal gastrointestinal disease that is usually complicated by myocardial injury and dysfunction. Due to the lack of understanding of the mechanism of SAP-associated cardiac injury (SACI), there is still no complete treatment. AIMS: To explore the alleviative effect and anti-ferroptosis mechanism against SACI of glycyrrhizin (GL), an inhibitor of oxidative stress. METHODS: The SAP model was established by perfusing 5% sodium taurocholate into biliopancreatic duct in rats. H&E staining and serum assays were used to assess the injury changes of pancreas and heart. Echocardiography was used to evaluate the cardiac function. Transmission electron microscopy (TEM) and oxidative stress assays were used to investigate the ferroptosis-related morphological and biochemical changes. Western blot and immunofluorescence were performed to analyzed the expression of ferroptosis-related proteins. RESULTS: Significant myocardial impairment was found in SAP rats according to increased histopathological scores, serum creatine kinase-MB (CK-MB) and cardiac troponin-I (cTnI) levels, and a decreased fractional shortening and ejection fraction. The decreased mitochondrial cristae and significant expression changes of ferroptosis-related proteins confirmed the presence of ferroptosis in SACI. GL treatment attenuated above-mentioned cardiac tissues damage by inhibiting ferroptosis via restoring the expression of Nrf2 and HO-1 in vivo and in vitro. Treating with ML385 (a Nrf2 inhibitor) or transfecting with siRNA-Nrf2 reversed the protective effect of GL. CONCLUSIONS: Our findings demonstrate the involvement of ferroptosis in SACI and suggest a potential role for GL in the treatment of SACI by supressing ferroptosis via Keap1/Nrf2/HO-1 pathway.
Asunto(s)
Ferroptosis , Ácido Glicirrínico , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Pancreatitis , Animales , Factor 2 Relacionado con NF-E2/metabolismo , Ferroptosis/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratas , Masculino , Ácido Glicirrínico/farmacología , Ácido Glicirrínico/uso terapéutico , Pancreatitis/metabolismo , Pancreatitis/tratamiento farmacológico , Pancreatitis/patología , Hemo Oxigenasa (Desciclizante)/metabolismo , Transducción de Señal/efectos de los fármacos , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Estrés Oxidativo/efectos de los fármacos , Lesiones Cardíacas/metabolismo , Lesiones Cardíacas/tratamiento farmacológicoRESUMEN
Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Caspasa 8/metabolismo , Conexinas/metabolismo , Neoplasias Hepáticas/metabolismo , Necroptosis/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Transducción de Señal/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Conexinas/genética , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naftoquinonas/administración & dosificación , Necroptosis/efectos de los fármacos , Coactivador 1 de Receptor Nuclear/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transducción de Señal/efectos de los fármacos , Transfección , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Proteína beta1 de Unión ComunicanteRESUMEN
BACKGROUND: Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear. METHODS: The expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice. RESULTS: Our study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression. CONCLUSION: The present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Conexinas/metabolismo , Receptores ErbB/metabolismo , Neoplasias Hepáticas/genética , Adulto , Anciano , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Persona de Mediana Edad , Transducción de Señal , Transfección , Adulto Joven , Proteína beta1 de Unión ComunicanteRESUMEN
Gap junctions (GJs) are novel molecular targets for pain therapeutics due to their pain-promoting function. INI-0602, a new GJ inhibitor, exerts a neuroprotective role, while its role in neuropathic pain is unclear. The objective was to investigate the analgesic role and mechanisms of INI-0602 in neuropathic pain induced by spared nerve injury (SNI), and whether INI-0602 attenuated pain-induced depression-like behaviors. Rats were randomly assigned to saline treatment groups (sham+NS and SNI+NS) or INI-0602 treatment groups (sham+INI-0602 and SNI+INI-0602). The von Frey test was used to assess pain behavior, and the sucrose preference test, the forced swimming test, and the tail suspension test were used to assess depression-like behaviors. Gap junction intercellular communication (GJIC) was measured by parachute assay. Western blots were used to determine the protein expression. In vitro, INI-0602 significantly suppressed GJIC by decreasing connexin43 and connexin32 expression. In vivo, INI-0602 significantly suppressed mechanical allodynia during initiation (7 days after SNI) and the maintenance phase (21 days after SNI) and simultaneously attenuated accompanying depression-like behaviors. Furthermore, INI-0602 markedly suppressed the activation of astrocytes and microglia on days 7 and 21 by reducing GJIC. Finally, INI-0602 reversed the changes in the brain-derived neurotrophic factor and Nr2b subunits of the N-methyl-D-aspartate receptor in SNI rats, suggesting that these effects of INI-0602 were related to its analgesic effect. Our findings demonstrated that blocking GJs with INI-0602 attenuated mechanical pain hypersensitivity and related depression-like behaviors in SNI rats by reducing glial activation.
Asunto(s)
Analgésicos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neuralgia/fisiopatología , Animales , Depresión/etiología , Uniones Comunicantes/efectos de los fármacos , Hiperalgesia/etiología , Masculino , Neuralgia/etiología , Traumatismos de los Nervios Periféricos/complicaciones , Traumatismos de los Nervios Periféricos/fisiopatología , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Fluid resuscitation is the first-line antishock treatment in severe acute pancreatitis (SAP). Currently, although mentions of complications related to aggressive fluid resuscitation are very frequent, a lack of proper handling of complications remains. One of the most important complications is intestinal barrier injury, including intestinal ischemia-reperfusion injury following aggressive fluid resuscitation. Once injured, the intestinal barrier may serve as the source of additional diseases, including systemic inflammatory response syndrome and multiple organ dysfunction syndrome, which aggravate SAP. This study focused on the underlying mechanisms of gut barrier dysfunction in rats induced by aggressive fluid resuscitation in SAP. This study further indicated the important role of necroptosis in intestinal barrier injury which could be relieved by using necroptosis-specific inhibitor Nec-1 before aggressive fluid resuscitation, thus reducing intestinal barrier damage. We also found pancreas damage after intestinal ischemia/reperfusion challenge and indicated the effects of high mobility group protein B1 in the vicious cycle between SAP and intestinal barrier damage.
Asunto(s)
Mucosa Intestinal , Necroptosis , Pancreatitis , Daño por Reperfusión , Resucitación , Animales , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Pancreatitis/etiología , Pancreatitis/metabolismo , Pancreatitis/patología , Pancreatitis/terapia , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/complicaciones , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/terapiaRESUMEN
Ovarian cancer is the most lethal gynecologic malignancy, and cisplatin is one of the first-line chemotherapeutic agents. However, acquired cisplatin resistance prevents the successful treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. In this study, we established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP. Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in chemoresistance.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisplatino/administración & dosificación , Resistencia a Antineoplásicos , Uniones Comunicantes/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Uniones Comunicantes/efectos de los fármacos , Humanos , Neoplasias Ováricas/patología , Sistemas de Translocación de Proteínas/efectos de los fármacos , Sistemas de Translocación de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
OBJECTIVE: To investigate the effects of Bazhen decoction (BZD), Siwu decoction (SWD) and Sijunzi decoction (SJZD) in mice with anemia induced by 5-fluorouracil (5-FU) and discussed the possible pharmacological hematopoietic mechanism to provide experimental evidence for the clinical use of the three classical prescriptions in the treatment of anemia. METHODS: Anemia was induced by intravenous injection of 5-FU and 80 female Kunming mice were randomly, assigned to oral administration of SWD, SJZD, or BZD daily for 10 days. Peripheral blood cells count and bone marrow cell cycle were monitored to evaluate anti-anemia effects. Serum cytokines, interferon-γ (IFN-γ), interleukin-3 (IL-3), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) were assayed. EPO mRNA expression was assayed in kidney and liver tissue homogenates. RESULTS: BZD and SWD significantly increased the number of red blood cells, hemoglobin concentration, and hematocrit, promoted bone marrow cells to enter the cell cycle, proliferate and differentiate, significantly increased IL-3 secretion, and significantly inhibited IFN-γ secretion. BZD stimulated transcription of EPO mRNA in the kidney and liver and enhanced serum EPO expression. A therapeutic effect of SJZD was not observed. CONCLUSION: BZD and SWD treatment specifically enhanced hematopoietic function and mediated myelopoiesis by altering serum cytokines levels and accelerating entry of bone marrow cells into the cell cycle. Better curative effects were achieved via nourishing both Qi and blood (BZD) than by enriching the blood (SWD) or invigorating Qi (SWZD) alone.