RESUMO
Cisplatin-based chemotherapy is the standard treatment for bladder urothelial carcinoma (UC). Most patients experience chemoresistance, the primary cause of treatment failure, which leads to disease relapse. The underlying mechanism of chemoresistance involves reduced apoptosis. In this study, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC. Deubiquitinase (ubiquitin-specific protease 14 (USP14) and USP21) immunohistochemical staining demonstrated that deubiquitination is related to chemoresistance in patients with metastatic UC and may be a target for overcoming chemoresistance. Cytotoxicity and apoptosis were assessed using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, and PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with the concurrent suppression of c-Myc expression in T24/R cells. Moreover, the expression of c-Myc was upregulated in human bladder UC specimens from patients with chemoresistance. Experiments in a xenograft nude mouse model confirmed that PR-619 enhanced the antitumor effects of cisplatin. These results are promising for the development of therapeutic strategies to prevent UC chemoresistance through the combined use of chemotherapeutic agents/deubiquitination inhibitors (PR-619) by targeting the c-Myc pathway.
Assuntos
Aminopiridinas/uso terapêutico , Carcinoma/tratamento farmacológico , Enzimas Desubiquitinantes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Tiocianatos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Aminopiridinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Humanos , Camundongos Nus , Tiocianatos/farmacologia , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.
Assuntos
Ciclo Celular/genética , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Chaperonas de Histonas/biossíntese , Chaperonas de Histonas/genética , Histonas/genética , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas do Grupo Polycomb/biossíntese , Fatores de Transcrição/biossínteseRESUMO
Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces, inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA's cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.
Assuntos
Carcinoma de Células de Transição/tratamento farmacológico , Inibidores de Histona Desacetilases/administração & dosagem , Ácidos Hidroxâmicos/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Paclitaxel/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Paclitaxel/farmacologia , Resultado do Tratamento , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma (UC). Most patients inevitably encounter drug resistance and resultant disease relapse. Reduced apoptosis plays a critical role in chemoresistance. Trifluoperazine (TFP), an antipsychotic agent, has demonstrated antitumor effects on various cancers. This study investigated the efficacy of TFP in inhibiting cisplatin-resistant bladder UC and explored the underlying mechanism. Our results revealed that cisplatin-resistant UC cells (T24/R) upregulated the antiapoptotic factor, B-cell lymphoma-extra large (Bcl-xL). Knockdown of Bcl-xL by siRNA resensitized cisplatin-resistant cells to the cisplatin cytotoxic effect. TFP (10-45 µM) alone elicited dose-dependent cytotoxicity, apoptosis, and G0/G1 arrest on T24/R cells. Co-treatment of TFP potentiated cisplatin-induced cytotoxicity in T24/R cells. The phenomenon that TFP alleviated cisplatin resistance to T24/R was accompanied with concurrent suppression of Bcl-xL. In vivo models confirmed that TFP alone effectively suppressed the T24/R xenograft in nude mice. TFP co-treatment enhanced the antitumor effect of cisplatin on the T24/R xenograft. Our results demonstrated that TFP effectively inhibited cisplatin-resistant UCs and circumvented cisplatin resistance with concurrent Bcl-xL downregulation. These findings provide a promising insight to develop a therapeutic strategy for chemoresistant UCs.
Assuntos
Antipsicóticos/farmacologia , Carcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Trifluoperazina/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteína bcl-X/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antipsicóticos/uso terapêutico , Apoptose , Carcinoma/metabolismo , Linhagem Celular , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Regulação para Baixo , Humanos , Camundongos , Trifluoperazina/uso terapêutico , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , Urotélio/patologia , Proteína bcl-X/genéticaRESUMO
Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stressâ»related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stressâ»related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stressâ»related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Condrossarcoma/tratamento farmacológico , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Ciclopentanos/uso terapêutico , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Proteína NEDD8/antagonistas & inibidores , Proteína NEDD8/metabolismo , Fosforilação/efeitos dos fármacos , Pirimidinas/uso terapêutico , Fator de Transcrição CHOP/metabolismo , Transplante HeterólogoRESUMO
Chondrosarcoma, a treatment-resistant cancer with limited therapeutic options, lacks significant advancements in treatment methods. However, PR-619, a novel inhibitor of deubiquitinating enzymes, has demonstrated anti-tumor effects in various malignancies. This study aimed to investigate the impact of PR-619 on chondrosarcoma both in vitro and in vivo. Two human chondrosarcoma cell lines, SW11353 and JJ012, were utilized. Cell viability was assessed using an MTT assay, while flow cytometry enabled the detection of apoptosis and cell cycle progression. Western blotting analyses were conducted to evaluate apoptosis, cell stress, and endoplasmic reticulum (ER) stress. Furthermore, the in vivo anti-tumor effects of PR-619 were examined using a xenograft mouse model. The results revealed that PR-619 induced cytotoxicity, apoptosis, and cell cycle arrest at the G0/G1 stage by activating caspases, PARP cleavage, and p21. Moreover, PR-619 increased the accumulation of polyubiquitinated proteins and ER stress by activating IRE1, GRP78, caspase-4, CHOP, and other cellular stress responses, including JNK activation. In vivo analysis demonstrated that PR-619 effectively inhibited tumor growth with minimal toxicity in the xenograft mouse model. These findings provide evidence of the anti-tumor effects and induction of cellular and ER stress by PR-619 in human chondrosarcoma, suggesting its potential as a novel therapeutic strategy for in human chondrosarcoma.
RESUMO
Epstein-Barr Virus (EBV) DNase (BGLF5) is an alkaline nuclease and has been suggested to be important in the viral life cycle. However, its effect on host cells remains unknown. Serological and histopathological studies implied that EBV DNase seems to be correlated with carcinogenesis. Therefore, we investigate the effect of EBV DNase on epithelial cells. Here, we report that expression of EBV DNase induces increased formation of micronucleus, an indicator of genomic instability, in human epithelial cells. We also demonstrate, using gammaH2AX formation and comet assay, that EBV DNase induces DNA damage. Furthermore, using host cell reactivation assay, we find that EBV DNase expression repressed damaged DNA repair in various epithelial cells. Western blot and quantitative PCR analyses reveal that expression of repair-related genes is reduced significantly in cells expressing EBV DNase. Host shut-off mutants eliminate shut-off expression of repair genes and repress damaged DNA repair, suggesting that shut-off function of BGLF5 contributes to repression of DNA repair. In addition, EBV DNase caused chromosomal aberrations and increased the microsatellite instability (MSI) and frequency of genetic mutation in human epithelial cells. Together, we propose that EBV DNase induces genomic instability in epithelial cells, which may be through induction of DNA damage and also repression of DNA repair, subsequently increases MSI and genetic mutations, and may contribute consequently to the carcinogenesis of human epithelial cells.
Assuntos
Desoxirribonucleases/metabolismo , Instabilidade Genômica , Proteínas Virais/metabolismo , Linhagem Celular , Aberrações Cromossômicas , Quebras de DNA , Dano ao DNA , Reparo do DNA/genética , Células Epiteliais/química , Células Epiteliais/metabolismo , Humanos , Micronúcleos com Defeito Cromossômico , Instabilidade de Microssatélites , Mutação , Biossíntese de Proteínas , Transcrição Gênica , Raios UltravioletaRESUMO
Urothelial carcinoma (UC) comprises the majority of bladder cancers. Standard platinum-based chemotherapy has a response rate of approximately 50%, but drug resistance develops after short-term treatment. Deubiquitinating (DUB) enzyme inhibitors increase protein polyubiquitination and endoplasmic reticulum (ER) stress, which might further suppress cancer stemness and overcome cisplatin resistance. Therefore, we investigated the cytotoxic effect and potential mechanisms of b-AP15 on urothelial carcinoma. Our results revealed that b-AP15 induced ER stress and apoptosis in BFTC905, T24, T24/R (cisplatin-resistant), and RT4 urothelial carcinoma cell lines. Inhibition of the MYC signaling pathway and cancer stemness by b-AP15 was confirmed by RNA sequencing, RT-PCR, immunoblotting, and sphere-forming assays. In the mouse xenograft model, the combination of b-AP15 and cisplatin showed superior therapeutic effects compared with either monotherapy.
RESUMO
Urothelial carcinoma (UC) is the most common type of bladder cancer, with a 5-year survival rate of only 4.6% in metastatic UC. Despite the advances related to immune-checkpoint inhibitor therapy, chemotherapy remains the standard of care for metastatic diseases, with a 50% response rate. The covalent cyclin-dependent kinase 7 (CDK7) inhibitor THZ1 interferes with transcription machinery and is reported to be effective in cancers without targetable mutations. Therefore, we investigated the therapeutic effect of THZ1 on UC and examined possible mechanisms underlying its effects in both chemonaïve and chemosensitive cancers. CDK7 expression is increased in bladder cancer tissues, especially in patients with chemoresistance. THZ1 induced apoptosis and decreased viability in RT4, BFTC905, HT1376, T24, and T24/R UC cell lines. RNA-sequencing, immunoblotting, and sphere-formation assays confirmed that THZ1 suppressed cancer stemness. In the mouse xenograft model, THZ1 suppressed both chemonaïve and chemoresistant tumors. These results indicate that CDK7 inhibition-related cancer stemness suppression is a potential therapeutic strategy for both chemonaïve and chemoresistant UC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Urotélio/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma/enzimologia , Carcinoma/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Urotélio/enzimologia , Urotélio/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Chemotherapy with gemcitabine plus cisplatin remains the mainstay of treatment for metastatic urothelial carcinoma (UC); however, drug resistance occurs in most patients and eventually leads to treatment failure. In this study, we investigated the role of cyclin-dependent kinase 7 (CDK7) regulation in the treatment of human UCs. Moreover, we studied the effect of THZ1, a CDK7 inhibitor, alone and in combination with gemcitabine, on UCs and explored the underlying mechanism. Immunohistochemical staining showed that CDK7 expression was significantly higher in UC tumors than in counterpart urothelium. THZ1 elicited dose-dependent cytotoxicity and apoptosis in two high-grade UC cells (BFTC905 and T24). THZ1 co-treatment potentiated gemcitabine-induced cytotoxicity with suppression of B-cell lymphoma 2 (Bcl-2). Studies with a xenograft nude mouse model also confirmed that THZ1 enhanced the antitumor effect of gemcitabine on UC. These findings provide important pilot data to target CDK7 or Bcl-2 for the treatment of UCs and for overcoming chemoresistance in UCs.
RESUMO
Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C-dependent and -independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-kappaB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Trombomodulina/química , Trombomodulina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidade , Ligantes , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sepse/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Trombomodulina/administração & dosagemRESUMO
Renal cell carcinoma (RCC) is a major cancer of the kidney. The 5-year survival rate is overall 74% and only 8% for Stage 4 cancers. Several agents including tyrosine kinase inhibitors, mTOR inhibitors, and immune checkpoint inhibitors are available as first- or second-line therapy for metastatic RCC. However, the survival benefits are limited. Recently, THZ1 has been identified as a cyclin-dependent kinase 7 (CDK7) inhibitor that interferes with the transcriptional machinery. Although it is apparently effective in various cancer models, the data for RCC has never been reported. In this study, we demonstrated the impact of CDK7 expression on tumor progression and patient survival in a clinical cohort. We found that THZ1 induced apoptosis and cell cycle arrest in RCC cells, thereby reducing cell viability. Furthermore, THZ1 acted synergistically with temsirolimus in vitro, probably by inhibiting autophagy. Moreover, compared to either THZ1 or temsirolimus used individually, the combination treatment further suppressed tumor growth in vivo. These results indicate that CDK7 is associated with the progression and prognosis of RCC, and is a potential therapeutic target for overcoming drug resistance in this cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Sirolimo/análogos & derivados , Animais , Autofagia/efeitos dos fármacos , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Quinases Ciclina-Dependentes/biossíntese , Sinergismo Farmacológico , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Fenilenodiaminas/administração & dosagem , Pirimidinas/administração & dosagem , Distribuição Aleatória , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cullin-RING E3 ligases are involved in the ubiquitination of substrates that regulate important biological processes and are a potential therapeutic target in many types of cancer. MLN4924, a small molecule of NEDD8-activating enzyme inhibitor, inactivates CRL by blocking cullin neddylation and has been reported to elicit anti-tumor effect. In this study, In this study, we aimed to investigate the effects of MLN4924 on angiogenesis in human umbilical vascular endothelial cells (HUVECs) and four types of cancer cells. Our results showed that MLN4924 inhibits cell viability and induced apoptosis in HUVECs in a dose-dependent manner. MLN4924 inhibits proliferation and interferes with the cell cycle checkpoint regulators, p21, p27, and phospho-histone H3. Vascular endothelial growth factor (VEGF) treatment increased the level of UBC12 in HUVECs, indicating that neddylation pathway is involved in VEGF-activated angiogenesis. MLN4924 decreased VEGF-activated cell proliferation via neddylation inhibition. MLN4924 inhibited VEGF-activated cell migration, capillary tube formation and VEGF-mediated Erk1/2 activation in HUVECs. We also examined antitumor effect of MLN4924 using xenograft SCID mouse models of four different types of cancer cells. The in vivo results showed MLN4924 inhibited tumor growth in all four types of cancers with decreasing CD31 expression in xenograft tumor. In conclusion, MLN4924 inhibited viability, migration, and VEGF-promoted angiogenic activity in HUVECs; consistently, MLN4924 inhibited tumor growth in four types of cancers with suppression of angiogenesis. These findings provide evidence to develop therapeutic strategy for cancer treatment through anti-angiogenesis through neddylation inhibition.
RESUMO
Cancer cells rely on aberrant transcription for growth and survival. Cyclin-dependent kinases (CDKs) play critical roles in regulating gene transcription by modulating the activity of RNA polymerase II (RNAPII). THZ1, a selective covalent inhibitor of CDK7, has antitumor effects in several human cancers. In this study, we investigated the role and therapeutic potential of CDK7 in regulating the angiogenic activity of endothelial cells and human renal cell carcinoma (RCC). Our results revealed that vascular endothelial growth factor (VEGF), a critical activator of angiogenesis, upregulated the expression of CDK7 and RNAPII, and the phosphorylation of RNAPII at serine 5 and 7 in human umbilical vein endothelial cells (HUVECs), indicating the transcriptional activity of CDK7 may be involved in VEGF-activated angiogenic activity of endothelium. Furthermore, through suppressing CDK7 activity, THZ1 suppressed VEGF-activated proliferation and migration, as well as enhanced apoptosis of HUVECs. Moreover, THZ1 inhibited VEGF-activated capillary tube formation and CDK7 knockdown consistently diminished tube formation in HUVECs. Additionally, THZ1 reduced VEGF expression in human RCC cells (786-O and Caki-2), and THZ1 treatment inhibited tumor growth, vascularity, and angiogenic marker (CD31) expression in RCC xenografts. Our results demonstrated that CDK7-mediated transcription was involved in the angiogenic activity of endothelium and human RCC. THZ1 suppressed VEGF-mediated VEGFR2 downstream activation of angiogenesis, providing a new perspective for antitumor therapy in RCC patients.
Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neovascularização Patológica/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neoplasias Renais/tratamento farmacológico , Camundongos , Neovascularização Patológica/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
After chemotherapy for the treatment of metastatic bladder urothelial carcinoma (UC), most patients inevitably encounter drug resistance and resultant treatment failure. Deubiquitinating enzymes (DUBs) remove ubiquitin from target proteins and play a critical role in maintaining protein homeostasis. This study investigated the antitumor effect of PR-619, a DUBs inhibitor, in combination with cisplatin, for bladder UC treatment. Our results showed that PR-619 effectively induced dose- and time-dependent cytotoxicity, apoptosis, and ER-stress related apoptosis in human UC (T24 and BFTC-905) cells. Additionally, co-treatment of PR-619 with cisplatin potentiated cisplatin-induced cytotoxicity in UC cells and was accompanied by the concurrent suppression of Bcl-2. We also proved that Bcl-2 overexpression is related to the chemo-resistant status in patients with metastatic UC by immunohistochemistry (IHC) staining. In a xenograft mice model, we confirmed that PR-619 enhanced the antitumor effect of cisplatin on cisplatin-naïve and cisplatin-resistant UCs. Our results demonstrated that PR-619 effectively enhanced the cisplatin-induced antitumor effect via concurrent suppression of the Bcl-2 level. These findings provide promising insight for developing a therapeutic strategy for UC treatment.
Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tiocianatos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In Fig. 1b, upper part, the cell viability counts after treatment with cisplatin and TSA in T24 cells was by mistake a duplication of the image for NTUB1 on the left. In the corrected version of Fig. 1, the image was replaced appropriately.
RESUMO
In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Neoplasias Urológicas/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Sinergismo Farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/administração & dosagem , Neoplasias Urológicas/genética , GencitabinaRESUMO
Chondrosarcoma is a malignant primary bone tumor. Sirtuin-1 (SIRT1), which is a member of sirtuin family, plays a dual role either in cancer promotion or suppression. There is no report about the role of SIRT1 in the human chondrosarcoma cells. Resveratrol is a potent activator of SIRT1. However, its effects on chondrosarcoma have not been extensively studied. Here, we investigated the role of SIRT1 induction by resveratrol in human chondrosarcoma cell growth and tumor progression. Resveratrol significantly decreased cell viability and induced cell apoptosis in human chondrosarcoma cells in a dose-dependent manner. The protein expression and activity of SIRT1 were activated after treatment with resveratrol. Resveratrol significantly inhibited NF-κB signaling by deacetylating the p65 subunit of NF-κB complex, which could be reversed by siRNA-SIRT1 transfection or deacetylation inhibitor MS-275. Resveratrol induced-apoptosis involved a caspase-3-mediated mechanism. Both siRNA-SIRT1 transfection and MS-275 significantly inhibited the resveratrol-induced caspase-3 cleavage and activity in human chondrosarcoma cells. Moreover, in vivo chondrosarcoma xenograft study revealed a dramatic reduction in tumor volume and the increased SIRT1 and cleaved caspase-3 expressions in tumors by resveratrol treatment. These results suggest that resveratrol induces chondrosarcoma cell apoptosis via a SIRT1-activated NF-κB deacetylation and exhibits anti-chondrosarcoma activity in vivo.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Condrossarcoma/tratamento farmacológico , Resveratrol/administração & dosagem , Sirtuína 1/genética , eIF-2 Quinase/genética , Acetilação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma/genética , Condrossarcoma/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: To investigate the protective effect of epigallocatechin gallate (EGCG), a green tea extract, on partial bladder outlet obstruction (pBOO)-induced bladder injury in a rat model. METHODS: The female Sprague-Dawley rats underwent sham or BOO procedures, and were divided into several groups (sham with saline injection, sham with EGCG treatment, BOO with saline injection, and BOO with EGCG treatment). The rats in each group were randomized into 2 groups (48 hours and 30 days after the BOO procedure) for when their bladders were harvested. EGCG (4.5 mg/kg/day) and saline were administered via intraperitoneal injection after the BOO procedure during the study period. Bladder tissue was examined for inflammation, endoplasmic reticulum (ER) stress-related apoptotic markers by Western blot, and histological staining. RESULTS: BOO induced acute bladder injury (hemorrhage, edema, and neutrophil infiltration) after 48 hours. In addition, cystometry showed a decrease in micturition pressure and intercontractile interval. We also observed increased expressions of cyclooxygenase-2, poly(ADP-ribose) polymerase at 48 hours, as well as ER stress markers such as caspase-12 and CCAAT/-enhancer-binding protein homologous protein (CHOP). Treatment with EGCG significantly improved pBOO-induced histologic changes, bladder dysfunction, and the overexpression of cyclooxygenase-2, CHOP, and caspase-12 at 48 hours. Similarly, EGCG treatment for 30 days effectively recovered compliance and intercontractile interval, submucosal ER stress-related apoptosis (CHOP and caspase-12) at 30 days after pBOO. CONCLUSIONS: EGCG alleviate pBOO-induced bladder injury and dysfunction via suppression of inflammation and ER stress-related apoptosis.
Assuntos
Antioxidantes/uso terapêutico , Apoptose , Catequina/análogos & derivados , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Obstrução do Colo da Bexiga Urinária/tratamento farmacológico , Animais , Antioxidantes/farmacologia , Catequina/farmacologia , Catequina/uso terapêutico , Feminino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC.